IV. Molecular biology of S-layers

Jose manuel Hernandez berenguer

IV. Molecular biology of S-layers

1997, FEMS Microbiology Reviews

FEMS Microbiology Reviews 20 (1997) 47^98 IV. Molecular biology of S-layers 1 è e Gulik-Krzywicki e , Emanuel Shechter c , Agne è s Fouet 3;f , Geèrard Leblon d , Thadde Steèphane Mesnage f , Evelyne Tosi-Couture g , Pierre Gounon g , Micheéle Mock f , Everly Conway de Macario 3;h , Alberto J.L. Macario h , è Berenguer 3;i , è ndez-Herrero i , Garbin ì e Olabarr|èa i , Jose Luis A. Ferna è ra k , Martin J. Blaser 3;j , Beatrix Kuen b , Werner Lubitz b , Margit Sa Peter H. Pouwels 3;l , Carin P.A.M. Kolen m , Hein J. Boot m , Airi Palva 3;n , Michaela Truppe b , Stephan Howorka b , Gerhard Schroll b , Sonja Lechleitner b , Stephanie Resch 3;b a Fachbereich Biologie, Mikrobiologie, Universitaët Rostock, D-18051 Rostock, Germany Institut fuër Mikrobiologie und Genetik, Universitaët Wien, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria c Laboratoire des Biomembranes, URA 1116 CNRS, Universiteè de Paris-Sud, F-91405 Orsay, France d Laboratoire de Biologie Moleèculaire des Corynebacteèries, URA 1354 CNRS, Universiteè de Paris-Sud, F-91405 Orsay, France e Centre de Geèneètique Moleèculaire, CNRS, F-91190 Gif sur Yvette, France f Laboratoire de Geèneètique Moleèculaire des Toxines (CNRS URA 1858 CNRS), Institut Pasteur, 28, rue du Dr Roux, F-75724 Paris Cedex 15, France g Station Centrale de Microscopie Electronique, Institut Pasteur, 28, rue du Dr. Roux, F-75724 Paris Cedex 15, France Wadsworth Center, Division of Molecular Medicine, New York State Department of Health and Department of Biomedical Sciences, School of Public Health, The University at Albany, Albany, NY 12201-0509, USA i Centro de Biolog|èa Molecular `Severo Ochoa', Universidad Autoènoma de Madrid, E-28049 Madrid, Spain j Division of Infectious Diseases, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA k Zentrum fuër Ultrastrukturforschung und Ludwig Boltzmann-Institut fuër Molekulare Nanotechnologie, Universitaët fuër Bodenkultur, A-1180 Vienna, Austria l TNO Nutrition and Food Research Institute, P.O. Box 5815, NL-2280 HV Rijswijk, The Netherlands m BioCentrum Amsterdam, University of Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The Netherlands n Agricultural Research Centre of Finland, Food Research Institute, FIN-31600 Jokioinen, Finland b h 1 This review is part of a series of reviews dealing with different aspects of bacterial S-layers ; all these reviews appeared in Volume 20/ 1^2 (June 1997) of FEMS Microbiology Reviews, thematic issue devoted to bacterial S-layers. 2 Guest Editors. 3 Corresponding authors. Dr. N. Bayan : Tel. : +33 (1) 69157065 ; Fax : +33 (1) 69853715. Prof. Dr. J. Berenguer : Tel. : +34 (1) 3978099 ; Fax : +34 (1) 3978087 ; E-mail : jberenguer@trasto.cbm.uam.es. Prof. Dr. M. Blaser : Tel. : +1 (615) 322-2035 ; Fax : +1 (615) 343-6160 ; E-mail : martin.blaser@mcmail.vanderbildt.edu. Prof. Dr. E. Conway de Macario : Tel./Fax : +1 (518) 474-1213 ; E-mail : everlym@wadsworth.org. Dr. A. Fouet : Tel : +33 (1) 45 68 86 54 ; Fax : +33 (1) 45 68 89 54 ; E-mail : afouet@pasteur.fr. Dr. A. Palva : Tel. : +358 (3) 4188277 ; Fax : +358 (3) 4188444 ; E-mail : airi.palva@mtt.fi. Prof. Dr. P.H. Pouwels : Tel. : +31 (30) 69 444 66 ; Fax : +31 (30) 69 444 62 ; E-mail : Pouwels@voeding.tno.nl. Dr. S. Resch : Tel. : +43 (1) 79515-4125 ; Fax : +43 (1) 7986-224 ; E-mail : Stefanie@gem.univie.ac.at. Dr. H. Scholz : Tel. : +43 (1) 79515-4125 ; Fax : +43 (1) 7986-224 ; E-mail : holger@gem.univie.ac.at. 0168-6445 / 97 / $32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S0168-6445(97)00050-8 Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Hubert Bahl 2;a , Holger Scholz 2;3;b , Nicolas Bayan 3;c , Mohamed Chami c , H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 48 Abstract In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins form the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been thermophilus. Thermus One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7^8 S-layer proteins with a high degree of Bacillus homology at the 5P and 3P ends were found. Environmental changes influence the surface properties of stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology. Keywords: Corynebacterium glutamicum ; cspB gene ; PS2 protein ; S-layer ; Bacillus anthracis ; EA1 protein ; Sap protein ; SLH motif ; Methanogenic archaea ; Antigenic diversity ; S-layer protein ; ABC transporter ; Methanosarcina mazei ; Thermus thermophilus ; Gene regulation ; Bacillus stearothermophilus ; S-layer variation ; Oxygen stress ; Lactobacilli ; slp gene ; Lactobacillus brevis ; slpA gene ; Secretion of L-lactamase ; S-layer gene ; sbs gene ; Heterologous expression ; S-layer structure-function relationship ; SbsA/SbsB fusion protein Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2. The crystalline surface layer of 50 3. Corynebacterium glutamicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Freeze-etching electron microscopy of C. glutamicum cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2.2. Isolation and chemical characterization of the S-layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2.3. Cloning and sequencing of the S-layer protein corresponding gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 2.4. Attachment to the cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 2.5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3.1. Genetic analysis of the S-layer components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Bacillus anthracis S-layer 3.1.1. Cloning of the S-layer genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3.1.2. Sequence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.2. Protein analysis of the S-layer components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 3.3. Phenotypic analysis of wild-type and mutant strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.3.1. Morphological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.3.2. Array structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.4. In vivo expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 4. S-layer and ABC transporter genes in methanogenic archaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 4.1. Universality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 4.2. Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.3. Mosaic complexity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.4. Pleomorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.5. S-layer genes and proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 4.6. Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 4.7. ABC transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 4.8. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5. The S-layer of Thermus thermophilus HB8 : structure and genetic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 6. 8. 9. 10. 65 65 65 66 66 66 67 68 68 68 69 69 70 71 71 75 75 76 76 77 78 78 78 79 79 79 80 81 82 82 82 83 83 84 84 85 85 85 85 87 88 88 89 89 90 90 90 91 91 91 Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 7. 5.1. The expression of SlpA is a well regulated process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Searching for transcriptional repressors in heterologous systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. SlrA is a transcriptional repressor of slpA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Coregulation in the expression of regular proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Does SlpA regulate its own translation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular genetics of variation of S-layer proteins of Campylobacter fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Structure of the sapA homologues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Physical arrangement of the sapA homologues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Rearrangement of the sapA homologues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S-Layer variation in Bacillus stearothermophilus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Investigation of sbsA in the S-layer de¢cient variant T5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Oxygen-triggered change in S-layer protein synthesis and isolation of the p2 variant . . . . . . . . . . . . . . . . 7.2.1. Physiological and morphological changes during variant formation . . . . . . . . . . . . . . . . . . . . . . . 7.2.2. Cloning, sequencing, and expression of sbsA and sbsB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3. Detection of sbsA in the p2 variant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.4. Detection of the second S-layer gene sbsB in the wild-type strain PV72 . . . . . . . . . . . . . . . . . . . . 7.2.5. Detection of sbsB in the S-layer de¢cient variant T5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Identi¢cation of plasmid located sbsA homologues in B. stearothermophilus PV72 and the variant T5 . . 7.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S-layer protein genes of Lactobacillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Relationship between the presence of an S-layer and adherence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. S-layer protein of L. acidophilus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. S-layer protein genes of L. acidophilus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Genetic organization of S-layer protein region of L. acidophilus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. Occurrence of two slp genes in other lactobacilli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. Regulation of expression of slpA gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. Expression of L. acidophilus S-layer protein in a heterologous host . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8. Antigenic variation in L. acidophilus? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9. Potential applications of lactobacilli as vehicles for antigen presentation . . . . . . . . . . . . . . . . . . . . . . . . . Molecular biology of the Lactobacillus brevis S-layer gene (slpA) and utility of the slpA signals in heterologous protein secretion in lactic acid bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Characterization of the L. brevis S-layer protein and gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. In vivo expression of the slpA gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1. mRNA analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2. S-layer synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Heterologous protein secretion with the L. brevis S-layer signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.1. Construction of a secretion vector based on the slpA signals . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2. Expression and secretion of Bla by the slpA secretion cassette . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.3. pH controlled cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biotechnological applications of recombinant S-layer proteins rSbsA and rSbsB from Bacillus stearothermophilus PV72 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. Heterologous expression of sbsA and sbsB in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Heterologous expression of sbsA in Bacillus subtilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Structural/functional analysis of SbsA and SbsB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4. Construction of SbsA/SbsB fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.1. Insertion of streptavidin in SbsA/SbsB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.2. Insertion of Bet v 1 in SbsA/SbsB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.3. Insertion of pseudorabies virus antigens into SbsA/SbsB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.4. Insertion of PHB synthase (PhbC) of Alcaligenes eutrophus H16 into SbsA . . . . . . . . . . . . . . . . 49 50 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 10.5. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction 2. The crystalline surface layer of Corynebacterium glutamicum Nicolas Bayan3 , Mohamed Chami, è Gerard Leblon, Thaddeèe Gulik-Krzywicki and Emanuel Shechter C. glutamicum is a Gram-positive bacterium widely used for the industrial production of amino acids such as glutamic acid. Our knowledge concerning the metabolism and the molecular biology of this microorganism is now well developed. In contrast, little is known about the structure and composition of the cell wall. In this article we report on the information available about the crystalline surface layer of corynebacteria. 2.1. Freeze-etching electron microscopy of C. glutamicum cells The various levels of the cell wall of C. glutamicum were visualized by electron microscopy using freezefractured and deep-etched preparations of whole untreated cells [1]. Under our experimental conditions, the main fracture plane propagated within the cell wall of the bacterium, close to the cell surface (SL) (Fig. 1A), and produced two fracture surfaces named F1, the convex fracture surface, and F2, the concave fracture surface (Fig. 1B). In some rare instances, the fracture was propagated within the cytoplasmic membrane (CM) (Fig. 1C). This unusual behavior, which was also observed by Richter et al. [2], suggests the presence of a hydrophobic layer in the cell wall. The cytoplasmic membrane (CM) was densely covered with particles representing integral membrane proteins. The cell surface as well as both fracture surfaces, F1 and F2, were covered with ordered arrays undoubtedly representing the so called S-layers found in nearly all taxonomic groups [3,4]. Optical di¡raction of these lattices (Fig. 1A, inset) showed a hexagonal symmetry with a cell unit diî . Surprisingly, for some strains, mension of 132 A the area occupied by the ordered arrays on the cell Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Since the discovery of S-layers in 1953 of a Spirillum sp. by Houwink, the structure of hundreds of di¡erent S-layer proteins and their morphological properties in a wide range of microorganisms have been studied in detail. In the past years genetic investigations of bacterial S-layer genes have become an important, additional contribution for a better understanding of S-layer protein and gene organization. However, little is still known about the regulatory mechanisms of transport, biosynthesis and the domains which are responsible for intra- and/or intermolecular interactions of layer proteins. Although all S-layers share the identical feature of a two-dimensional (glyco-)protein layer covering the bacterial cell, the analysis of various S-layer proteins revealed the existence of a great diversity in their amino acid composition. The majority of S-layer genes sequenced so far have only little sequence identity, suggesting an analogue rather than a homologue evolutionary process of these structures. An important contribution to S-layer diversity and the understanding of S-layer gene regulation represents the ability of an individual bacterial species to express di¡erent S-layer genes under altered environmental conditions. The investigation of S-layer variation at the molecular level will allow us to get a better insight into gene regulation of bacteria. During the last years S-layers have been shown to have considerable application potentials. Beside the chemical modi¢cation of S-layers, like the immobilization of functional molecules, the construction of recombinant S-layers by insertional mutagenesis opens a new way in vaccine development. The speci¢c properties of the S-layer protomers in combination with molecular techniques will allow the synthesis of recombinant S-layers with high potential in biotechnical applications. The following articles will give an overview of the knowledge we have today about S-layer gene expression, variation and the construction of recombinant S-layer genes. 91 91 92 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 C. glutamicum !" # # $%& ' ( ) * % + ,# - !" # # . !" + # $%& + ( ( + .( #" ""/ - W" H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 # !" !# 6 )$ $ ) * + 8 9;7<3 Bacillus 2.2. Isolation and chemical characterization of the S-layer brevis ,- - Corynebacterium diphtheriae %, . Aquaspirillum serpens ( / Aquaspirillum 0 1 "2 Nitrosocystis oceanus "" Lampropedia hyalina "# 7 8 ! # * ( !" !# % $ ' !" !# !" & 9 : C. glutamicum 2 W : 8 Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Bacillus thuringiensis H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 53 to almost all S-layers, denaturing agents such as urea would be strictly dependent on the presence of the or guanidine hydrochloride were unable to detach PS2 lattice. In this case, the underlying lattice is the S-layer from the cell. The release of large sheets probably not a protein since PS2 is the only one of S-layer required incubation of the bacteria with found in S-layer sheets isolated with SDS. Its nature 2% SDS indicating that strong hydrophobic interac- remains tions are involved. Under these conditions, the lattice S-layer of was not disrupted unless the preparation was heated PS2 lattice, then one has to assume that the protease to 100³C indicating that the bonds between lattice cleaved part of the polypeptide chain of PS2, respon- subunits are stronger than those with the underlying sible for the formation of the inner face of the cell wall material. Such stability for an S-layer has S-layer. durans (formerly meister et al. [13]. determined. Alternatively, if the is composed of a single 2.3. Cloning and sequencing of the S-layer protein corresponding gene The large sheets of S-layer released upon SDS treatment were recovered after di¡erential centrifu- be C. glutamicum The cspB gene encoding PS2 was cloned in V gt11 gation. These sheets display two di¡erent faces (Fig. by immunological screening [16]. Analysis of the nu- 2A). One resembles the ordered arrays seen on the cleotide sequence revealed an open reading frame of cell surface ; the other resembles those observed on 1533 nucleotides. The deduced 510 amino acid poly- the F2 fracture surface. Biochemical analysis of these peptide has a calculated molecular mass of 55 426. sheets indicated the presence of a single protein (mo- No signi¢cant homologies were found with other lecular mass 63 kDa) corresponding to PS2, the ma- proteins but PS2 shares several features with surface jor cell wall protein of layer proteins. The similarities include a high content C. glutamicum (Fig. 2A, in- set). According to our data, PS2 is, in contrast to of hydrophobic amino acids (45.2%), a higher pro- several S-layer proteins, not glycosylated [14,15]. In- portion of acidic amino acids (17.7%) as compared cubation of the cells in the presence of non-speci¢c to basic amino acids (8.3%), a very low content of proteases also led to the release from the cells of sulfur-containing amino acids (totally absent in the large sheets of S-layers. However, these protease mature form of PS2) and the presence of putative sheets displayed symmetrical faces resembling the or- N-glycosylation dered arrays seen on the surface of the bacterium quence homology of PS2 with other known surface (Fig. 2B). SDS-PAGE analysis revealed the presence layer proteins is not unexpected as surface layer pro- of a slightly truncated form of PS2 (molecular mass teins display little sequence homology between them- 51 kDa) in this preparation (Fig. 2B, inset). If the selves [17]. S-layer of C. glutamicum is composed of two distinct sites (seven). The absence of a se- S-layers, which are the outermost envelope com- lattices, then the protease may have cleaved the do- ponent, represent an important interface between the main of PS2 involved in the association with the cell and its environment. They probably act as a second barrier controlling the exchange of molecules, as pro- lattice of the S-layer whose organization Fig. 3. A simpli¢ed sequence of PS2. The putative signal sequence and the hydrophobic C-terminal sequence are in bold. Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Deinococcus radioMicrococcus radiodurans) by Bau- also been described in the case of to H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 "7 cspB aphIII # C. glutamicum ! Streptococcus faecalis "P $ % ! # ' ) -. C. glu- ) $ '12 L Clostridium thermocellum Streptomyces limosus ! 3 C. glutamicum , 2 $ ! % E. coli % ( ! 4 7 fetus - + ! 80/ "9 % Caulobacter crescentus ! ) - 4 ! ) $ , ! ! 7/ $ $ $ $ 4 C. glutamicum % ) ) ,/ ' ! 0! + ! ,/ ! ! -. ! # -. tamicum * + C. glutamicum ( $ & ! Campylobacter / 5 80 4 ! ) + tamicum ( ) $ * ! C. glu- !! % 2.4. Attachment to the cell $ ! ! % 7 ! ' ( ( 2 ' $ 4 - 56 / ! . 7 ;$(<1 C. glutamicum $ - 4<8,"/ / =0 8 ! '4( # - 4<87/ 8 ;A 4A - 4<8,"/ 8 1 A " " ! 8 = A 4 4 4<8 @ ! >;650 ? 7 - ( A 2 - 4<87/ - 4<87/ " " 4<8 % 8 4<8 4<8 4<8 " " 4<8 " - 4<8,"/ Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 ! ) $ H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 2.5. Conclusions and perspectives The presence of an S-layer at the surface of C. glutamicum has been clearly demonstrated. However, the exact organization of this structure on the surface of the bacterium is still unclear. Indeed, freeze-etching electron microscopy studies did not allow us to discriminate between the presence of one or two superimposed lattices. Additional structural studies are required. Yet, at this stage, ultrathin sections or negatively stained preparations of isolated S-layer sheets have not given additional information. However, it is clear that PS2 is responsible for the formation of this S-layer. This protein is constituted of two domains. The N-terminal domain involves the major part of the polypeptide and is responsible for monomer interactions. The C-terminal domain is responsible for the attachment of the protein to the cell wall. In this latter domain the terminal hydrophobic stretch of amino acids is absolutely required. Such distinct domains have also been described for some other S-layer proteins [37,38], but the presence of a hydrophobic terminal sequence is not frequent. Its presence in corynebacteria may be related to that of mycolic acids which are responsible for the formation of a hydrophobic barrier as is the case in mycobacteria [39,40]. This remains to be determined. 3. Bacillus anthracis S-layer Agneés Fouet3 , Steèphane Mesnage, Evelyne Tosi-Couture, Pierre Gounon and Micheéle Mock Bacillus anthracis is the etiologic agent of anthrax, a disease which occurs in many animals including man. It is a Gram-positive spore-forming bacterium. The major virulence factors are two toxins and a poly-Q-D-glutamic acid capsule; their corresponding genes reside on two plasmids [41,42]. The surface of non-capsulated vegetative bacilli appears layered [43], and fragments display a highly patterned ultrastructure [44]. S-layers are found ubiquitously [45], and they may be an important virulence factor [4]. Therefore, we investigated the S-layer of this pathogenic organism in a strain cured for both plasmids. 3.1. Genetic analysis of the S-layer components 3.1.1. Cloning of the S-layer genes We initially hypothesized that a major bacterial protein, which is often found in high abundance in the B. anthracis culture supernatant, was an S-layer component. This protein (molecular mass 94 000) is produced by various B. anthracis strains, including strains without plasmids, and must therefore be chromosomally encoded. The N-terminal sequence of this protein (Sap, surface array protein) was determined as well as those of polypeptides obtained Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 phobic anchoring signal seems to be very speci¢c since no similar sequences are found in all known S-layers except for Halobacterium halobium [29], Haloferax volcanii [30] and Rickettsia prowazekii [31]. For R. prowazekii, a Gram-negative bacterium, the C-terminal hydrophobic sequence could be anchored in the outer membrane, while for halobacteria it was proposed that the C-terminal hydrophobic sequence is anchored in the cytoplasmic membrane [32]. In the case of C. glutamicum, it is di¤cult to assume an insertion of this sequence in the cytoplasmic membrane since the cell wall of corynebacteria is very thick (Fig. 1). The S-layer is thus located far above the plasma membrane. Alternatively, PS2 could be anchored in a hydrophobic layer of the cell wall. The existence of such a layer is suggested by the recent ¢nding of porins in Gram-positive bacteria [33] and more particularly in corynebacteria [34]. This layer may be composed of mycolic acids known to be present in the cell wall of C. glutamicum [35]. As we mentioned, PS2 deleted from its hydrophobic anchor is shed in the medium as monomers suggesting that the formation of the S-layer lattice requires the interaction of PS2 with the cell. The association of PS2 with the cell surface by the C-terminal hydrophobic sequence may favor interactions between monomers by lateral di¡usion on the surface of the bacterium. The surface of the cell would act as a matrix for crystallization, a phenomenon similar to that leading to the crystallization of proteins on lipid monolayers [36]. 55 0 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 7 0 ' B. anthracis ' " Campylobacter fetus . / ,-" 0 3.1.2. Sequence analysis - 1 23304 23#4 0# sap eag % B. brevis cwp ' * ' , 1 05 "1 6 b eag ' $.# & !"# $.5 & $7 0& ) 1 03 / '8 8 50 / 5 9 : *: 7 % 9 '8 9 " + <' $= "& B. licheniformis Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 sap !"# $ % #& vsap ' ' ( !"# ) sap eag !"# eag ) sap eag % * eag sap !"# sap ( + Bacillus brevis ' * ' sap eag B. anthracis ,-" ' + % ( sap eag " ' H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 57 tity decreasing to 22%. Thus these proteins could be but also an original poly-Q-D-glutamic composed of two domains ; the ¢rst one, constituted [56]. Selective pressure could by the SLH motifs, could be the consequence of a curred duplication the S-layer event. This SLH harboring domain to minimize changes protein amino therefore in the capsule have oc- sequence acid residues of interact- could permit the anchoring of these proteins to the ing with the capsule. Comparison of these two or- peptidoglycan containing sacculus [25,26,54]. ganisms' cell wall structures would be very informa- Further research in the protein data banks showed very little similarity for Sap outside the SLH motifs. This is in contrast to EA1 which is very similar to the sequence of the Bacillus licheniformis [54a]. The ¢rst 200 residues share The presence of two S-layer components could be 93% due to the simultaneous synthesis of both proteins, still or to the activation of a second gene in the deleted very high, 63% and 76% respectively, for the rest mutant. The protein content of the wild-type strain of the proteins [46a]. Since a high level of simi- was analyzed by SDS-PAGE, and, after obtaining larity, such as that found between EA1 and RS, speci¢c antibodies to EA1 and Sap, by Western blots is infrequently encountered in S-layer proteins, it and by immunoelectron microscopy. The results in- can be hypothesized that they are derived from the dicated that both proteins are synthesized in the same and wild-type strain. EA1 is nearly exclusively cell asso- possess not only an S-layer [55] ciated, whereas Sap is equally cell associated and in identity and 97% ancestral B. licheniformis similarity ; protein. the Also, scores are B. anthracis Fig. 6. Sedimentation properties of wild-type and S-layer mutant strains. Shaking was stopped 15 min before this photo was taken. Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 5) 3.2. Protein analysis of the S-layer components RS (OlpA) S-layer protein (accession number U38842) (Fig. tive. H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 Aquaspirillum serpens B. brevis ! Lactobacillus acidophilus C. fetus Thermus thermophilus Bacillus stearothermophilus L. acidophilus " # $ '% philus ! $ C. fetus & T. thermophilus )* % & ! ( ! B. stearothermo- " $ + , & 0 & B. anthracis 8 935 $ / $ ( ( vsap veag $ vsap veag , 3 3 ! # . ( /0 )1 veag $ vsap - 3.3.2. Array structure 2 " $ - ) 7 , * 1 2 3 Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 3.3.1. Morphological aspects 3.3. Phenotypic analysis of wild-type and mutant strains H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 3.4. In vivo expression The in vivo expression of each protein was analyzed by Western blots, using sera from mice injected with an EA1 Sap strain. A strong signal was obtained with EA1, but Sap was barely visible. Thus, EA1 is synthesized in vivo, whereas Sap is either weakly produced or not antigenic. In fact, EA1 appeared as a major antigen. Our data support the conclusion that the previously described extractable antigen 1 [63] and the S-layer component that we have studied are the same protein. The role of the S-layer in B. anthracis is not easy to conceptualize. Fully virulent bacilli are also capsulated, and the capsule could mask the S-layer from the host macromolecules. The B. anthracis S-layer(s) is composed of two simultaneously synthesized proteins with a molecular mass of 94 000. The N-terminal sequences, composed of three SLH motifs, are highly similar. Re¢ning the array structure should enable us to de¢ne whether there are one or two S-layers. The in vivo expression of both components can also be further analyzed. The contribution of the relevant proteins in virulence will then be studied. We will assay the possible advantages provided by this structure to the bacterium, such as in vivo survival or development. After injecting mutants constructed during the course of this work and others from a capsulated strain, protection against infection and antigenic responses will be assayed. 4. S-layer and ABC transporter genes in methanogenic archaea Everly Conway de Macario3 and Alberto J.L. Macario Immunologic analyses have revealed that archaea are immunogenic, and that while their antigenic relationships are coherent within each group and parallel their phylogenetic classi¢cation, their antigenic mosaics are quite diverse [64]. This antigenic diversity surely re£ects diversity and variability of surface envelope structures. Among these structures, those belonging to the S-layer must contribute signi¢cantly to the antigenic mosaic [4,45,65,66]. This brief review will focus on methanogenic archaea (methanogens), and will proceed from the picture uncovered by immunologic analyses to the data currently available on the genes encoding surface structures, with comments on what these data suggest about possible mechanisms responsible for generating molecular (antigenic) diversity. A recently discovered gene cluster, encoding proteins of the ABC transporter system which are important structural and functional components of the cell's envelope, will also be discussed. Due to strict space limitations, the scope will be reduced to essentials. 4.1. Universality Methanogens have been found in many di¡erent ecosystems. For example, immunologic analyses along with other studies have demonstrated the presence and variety of methanogens in human and ani- Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 wild-type and the three mutant strains. B. anthracis cell envelopes were obtained after disruption and negatively stained (Fig. 7). Preliminary optical diffraction analysis suggests that both the wild-type and the vsap mutant envelope micrographs exhibit di¡raction patterns, whereas no di¡raction pattern was observed in the double mutant. Thus EA1 is indicated to be an S-layer component. The di¡raction patterns obtained with micrographs from wildtype and vsap envelopes seem slightly di¡erent, indicating that Sap is also an S-layer constituent. The slight di¡erence suggests that the EA1 lattice could determine the main pattern of the architecture of the B. anthracis S-layer. However, we failed to visualize an array in the veag mutant. Thus, either there are two S-layers, with Sap forming a more fragile structure, requiring the EA1 array to be stable, or there is a single S-layer, with EA1 and Sap forming a protein complex in an array. Although most S-layers result from the assembly of a single protein [45], certain S-layers contain two abundant surface proteins which are synthesized simultaneously. Some organisms, such as A. serpens, seem to have two superimposed S-layers composed of one protein each [57]. For B. brevis, the arrays are di¡erent with either one or both proteins [58]. Alternatively, in Clostridium perfringens, the S-layer is composed of two proteins in equal amounts; no regular array is observed with the self-assembly of each protein alone [62]. 59 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 ! "# " $ % " & % " $ # " % % ' ( " " # # " ) ' * # +# , "' 4.2. Diversity - ' . / " # 0 # ' 1 # # / # +'' " $ # , " 0 ' * # # " 0 # " # ' 2 # # 0 # # +'' " 3 , + # . ",' * " # Methanobrevibacter smithii # # & " " # 4. %!' 2/ & M. smithii " # " & + , " # +(" ,' 4.3. Mosaic complexity 5 (" # 4. / / 0 + , #' ( # + 1, ' ( # # ' 2 / # Methanobacterium thermoautotrophicum v) / # " " # $ % ' . # # / % ' M. smithii 4.4. Pleomorphism 6 0 # ' 2 / Methanosarcina mazei . Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 (" * / # Methanobrevibacter smithii # " " 1a 5 b 9c : " +;, " # (< d 1 ! +, = = = = = = 11 +, 3 = = = = = ! 111 % + , 3 3 = = = = 1> +, 3 = 3 3 = = > +, 3 3 = 3 = = >1 + , 3 3 3 = = = >11 +, 3 3 3 3 = = >111 +, 3 3 3 = 3 3 1? +!, 3 3 3 3 3 3 ( !e 4 % a 2/ 3 Methanobrevibacter smithii " # %%!' b 7 # 3 " " " * 2 ' c 1 $ " " " * 2 +'' # 11 " # $ ,' d ( & # 3 " # " " * 2 # +1 1?,' e 2 +'' # , "' H. Bahl et al./FEMS Microbiology Reviews 20 (1997) 47^98 $ & $ & %.1 $% $ $ )'* !1 6 - K 2 .P $ " & (1. $ " L orf $ & 0 $ & 8 M. fervidus 2 !' "& $ & $ $ # $% $ " & $ " - M. mazei 7))P $ " )7. !' M. sociabilis M. mazei orf orf orf +%.!( / +!1 $ & $ " $ & $% ncr3 # # , $ ncr1 ncr2 !' "" 3 ! - - & $% # ++* : !( $% Methanothermus fervidus Methanothermus sociabilis M. mazei $ & " $ ! M. mazei .1% %.1 4.5. S-layer genes and proteins slg M. mazei !' M. mazei " # 9-9 ).1 +7! 9 - Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 3 $ %( 62 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 4.6. Mechanisms The mechanism(s) involved in generating the observed antigenic (surface) diversity of methanogens has not been elucidated. Perhaps, the organization and structure of the genes described in the preceding section play a role in the generation of surface diversity. It is possible that mechanisms similar to those postulated to operate in Bacteria and/or Eucarya for generating phase and antigenic variation, and other antigenic switches [93,94], are also operative in archaea. Expression, or absence thereof, of a particular gene coding for a protein antigen, and expression of either one or the other of a series of versions of the same gene-protein antigen, may be part of the general mechanism that generates di¡erent envelopes and cell surfaces. Moreover, expression of a protein, or glycoprotein, not always present, may mask one or more antigens or antigenic determinants, and thereby render them unavailable for reaction with antibodies. This masking phenomenon can lead to the erroneous conclusion that a given protein, or determinant in it, is not present in the envelope when in fact it is just hidden. Antigen masking adds to the complexity of the phenotype as detected by antibodies. The molecular genetic mechanisms leading to phase and antigenic variation in pathogens are believed to operate at the pre- and posttranscription levels, as well as during transcription itself. Phase variation, exempli¢ed by the absence or presence of a protein antigen, may be caused by a pretranscriptional deletion of a gene (or part thereof), or translocation of the gene to a place where there is no functional promoter, or removal (or mutation) of the promoter. Transcriptional ablation or down-regulation may be due to a number of processes involving repressors, absence of activators (lack of them, or of their activation), and failure of the signal transduction pathway (if the gene in question is inducible by outside factors). Posttranscriptional down-regulation may involve changes in mRNA stability-degradation and processing, inhibition of translation (ribosome binding and/or elongation), and alteration of protein (antigen) translocation to the cell's surface. Any one of the above processes can cause the absence of a protein antigen, properly folded and postsynthetically processed, from the cell's surface. Antigenic variation pertains to a single antigen molecule which is always expressed but in di¡erent versions of itself, e.g. longer or shorter than usual, with or without modi¢cations of its amino acid sequence (e.g. amino acid substitutions). One or more of these structural changes will result in antigenic variation detectable by antibodies. The mechanisms that cells use to express on their surface either one or another of several versions of the same protein antigen are varied. For example, a cell may use a single gene with repeats or modules to generate proteins with di¡erent lengths depending on how many mod- Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 are very similar to each other (Fig. 8). Four shorter repeats, named 1^4 from the N- towards the C-terminus, each 56 amino acids long, occur in the same region of the molecule, mostly overlapping the long repeats [90]. These shorter repeats are quite similar to each other, particularly repeats 2 and 4. Interestingly, repeats also occur in other open reading frames (orfs) adjacent to the gene that encodes SlpB (unpublished results), and in another cluster of genes that also encode envelope proteins (Fig. 9) [92]. In this cluster, three genes, 5P-orf492-orf375-orf783-3P, encode the proteins ORF492, ORF375, and ORF783, respectively. ORF492 (i.e. the protein encoded by the gene at the 5P end of the cluster) has a putative signal sequence, as SlpA and SlpB do [91], and the mature protein is made up of repeats (AB), two half repeats (A and B), a segment C (which is a repeat in ORF783), and a hydrophobic stretch. Very similar AB repeats, A and B half repeats, C segment, and hydrophobic stretch are present in ORF375. The protein ORF783 is truncated (no sequence is available for the 3P end of orf783), and it is constituted of C repeats exclusively, which are very similar to the C segments present in the other two ORFs. The AB and C repeats average 42 and 85 amino acids, respectively. The AB repeats are quite similar to each other, and so are the C repeats among themselves. However, the latter can be grouped into families or clusters composed of very similar repeats each (unpublished results). The AB repeats contain the SLH domain found in many S-layers [26]. These genes were expressed in vivo [90,92]. H. Bahl et al./FEMS Microbiology Reviews 20 (1997) 47^98 M. ma- zei 4.7. ABC transporters Two genes, orfD and orfF encoding the proteins OrfD and OrfF, respectively, were discovered in the genome of M. mazei S-6, which are homologues of the nucleotide binding components D and F of bacteria and eukaryotes [95]. The signatures typical of these components are present in the archaeal proteins (Table 2). Shorter motifs were identi¢ed that are the most conserved within the signatures or boxes. These short motifs are the amino acid triad GKS for box A, SGG for the ABC transporter family signature, DEP for box B, and HD for box C. Exceptionally, GKT replaces GKS, DDA or DDP occurs instead of DEP, and HR or HK substitutes for HD. SGG is a highly conserved triad, which is present even when the rest of the ABC transporter family signature is hardly discernable at the expected location (see Table 2). Recently, the other components of the archaeal ABC transporter system were cloned and sequenced [96]. The gene cluster is 5PA- B- C- D- F-3P (Fig. 10). This organization is typical but not universal for bacterial systems. In some cases the D and F genes are upstream of B and C, while the A gene is at the 3P end of the cluster (see Fig. 10). A rather common feature is the overlapping of adjacent genes over stretches encompassing between 1 and 20 bases for example. In eu- orf orf orf orf orf Table 2 Comparison of the sequence and structural features of the archaeal nucleotide binding molecules OrfD and OrfF with those of bacterial and eucaryal homologuesa Proteind Length (aa) Percent aac I S Motif startb ATP/GTP binding box A (8 aa) OrfD Bacterial range ( = 9) Eucaryal range ( = 6) N-terminal half 317 253^358 1276^1582 n.a. 45 n.a. 31.2^44.7 52.2^62 36^54 21.0^26.3 41.9^52.5 426^459 (n = 4)f OrF Bacterial range ( = 9) Eucaryal range ( = 6) C-terminal half 232 268^335 1276^1582 n.a. n.a. 44 31.7^40.0 58.3^64.0 41^57 22.1^30.5 48.9^54.4 1066^1379 n n n n e ABC transporter (15 aa) 158 141^167 530^549 ( = 4)g n 150 151^165 1172^1193 ( = 2)h n Data from [95] in which the names of the Bacterial and Eucaryal species compared and the data base accession numbers and references for the proteins are given. Reproduced with permission of the copyright owner. b Molecular signatures de¢ned by the GCG program Motifs. The amino acid position at which the motif begins in the sequence is indicated. c aa, amino acid(s); I, identity; S, similarity (identities plus conservative substitutions). d Data base accession numbers and literature references are listed in [95]. e Not applicable. f The sulfonylurea receptor proteins examined have a box A in a di¡erent location, beginning at position 713. g The sulfonylurea receptor proteins examined do not have this motif. h The cystic ¢brosis transmembrane conductance regulator and sulfonylurea receptor proteins examined do not have this motif. a Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 ules are transcribed, or to generate proteins not only di¡ering in length but also in sequence depending on how the modules are rearranged before transcription. Modules are usually similar but not identical to each other. Thus, depending on which repeats are brought together in the coding region and then transcribed, the ¢nal protein product will vary. If, in addition, genes with similar repeats or modules in a cluster (e.g. operon) are used by a cell in a coordinated fashion, the possibilities for generating variability multiply. The genes encoding the S-layer proteins in S-6 described in the preceding section show repeats or modules. These genes are organized in clusters with a putative promoter of the archaeal type in the 5P £anking region of each cluster. Some of the features of the genes' nucleotide sequences and of the deduced mRNA sequences suggest that regulation at the levels of transcription and translation is possible. The presence of modules in all genes and the sharing of some of these modules between the genes indicate that rearrangements and module exchanges may have occurred during evolution, and probably do still occur frequently enough to generate surface diversity. Future research ought to address these points and elucidate the mechanism(s) involved in gene and module rearrangements, and in the regulation of the rearranged genes. 63 &F H. Bahl et al./FEMS Microbiology Reviews 20 (1997) 47^98 M. mazei Lactococcus lactis Salmonella typhimurium, Bacillus subtilis !"# $ %&# ' (' ) (' # $ ) ' * + # M. mazei 4.8. Perspectives , # # ' - " . / " / " " (' / " 0 / " ' 1 # 5. The S-layer of Thermus thermophilus HB8: structure and genetic regulation 2 ' 3 14 5 6 7 83 93 : T. thermophilus 4; ) % <== ===" % ' >?@<==A# % ' ) + ><=<<=!A# 4) 0 % ' ><=<A# , %% >?;A# $ Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 # <=# % ' 1 CP :P " %& # * ) D # 0 ) # 4 ) # $ E ) )# E >?&A# ( # H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 65 the structure of the tetragonal array was similar to are usually found as the binding sites for proteins that of bacterial porins, supporting the existence of that inhibit or activate the transcription of the target slpA an ancient phylogenetic relationship between these gene [112]. Furthermore, proteins [103]. long leader mRNA which could play a role in its In addition to the strong horizontal interactions is transcribed with a stability, similar to that described for other S-layer E. coli. within SlpA molecules, there is evidence supporting genes [113], and for the OmpA protein of the existence of a speci¢c binding to the underlying However, alternative roles in the control of expres- peptidoglycan layer. In fact, solubilization of the sion of SlpA are also possible. S-layer protein by neutral detergents requires the digestion of the peptidoglycan layer [98]. Moreover, 5.2. Searching for transcriptional repressors in heterologous systems terminus of SlpA resulted in the inability to bind peptidoglycan fragments in a Far-Western [26]. The functionality of the PslpA in E. coli allowed us to develop a genetic system for the detection of 5.1. The expression of SlpA is a well regulated process putative repressor genes among an expression library of T. thermophilus HB8 DNA [60]. In the system, we used a transcriptional fusion between the PslpA proThere is evidence supporting the existence of tight controls on the expression of the SlpA protein : (i) There are no stocks of SlpA in the cell, nor can any secreted S-layer protein be detected in the culture media. Moreover, when T. thermophilus slpA was cloned in HB8 using a multicopy vector [104] there was no overexpression of SlpA. peptidoglycan [105], is encoded in a divergent tran- slpA [110]. From around 30 000 clones checked, three genes whose expression in E. coli [106]. The presence repressed the expression of the reporter gene were reproducibly cloned. One of the genes, termed (ii) GlmS, an essential enzyme in the synthesis of scriptional unit upstream of moter and a reporter gene (lacZ). Speci¢city controls were carried out with other transcriptional fusions cytoplasmic slrA, protein. was shown to encode a Surprisingly, the genes were identi¢ed as 5P fragments of slpA other slpM two and [110,111]. of the genes encoding GlmS and SlpA clustered in the chromosome and transcribed from divergent pro- 5.3. SlrA is a transcriptional repressor of slpA moters suggests the existence of mechanisms that coordinate their expression, as has been demon- The slrA gene codes for a cytoplasmic protein strated in a number of cases of similar genetic archi- (molecular mass 27 000) with no homologous pro- tecture [107]. teins in the gene banks. Nevertheless, the overall (iii) The deletion of slpA causes severe defects in properties of SlrA, especially its size and basic iso- cell growth and division [108], and leads to the over- electric point, were in agreement with a putative role expression of a regular array built up by the SlpM as DNA binding protein. This possibility was clearly protein [60]. The overexpression of slpM supports shown by South-Western blots, in which a labeled the existence of a genetic control in like manner to DNA fragment containing the PslpA promoter was what has been described for bacterial porins [109]. used as probe [110,111]. The ability of SlrA to bind The identi¢cation of SlpM as a regulator of SlpA speci¢cally to the PslpA promoter suggests a role as added transcription factor of the further arguments to these relationships [110,111]. (iv) A last argument comes from the structure and transcription of the PslpA promoter. The is transcribed slpA gene for this protein. More convincing evidence was further obtained by from a complex slpA promoter gene in vivo gene replacement [111]. Under the optical microscope, slrA : : kat mutants were indistinguish- which able from wild-type cells. However, lower growth presents inverted repeated sequences separated by rates were detected in liquid cultures, suggesting two complete helix turn distances within a bent some defects in cell metabolism or division. Further- DNA region [100]. As is well known, these structures more, cells started to grow much later than the wild- Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 the deletion of an SHL domain [25] from the N- H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 ?? 4! $% ! & "# ! 5.4. Coregulation in the expression of regular proteins & slp' slp E. coli & & ' !( ! ! ! !( . ! 4! $% & # !lac6 $% % ! 4! $%# slp $% )***- . ! ( 7 4! 5.5. Does SlpA regulate its own translation ? slp # ' . )*+,- # ! /0 # 4! . ! ' ! ! / **2 . ! ! $% 3 ! 1 $% ! ! slp & slp' kat T. thermophilus# ' 0 # 9 slp $% 3 ! slp ! # ! )**8- 5 # ! $% ! # # slp $% 4 # # ! "# % ! slp' kat slp' 5 # 2# slp' kat slp 5.6. Conclusions and perspectives *++ +++2# 1 # / 0 1 1 slp' kat /0 1 ! 2 3 0 slp' kat . ! ! Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 "# slr kat ! H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 !: slp !"# $ % & ' ( )) slp * ( )) ( + , $ 3 ( )) - + ' ( )) . + + ( / , / T. thermophilus -01 + 6. Molecular genetics of variation of S-layer proteins of Campylobacter fetus 2 0 Campylobacter fetus + + 3 + . + Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 ( )) T. thermophilus -01 T. thermo-01 . ;3 + < / 3 slp slp glm slr $ 3 slp ' ( slp slp ' $ slp 5=slpA < + + 3 !; 3=!; 3 philus 68 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 via antigenic variation [20,115^118]. Thus, the S-layer is a critical virulence factor for this organism. C. fetus strains have been divided into type A and type B, based on their lipopolysaccharide (LPS) characteristics; cells of each type are covered by either one of two parallel families of S-layer proteins (SLPs) that range in molecular mass from 97 000 to 149 000, and form hexagonal or tetragonal crystals [115,119]. The SLPs of the type A strains are encoded by sapA and seven or eight sapA homologues, each of which encodes a full length SLP open reading frame (ORF) [49,120,121]. Each homologue is conserved at the 5P end [49,120,121] and is unique at the 3P end. The middle region is semiconserved among the homologues [121]. A nearly identical pattern is found for the sapB homologues present in type B strains [119]. The major di¡erence between sapA and sapB is the 5P conserved region which although substantially di¡erent in primary sequence encodes deduced peptides that are similar in secondary structure. These regions are responsible for the type-speci¢c LPS binding of the SLP. Interestingly, sapA and sapB are identical except for the nucleotides encoding the ¢rst 190 amino acids of the protein. PCR and hybridization data indicate that sapA1 and sapB1, and sapA2 and sapB2 are homologous to one another as well. These data suggest that such similarities will be present for each of the sapA and sapB homologues [119]. In addition, non-coding regions both upstream and downstream of the ORFs are identical [119]. This strong conservation of non-coding sequences implies important functions for these regions as well. The conserved features upstream of the ORF include a putative RecBCD recognition (Chi) site, a CTTTT pentamer repeated three times and inverted repeats encompassing the ribosome binding site just upstream of the translation start site [121]. These features suggest importance of the region in S-layer transcriptional and translational regulation of Slayer protein synthesis. By pulse ¢eld gel electrophoresis and Southern hybridization using the conserved region of the sapA homologues as a probe, it has become clear that all the homologues are clustered in a small part of the genome representing less than 8% of the total [121]. Initial studies indicated that several of the homologues are arranged in tandem, one after another. After each homologue is an inverted repeat sequence that could serve as a b-independent transcription terminator, and then a 30^50 bp region that is highly conserved in all homologues studied [49,120,121]. However, more recent work has clari¢ed the physical relationship of the homologues to one another [50]. At least one homologue is oriented oppositely to the others and is separated by a 6.2 kb region that does not contain any homologues. 6.3. Rearrangement of the sapA homologues Previous work showed that sapA homologues can rearrange via reciprocal recombination [49]. This is a conservative process, in which cassettes are shu¥ed but never lost, nor are new cassettes being created, as occurs with gene replacement. We have now shown that SLP expression is governed by a single promoter in which silent gene cassettes are rearranged downstream of this unique promoter [50]. We have found that the promoter is present in the 6.2 kb region £anked by sapA homologues facing in opposite orientations. Using genetic techniques, we have shown that this 6.2 kb region is able to invert, with the consequence that the unique promoter inverts as well, permitting expression of one or the other £anking sapA homologue. The presence of inverted repeats just upstream of the ORFs is consistent with an inversion mechanism because invertases often use such repeats as recognition sites. Each of the homologues possesses such an upstream repeat, a structural feature that could facilitate a general rearrangement process other than a simple switching back and forth only involving the two £anking cassettes. From a teleologic standpoint, why would C. fetus maintain eight highly conserved cassettes from strain to strain, if it was using only two for SLP expression? Recent work has shown that not only can the promoter invert but the gene cassettes can be part of the inver- Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 6.1. Structure of the sapA homologues 6.2. Physical arrangement of the sapA homologues H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 sion process, so that SLP expression is not limited to 69 tending cell surface is completely covered with S- B. stearothermophilus the two £anking cassettes [122]. This appears to be a layer subunits [131,132]. When unique model for invertible DNA elements, and it PV72 was cultivated on synthetic PVIII medium at will be of interest to determine whether such a sys- moderate aeration at a dissolved oxygen concentra- tem governs SLP expression for other bacteria. tion (DO) of 20^30%, no change of the S-layer was observed. By changing these conditions, however, 6.4. Conclusions variation of the S-layer was induced. The amount of S-layer protein synthesized as well as the forma- The sapA homologue conservation, tractability, and strong phenotypic selection make C. fetus tion of an S-layer protein pool could be in£uenced by varying the speci¢c growth rate during continu- excellent model to examine the genetics of SLP re- ous cultivation [133]. Feeding of an amino acid mix- arrangements, and also to explore structure-function ture consisting of Gly, Ala, Val, Leu, Ile, Glu, Asp, relationships. The development of animal models of Gln and Asn to the continuous culture signi¢cantly infection [118,123] further accentuates the utility of stimulated S-layer protein synthesis, whereas the ad- this system for biological analysis. dition of aromatic or basic amino acids led to an irreversible loss of the ability to express the 7. S-Layer variation in Bacillus stearothermophilus sbsA gene. An S-layer de¢cient variant, designated T5, could be isolated from batch culture when the wild-type PV72 was grown for at least 10 passages, Holger Scholz 3 , Beatrix Kuen, Werner Lubitz è ra and Margit Sa inoculated in 24 h intervals, at 68³C instead of 57³C [61,134]. As shown by SDS-PAGE and electron microscopy, the amount of SbsA was signi¢cantly re- Variation of surface exposed proteins has been duced after 2^3 passages. However, by lowering the reported for many di¡erent microorganisms [94]. growth temperature back to 57³C, full SbsA expres- However, most of them have been described for sion was retained within the following passage. As pathogenic bacteria as a strategy to escape the de- shown by hybridization and PCR analyses, the var- sbsA struction by the immune system of the infected host. iant T5 exhibits an intact Bacillus stearothermophilus ever, a DNA rearrangement has occurred within the PV72 represents a strictly sbsA coding region. How- aerobic, non-pathogenic, thermophilic, S-layer carry- upstream region of ing organism, which was isolated from a Slovenian S-layer negative phenotype. Whether the layer de¢- beet sugar factory [124]. The organism can use glu- cient variant induced by adding aromatic or basic cose and maltose as carbon sources [124^126], has a amino acids to the medium and the variant arising growth optimum of 57³C and was cultivated on after temperature upshift have identical changes on complex SVIII medium [127] before the synthetic DNA level has to be investigated. When the oxygen PVIII medium was developed by applying the pulse supply was increased during continuous cultivation, and shift technique in continuous cultures [128]. The S-layer of the wild-type B. stearothermophilus a variant strain, which probably causes the B. stearothermophilus PV72/p2, oc- PV72 curred which was covered by an altered protein shows hexagonal symmetry with a center-to-center (SbsB), forming a p2 ordered surface layer. Protein spacing of the morphological subunits of 22.5 nm and sequence analyses revealed that SbsA and SbsB and is composed of identical protein subunits with are two di¡erent proteins encoded by di¡erent genes. molecular masses of 130 000 each [126]. The gene The exact mechanism which is used to switch from sbsA encoding the S-layer protein has been cloned SbsA to SbsB expression remains to be elucidated. and sequenced by Kuen et al. [129,130]. The S-layer The results of our investigation suggest, however, subunits consist of 1198 amino acids and possesses a that multiple recombination events are involved in signal peptide of 29 amino acids. Processing of the the switch from SbsA to SbsB expression. During S-layer protein occurs before the subunits appear in the course of our genetic analysis it could be shown the peptidoglycan containing layer in which an that the wild-type PV72/p6, the variant PV72/p2 and S-layer protein pool is stored to ensure that the ex- the S-layer de¢cient variant T5 also harbor huge Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 an 4- H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 (P sbs ), P sbs )!, Bacillus / B. stearothermophilus 564+ )*, $ )+, Hin''' -47 )! +, sbs sbs! " sbs 7.1. Investigation of sbsA in the S-layer de¢cient variant T5 # sbs $3 % & Hin''' " (P )% *+, & sbs P sbs *--- )% *+!, sbsA . / sbs # & sbs E. coli & Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 % *+ $ stearothermophilus 71 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 are in bold letters. The ATG start codon is written in capital letters, the putative rbs is underlined. come this problem the promoter probing vector pKK232-8 was used. This vector allows the cloning of strong promoters 5P to a promoterless CAT reporter gene. Selected positive clones were able to grow on a chloramphenicol concentration of 200 Wg/ ml, indicating that the sbsA upstream region has a strong promoter activity in E. coli. Sequencing as well as PCR analysis revealed that the size reduced signal of the variant is due to a DNA rearrangement but not to a deletion. The alignment of both sbsA upstream regions showed that they were identical up to position 188 5P of the ATG start codon, followed by a di¡erent nucleotide sequence (Fig. 13). Since the variant T5 has lost the ability to express SbsA, it can be assumed that the sbsA promoter is located further upstream of this position. Characterization of the sbsA promoter region of the wild-type will give further insight into the regulation of the gene. 3 7.2. Oxygen-triggered change in S-layer protein synthesis and isolation of the p2 variant 7.2.1. Physiological and morphological changes during variant formation Continuous cultivation of the wild-type strain on complex medium under glucose and oxygen double limitation (glucose in spent medium 0.1 g/l ; DO = 0%) or on synthetic PVIII medium when the 6 3188 DO was controlled at 20^30% resulted in a stable synthesis of SbsA. Variation of these conditions (complex medium : DO 0% ; synthetic PVIII medium = 50%) led to a oxygen triggered synchronized change in S-layer protein synthesis and variant formation [133,135]. In the following, the typical time course of variant formation on synthetic PVIII medium at a DO of 50%, T = 57³C and a dilution rate of 0.3/h is described. As shown in Fig. 14, an apparent steady state concerning the respiratory activity (stirrer speed for constant DO ; CO2 emission rate ; Fig. 14A), the redox potential (Fig. 14B), the OD and biomass dry weight (Fig. 14C) was observed 2 volume exchanges after starting continuous cultivation representing 10 h after inoculation. The only online measured signal which showed a steady increase during this ¢rst stage of continuous cultivation was the culture £uorescence determining the intracellular NADH2 level. After 2^3 further volume exchanges (16 h after inoculation) the redox potential and the OD showed a signi¢cant increase while the biomass dry weight stayed constant (Fig. 14B,C). At this time point the respiratory activity increased to a maximum (Fig. 14A). The lack of correlation between the OD and the biomass dry weight can be explained by changes in the morphology of the cells, the chain length and a slight sporulation observed during variant formation [133]. Biomass sample analysis by SDS-PAGE revealed that 15^16 h after inoculation, s Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Fig. 13. Sequence alignment of the sbsA upstream region of PV72 and the S3 strain T5. Identical nucleotides up to the position Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 89^74 )7991( 02 sweiveR ygoloiborciM SMEF / .la te lhaB .H H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 ) * Bacillus stearothermophilus +*' +* , -" . / !0 (- !2 3 6 ( 1 !" # Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 ! 4 Bacillus stearothermophilus +*' . 5 6 . 2! H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 ,9 3 W !"# 8 % & '"# ( *.# 0 -- # $ 45,3# 6 *. *.# Bacillus stearothermophilus ) ) * *.# 7 # +, ---. */# '$. # 0 # & Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 /# '# ( H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 7.2.2. Cloning, sequencing, and expression of sbsA and sbsB To answer the question whether sbsA and sbsB are two di¡erent genes encoding di¡erent proteins, they have both been cloned, sequenced and stably expressed in E. coli [129,130,138]. The alignment of both sequences revealed an overall low similarity of 48% at the DNA level and only 20% at the protein level [137]. Furthermore no signi¢cant stretches of sequence identity could be detected. However, an identical putative transcriptional termination signal is located 50 bp downstream of the TAA stop codon of both genes. The sbsA and sbsB upstream regions are di¡erent despite a stretch of 36 bp with a sequence identity of 90% which has high homology to several promoter structures of the genus Bacillus (data not shown). These ¢ndings indicate that sbsA and sbsB are two di¡erent genes and in addition that sbsA is probably not directly involved in recombination events to generate the sbsB coding sequence. The expression of SbsA and SbsB in E. coli led to the accumulation of stable, recrystallized sheet-like structures in the cytoplasm. These self-assembly products of SbsA and SbsB were arranged in parallel fashion at constant distance to each other following the curvature of the cylindrical part of the cells [130,138]. As observed for SbsA, SbsB was not translocated through the cell envelope of E. coli [130,138]. 7.2.3. Detection of sbsA in the p2 variant Like the S-layer de¢cient variant T5, the p2 variant does not express SbsA. To determine whether the same DNA rearrangements within the sbsA upstream region had occurred leading to the loss of the ability to express this protein, PCR analysis with sbsA speci¢c primers was carried out. In contrast to the variant T5, whose S-layer de¢cient phenotype is caused by DNA rearrangements within the sbsA upstream region, but not by an altered sbsA coding sequence, the sbsA gene in the p2 variant was modi¢ed. Only N- and C-terminal regions of the sbsA gene could be ampli¢ed with the correct size derived from the sbsA sequence. When internal parts of sbsA were ampli¢ed, larger or even multiple PCR products were obtained. The disruption of sbsA in the p2 variant might be responsible for the irreversibility to express this gene. Fig. 17. Agarose gel electrophoresis of ampli¢ed DNA fragments obtained with a sbsB speci¢c primer combination using di¡erent chromosomal DNA as templates during variant formation. Lane 1, wild-type PV72. Lane 2, sample 1. Lane 3, sample 2. Lane 4, p2 variant. To follow the switch from SbsA to SbsB expression templates from di¡erent time points during variant formation were taken for the PCR reaction. The appearance of the p2 speci¢c fragment in sample 2, indicated by an arrow, correlates with the expression of SbsB. However, no sbsB speci¢c band was observed before SbsB was expressed (PV72, sample 1). Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 variant were arranged as monomolecular layer (Fig. 16B). With increasing intensity of the S-layer protein band (molecular mass 97 000), extended patches with oblique lattice symmetry could be observed (Fig. 16C) which ¢nally completely covered the cell surface (Fig. 16D). The variant strain with the oblique Slayer lattice could be isolated from continuous culture and showed stable growth on complex and synthetic medium, under both oxygen limited and nonoxygen limited conditions. The oblique S-layer lattice of the variant strain was composed of non-glycosylated subunits with a molecular mass of 97 000. The S-layer protein of the wild-type strain PV72 and the p2 variant yielded di¡erent cleavage products upon peptide mapping with endoproteinase Glu-C, trypsin or chymotrypsin [136]. Polyclonal antiserum raised against either SbsA or SbsB showed no cross-reaction with the other type of S-layer protein [61], indicating that SbsA and SbsB were two di¡erent proteins. 75 7 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 7.2.5. Detection of sbsB in the S-layer de¢cient variant T5 2# -8# " !" * sbs * $ !" # = - # = # = , +# sbs + * # * # 7.2.4. Detection of the second S-layer gene sbsB in the wild-type strain PV72 sbs sbs !" # $ % & sbs ' ( )# * !" +P ,P sbs % -,.. * -/.. * # 0 sbs !" 1# * sbs * * # 2 * sbs * 1# sbs" sbs !" ! *" * 1 * + 1 789# : * * 1 *" * & +9 # 2 1 * 1 * # 0 1 *" + 1* sbs" * 1 * *" 1# % sbs + % * 1 *% # 1 1* * sbs" sbs # 2 + 1* sbs ' * (2# -8)# sbs sbs + $ % * !" # * *" + * # * + 1* sbs sbs" 1 * 1 *" *# 2 + * 1# Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 & $ # - & * * * *"3 * -4- * "56 (2# -+)# * * * - sbs * *" * 1 (2# -)# * *% ( )# ' * sbs !" sbs # H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 sbs Bam$ Eco6 " 0* 7& " * 8& ,3 " 7* 8& " 0* & - . ! " #$& + ! ! * * , 2 sbs #$ ! * ! , ! ( ) #$ % - sbs "/ 01*#& ! , " #$ %& '( )* ! ! ! * . sbs B. stearothermophilus "& 7.3. Identi¢cation of plasmid located sbsA homologues in B. stearothermophilus PV72 and the variant T5 (9 / ! . sbs 3 ) sbs ! + Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 / 01 , "#& 78 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 7.4. Discussion 8. S-layer protein genes of Lactobacillus Peter H. Pouwels3 , Carin P.A.M. Kolen and Hein J. Boot Lactic acid bacteria are widespread in nature and are generally used in the production and preserva- 8.1. Relationship between the presence of an S-layer and adherence Some Lactobacillus strains can colonize the gastrointestinal tract of humans and animals. These strains thus must possess the ability to adhere to the exposed surface of the epithelial cells. Adherence of colonizing lactobacilli is mediated by ¢mbrial adhesins, which interact with the epithelial cells [147]. The presence of an S-layer on the outside of strains of di¡erent Lactobacillus species has been reported by several research groups [148^150]. S-layer proteins of lactobacilli have a molecular mass between 40 000 and 55 000 and are, in general, non-glycosylated. The role of the S-layer protein in the interaction of lactobacilli and the epithelial cells of the host is unclear, as several Lactobacillus species which either do or do not contain an S-layer have been recovered from the gastrointestinal and female urogenital tracts of mammalian hosts. The type strain of L. acidophilus binds speci¢cally to intestinal lectins from chicken, although it is not clear whether the S-layer is involved in this binding [151]. Schneitz et al. reported that the S-layer of L. acidophilus strains is involved in the adhesion of these bacteria to avian intestinal epithelial cells [152]. Chemical removal of the S-layers of L. acidophilus, however, does not a¡ect the adhesion to Caco-2 cells [153]. Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 The mechanisms underlying S-layer variation so far include single recombination events with conserved and variable gene segments [49], the inversion of whole gene cassettes [59] or the inversion of promoters [139]. In each case the expressed and the silent gene ^ or homologues of the gene ^ share high sequence identity within de¢ned conserved regions. The S-layer genes sbsA and sbsB of B. stearothermophilus, however, have no signi¢cant identity. Therefore it can be assumed that sbsA is not directly involved in the generation of sbsB. The results of PCR analysis suggest that sbsB is separated into more than two fragments before it is expressed. On the other hand sbsA is separated into fragments after the switch from SbsA to SbsB expression. These observations indicate that multiple DNA rearrangements are involved in the switch from SbsA to SbsB expression. Interestingly the variant strain T5, which has neither SbsA nor SbsB expression, exhibits the full length sbsA and sbsB coding region. Since this variant can be induced to express either SbsA or SbsB it probably represents an intermediate state of the variant formation. The switch from SbsA to SbsB expression can be followed by PCR where the appearance of the sbsB speci¢c band occurs after induction of variant formation. PCR analysis and sequencing of the PCR products obtained with samples taken at short time intervals will give further insight into the mechanism underlying recombination. Plasmid located genes expressing other surface proteins than S-layers have been described previously [140,141]. To our knowledge, this is the ¢rst report of the presence of plasmid located S-layer sequences. All our ¢ndings together suggest a complex mechanism underlying the switch from SbsA to SbsB expression, whose exact course has to be elucidated in further experiments. tion of food and feed products like cheese, meat, yoghurt and silage [142]. Some strains of Lactobacillus are believed to also display health promoting activities for humans and animals when present in the gastrointestinal or female urogenital tract. Several e¡ects have been reported to be associated with the presence of lactobacilli in the gastrointestinal tract, e.g. stimulation of immunoglobulin production [143], induction of interferon expression in macrophages [144], hypocholesterolemic e¡ects [145], and the prevention of pathogenic bacteria like Salmonella typhimurium and Neisseria gonorrhoeae to epithelial cells [146]. These so-called probiotic properties and the potential of lactobacilli as vehicles to target compounds of interest, e.g. antigens or immunomodulators, to the mucosa have stimulated research on the role of surface proteins in adherence and adjuvanticity. H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 8.2. S-layer protein of L. acidophilus 8.3. S-layer protein genes of L. acidophilus The nucleotide sequence of the cloned S-protein gene showed 79% similarity to that of the L. helveticus slpA gene (GenBank X91199) but very little if any similarity to the L. brevis slpA gene [156]. Also the deduced amino acid sequences of the L. acidophilus and L. helveticus S-layer proteins are very similar (75% identical amino acids). The closer similarity of the S-proteins of L. acidophilus and L. helveticus compared to L. brevis is in agreement with the closer evolutionary relationship. The S-layer protein gene of L. acidophilus, like that of L. helveticus and L. brevis, encodes a pre-protein comprising a signal sequence with a predicted cleavage site after amino acid 24. Several direct repeats have been found in the DNA sequence of the slp genes of L. acidophilus and L. brevis which result in amino acid repeats. How- ever, no experimental data exist supporting the suggested structural function of the amino acid repeats [156]. S-proteins are expressed at a high level. As for many other species, a biased codon usage is also observed for Lactobacillus mRNAs that are translated at a high rate [157]. Over 50% of the S-protein of these organisms are encoded by seven triplets only. Random rearrangement of the S-protein encoding triplets of L. acidophilus and L. brevis yields about the same number of direct repeats, again resulting in amino acid residue repeats, arguing against a functional role for these repeats. All L. acidophilus strains investigated contain two slp genes, since two hybridizing bands of the same size are found, when the slpA gene is used to probe the chromosome [158]. The nucleotide sequence of the second gene (slpB) is highly similar to that of slpA. The 5P untranslated region and the sequence encoding the signal sequence up to the start of the mature S-layer protein are highly similar. The same holds for the 3P region of the two genes but the middle regions di¡er [155]. The two proteins encoded by the slp genes are also very similar. The C-terminal one-third regions are the same, except for 1 amino acid substitution, while the N-terminal and middle parts of the proteins are di¡erent. The conclusion that slpA encodes the Slayer protein whereas slpB is a silent gene is based on the following considerations. (i) slpA rather than slpB was isolated from an expression library probed with antibodies against the S-protein, (ii) the amino acid sequences of the N-terminal region and of tryptic peptides of the S-layer protein correspond to that of slpA, (iii) RNA is transcribed from splA but not from slpB, and (iv) a promoter sequence is present before the slpA gene but is lacking before slpB [155]. 8.4. Genetic organization of S-layer protein region of L. acidophilus By a combination of restriction enzyme analysis and PCR experiments slpA and slpB were found to be located, facing each other, on a 6 kb DNA fragment [59,113]. The nucleotide sequence of the region between the two slp genes has been determined. Four ORFs ( s 150 nt) are present in the region (3.0 kb) between slpA and slpB (Fig. 20). No transcriptional regulatory sequences (e.g. promoter, operator, termi- Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 To investigate the role of the S-layer or S-layer protein of lactobacilli in adhesion to epithelial tissues, we have started a research project aimed at the characterization of the S-protein of L. acidophilus and its mode of expression. The L. acidophilus ATCC 4356 type strain, which originates from human pharynx, was chosen because of its presumed adhering properties and because it had been shown to contain an S-layer. The amino acid composition of puri¢ed S-layer protein L. acidophilus is typical for S-layer proteins, i.e. a relative abundance of threonine, serine and hydrophobic amino acids, and absence of cysteine and methionine residues [154]. The molecular mass determined by electrospray ionization mass spectroscopy yielded a value of 43 639 þ 6. This result taken together with that of N-terminal sequence determination of the puri¢ed, mature protein and nucleotide sequence analysis (see below) allowed the conclusion that the S-layer protein of L. acidophilus is not glycosylated [155]. The isoelectric point of the L. acidophilus S-protein, calculated from the deduced amino acid sequence, is 9.4. The S-proteins of L. brevis and L. helveticus display similarly high values. These high values are in contrast with those of S-layer protein genes of other species which are in general negatively charged (pI 3^4). 79 80 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 nator) could be detected in this region. Preliminary results indicate that ORF-1 and ORF-2 are transcribed at a very low level, if at all, suggesting that ORF-1 and ORF-2 are part of a silent operon, together with slpB. The amino acid sequence encoded by ORF-1 shows considerable homology with IcaI of Staphylococcus epidermis and with HmsR of Yersinia pestis. IcaI has been implicated in the formation of the polysaccharide intercellular adhesin PIA of Staphylococcus epidermidis [159]. HmsR was postulated to be a transacting, positive regulator for expression of the hemin storage locus, hms [160]. The hms locus has been shown to be involved in transmission of Yersinia by £eas. Interestingly, expression of the hms genes is accompanied by a cell surface change of Yersinia, facilitating autoaggregation and blockage of the foregut of £eas [161]. The blockage is dependent on adherence to a speci¢c region of the £eas' gut. It is therefore tempting to assume that the silent SB operon of L. acidophilus may be involved in adhesion and/or heme binding. A homology search of the protein sequence encoded by ORF-2 yielded no signi¢cant homologies. Despite this lack of general homology, we found in this protein a region of amino acid residues which has a high degree of similarity with the active site of proteins belonging to the family of Din invertases (see below). The putative protein (79 amino acids) encoded by ORF-3 shows for the 3/5 C-terminal part homology with an ATP binding transporter protein, whose gene has been identi¢ed in the genome of Mycoplasma genitalium. For Aeromonas salmonicida it has been shown that an ATP binding domain was present in a protein which is involved in regulation of S-layer protein expression. This protein (AbcA) acts, in a heterologous system, as a positive regulator of one of the two promoters present in front of the S-protein encoding gene [162]. No homology with a known protein was found for ORF-4. 8.5. Occurrence of two slp genes in other lactobacilli Previously it had been observed that strains of group A (L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum) possess an S-layer, whereas group B (L. gasseri and L. johnsonii) strains lack such a structure [148]. We have con¢rmed these results and have shown that, in contrast to some reports, L. bulgaricus and L. fermentum strains do not harbor S-layer proteins. Antibodies raised against L. acidophilus ATCC 4356 strongly crossreact with S-layer proteins from L. amylovorus and L. helveticus, react weakly with S-layer protein from L. gallinarum, but do not cross-react with S-layer protein from L. crispatus [158]. Apparently, all L. acidophilus group A bacteria, except L. crispatus, have epitopes in common. The observation that L. crispatus S-layer protein does not cross-react with L. acidophilus antibodies, while other group A bacterial S-proteins and even L. helveticus S-layer L. acidophilus Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Fig. 20. Schematic representation of the 6 kb chromosomal slp segment of L. acidophilus ATCC 4356. ORFs larger than 150 nucleotides are depicted (arrows). Ribosome binding sites are represented by dots. Only two potential promoters (present upstream of slpA) are predicted by nucleotide sequence analysis. The 5P identity regions (dashed line, 280 nt) are used to invert the slp segment which is expected to lead to the expression of the SB protein instead of the SA protein. The 3P regions of identity (triangles, 430 nt) are not used as recombination regions during inversion of the slp segment. H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 amplify speci¢c DNA fragments. These rapid and cheap methods could replace the rather time consuming and thus costly methods currently in use for strain identi¢cation. 8.6. Regulation of expression of slpA gene As in many other bacteria, S-layer proteins of Lactobacillus are very e¤ciently expressed and secreted. Expression and/or secretion are probably tightly controlled. Bacteria are completely covered with S-layer protein indicating that S-layer protein expression and secretion keep pace with bacterial growth. The e¤cient expression is most probably due to a combination of expression signals that allow high rates of transcription, translation and secretion. The promoter of the L. acidophilus slpA gene is two times more e¤cient than that of the lactate dehydrogenase gene (ldh) of L. casei, considered to be one of the strongest promoters in many bacteria [114]. A high rate of translation of slp genes is achieved not only because of the use of a biased set of codons but also because slp mRNA is highly stable (L. acidophilus mRNA, half-life 15 min) [114]. The 5P untranslated leader of L. acidophilus mRNA can fold into a stemloop structure with an energy of 3191 kJ/mol, which most likely will protect mRNA from 5P-3P degradation. The role of secondary structure of the 5P untranslated region in regulation of gene expression was established by introduction of a deletion which removed approximately one half of the untranslated region. The inability to form a stem-loop structure resulted in a 2-fold reduction of the level of gene expression [114]. Two promoters are present in the region upstream of the slpA gene of L. acidophilus yet only the promoter closest to the slpA gene is being used under all growth conditions tested [114]. In L. brevis both promoters are equally e¤ciently used during the exponential phase of growth [156]. The sequence of the upstream promoter of L. acidophilus (P2) is the same as that of the downstream located promoter (P1) but the spacing between the 335 and 310 regions is 20 nucleotides, whereas for P1 the optimal spacing is 17 nucleotides. Although P2 does not conform to the optimal promoter sequence, promoters with a spacing of 20 nucleotides have been described before [164]. The suboptimal spacing may re£ect the need of an Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 protein do, is striking as L. crispatus is evolutionarily more closely related to L. acidophilus than L. helveticus. Moreover, the C-terminal regions of the L. helveticus and L. crispatus S-layer proteins are almost identical to that of the L. acidophilus S-layer protein. A possible explanation might be that the C-terminal part is buried inside the S-protein or is much less immunogenic than the less conserved N-terminal regions. By using probes that are speci¢c for di¡erent regions of the slp genes as well as probes that can di¡erentiate between slpA and slpB, we could establish that all A group bacteria contain two slp genes with a structure very similar to that of the L. acidophilus slp genes. L. acidophilus group B bacteria and L. bulgaricus lacked slp genes, whereas L. helveticus contained a second gene showing strong homology with the L. acidophilus genes at the 3P end only [158]. Apparently, L. helveticus contains a truncated gene lacking the 5P region or harbors a second gene with a non-homologous 5P region. A silent S-layer protein gene which lacks the 5P part of the coding region has also been described for Bacillus sphaericus [163]. The strong conservation of both 5P and 3P regions among di¡erent lactobacilli living in di¡erent environments suggests that these regions have important functions. The C-terminal region encoded by the 3P end might for instance be involved in interaction of S-proteins with the peptidoglycan layer. Peptidoglycan binding regions, also called SLH regions, have been found at either the N-terminal or the Cterminal end of several S-layer proteins [25]. Conservation of the 5P untranslated region probably has to be explained by constraints imposed on the nucleotide sequence regarding mRNA stabilization and as a target site for recombination (see below). Di¡erences in cross-reactivity with antibodies between group A bacteria and the complete lack of reaction of group B bacteria o¡er attractive possibilities for rapid identi¢cation of these organisms. An even more powerful method can be devised for identi¢cation of these species which is based on di¡erences in the structure of the slp genes. The presence of nearly identical 5P and 3P regions interspaced by more variable regions o¡ers the opportunity to unambiguously di¡erentiate between di¡erent isolates by PCR. Once the variable regions have been (partly) sequenced speci¢c PCR primers can be designed to 81 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 82 activator protein that binds to a nearby site in the DNA and facilitates the entry of RNA polymerase. A sequence of dyad symmetry, which is located immediately upstream of the 35 region of P-2, might play a role in binding of such a hypothetical activator. Regulation involving a transacting factor (AbcA) and a potential inverted DNA sequence near one of the promoters of an S-layer protein gene has recently been described for Aeromonas salmonicida [162]. 3 To study the role of the S-layer in the interaction of probiotic lactobacilli with receptors of the epithelial tissue of the host, we expressed the SA protein gene of L. acidophilus in L. casei, a bacterium which does not have an S-layer. Transformed L. casei bacteria produced and secreted the SA protein e¤ciently. However, secreted SA protein does not interact with the cell wall and can therefore not form an S-layer on the outside of this bacterium [165]. Our ¢ndings corroborate the results of earlier studies showing that in vitro reconstitution of S-layers is only possible on the cell wall of bacteria that normally carry an S-layer [166]. A comparison of the structure of the cell walls of L. acidophilus and L. casei may reveal which essential component is missing in the latter organism. 8.8. Antigenic variation in L. acidophilus? The presence of regions of near-identity at the 5P and 3P regions of L. acidophilus and of related strains suggests that these regions may be involved in genetic recombination. By a series of PCR experiments we have shown that inversion of the slp segment takes place, replacing the silent gene slpB by the active one and vice versa, in 0.3% of the bacteria [59]. Inversion of the slp segment occurs through homologous recombination which takes place in the 5P untranslated region of the S-layer protein genes. Interestingly, a stretch of 15 bp which has a high degree of similarity with the consensus recognition site for invertases of the Din family is present in the middle of this region, suggesting that inversion of the slp segment is catalyzed by a member of this family. A region in the 8.9. Potential applications of lactobacilli as vehicles for antigen presentation Lactobacillus strains have a number of properties which make them attractive candidates for oral vaccination purposes [169]. Lactobacilli have been used for centuries in food and feed, and are considered to be safe organisms, this in contrast to other live vaccine carriers used so far (e.g. Salmonella, Escherichia coli, Vaccinia) which cannot be classi¢ed as safe. Furthermore, in contrast to lactobacilli, the latter type of carriers are themselves highly immunogenic, possibly preventing repetitive use of the carriers with the same or other antigens. Forming the outermost layer of bacteria and being present in vast quantities (up to 105 molecules per bacterium), the S-layer protein is, in principle, an attractive candidate for fusion with antigenic determinants. The possibility to genetically manipulate lactobacilli and the availability of multiple S-layer genes of Lactobacillus opens new avenues for research aimed at presenting foreign proteins (antigens, ScFv, enzymes) at the surface of these bacteria. Further studies on the mechanisms triggering antigenic variation should shed light on the role of S-proteins in adherence. Such knowledge will facilitate screening of new isolates with probiotic properties. Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 8.7. Expression of L. acidophilus S-layer protein in a heterologous host hypothetical protein encoded by ORF-2 of the slp segment (Fig. 20) shows a striking conservation of amino acid residues which were shown to be involved in recognition of the DNA recognition site by the Hin invertase [167]. Because of the similarity in structure of the slp genes in bacteria that are evolutionarily related, we assume that inversion, leading to the interchange of the expressed and silent S-protein genes, will also take place in these organisms. These data strongly suggest that L. acidophilus and related organisms show antigenic variation. What the role of antigenic variation might be is at present not known. The S-layer protein of L. crispatus, which is closely related to L. acidophilus, has recently been shown to be involved in adherence to the extracellular matrix proteins, suggesting a role of antigenic variation in adherence [168]. H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 9. Molecular biology of the Lactobacillus brevis S-layer gene (slpA) and utility of the slpA signals in heterologous protein secretion in lactic acid bacteria Airi Palva chaebacterial species [4], also many Lactobacillus species are present, which are either widely used in food fermentations or found as part of the normal £ora of humans and animals. The DNA sequence of the lactobacillar S-protein gene (slp) has been published only from L. brevis, L. acidophilus and L. helveticus. Functions of the S-layer proteins in Lactobacillus are unknown, but they can be expected to play an essential role since inactivation of these genes has repeatedly failed [165]. Recently, the L. crispatus S-layer protein has been demonstrated to mediate adhesion to type IV collagen [168] and preliminary results indicate that also L. brevis S-layer protein can mediate binding to intestinal epithelial cells. Since for average sized cells 5U105 S-layer subunits per cell generation have to be synthesized in order to cover the entire cell surface with the S-layer proteins [170], the expression of a slp gene and the secretion machinery of a S-layer harboring cell may be expected to be very e¤cient. These properties are obvious targets for utilization of S-layers in biotechnological applications. To study heterologous protein production in lactobacilli, the L. brevis slpA has been chosen as a model. L. brevis is a heterofermentative lactic acid bacterium commonly found in vegetable fermentations, sour dough, silage and in the intestine of humans and animals [171,172]. In this review, the characterization of the L. brevis S-layer protein, gene, mRNA and in vivo expression are discussed along with the demonstration of heterologous protein secretion with the aid of the slpA signals. 9.1. Characterization of the L. brevis S-layer protein and gene In L. brevis (ATCC 8287/GRL1), the S-layer has previously been shown to consist of tetragonally arranged subunits, which are composed of a protein with a molecular mass of about 51 000 [173]. The subunits can be dissociated from the cell wall, e.g. with guanidine hydrochloride, and they can be reassembled into a native-like array in vitro [166]. From the intact L. brevis cells, boiled in Laemmli sample bu¡er and analyzed in SDS-PAGE, only one major band with an apparent molecular mass of 46 000 was detected [156]. To con¢rm its identity as the S-layer protein of L. brevis, the cells were treated with an Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 The Lactobacillus brevis S-layer protein is the major protein of the cell with a molecular mass of 46 000 in SDS-PAGE. Genetic characterization of the slpA gene has revealed two adjacent promoters (P1, P2) with the conserved hexanucleotide 310 and 335 regions typical of prokaryotic promoters, the consensus ribosome binding site, the ATG start codon and a signal sequence encoding a region of 90 nucleotides. The structural slpA gene has a coding capacity for a mature S-layer protein (molecular mass 45 000) followed by a strong transcription terminator sequence downstream of two translation stop codons. According to the mRNA size and the 5P end analyses the slpA gene forms a monocistronic transcriptional unit where both promoters are functional. Further characterization of in vivo expression of the L. brevis S-layer protein and determination of the usage of the two slpA promoters as a function of growth have been performed. The half-life of slpA transcripts has been shown to be 14 min. Functionality of slpA expression and secretion signals in heterologous protein secretion has been studied by using the E. coli L-lactamase (bla) as the reporter in a slpA based secretion cassette. Secretion studies performed with L. lactis, L. brevis, L. plantarum, L. gasseri and L. casei have shown that in all hosts tested the bla gene was expressed under the slpA signals and all detectable Bla activity was secreted into the culture medium. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and L. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivation with all lactic acid bacteria tested. When growing L. lactis and L. brevis under pH control, the Bla activity could also be stabilized at the stationary phase. L. lactis produced up to 80 mg/l of Bla which represents the highest amount of a heterologous protein secreted by lactobacilli so far. Among over 300 S-layer harboring eu- and ar- 83 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 84 3 3 3 3 [177]. The typical features of the amino acid composition of the S-layer protein deduced from the DNA sequence were the high number of hydrophobic amino acids, amino acids with hydroxyl groups (Thr (17.8%), Tyr (6.4%) and Ser (9.2%)) and the absence of cysteine. The amount of basic amino acids (lysine (8.1%) and arginine (1.7%)) was higher than that of acidic amino acids which is a notable exception to the general features of the other non-lactobacillar S-layer proteins [178]. A similar characteristic has also been described for L. acidophilus S-layer protein [154] and can be analyzed from the L. helveticus slpA sequence. The pI values predicted for the L. brevis, L. acidophilus and L. helveticus S-layers are 9.88, 9.84 and 10.08, respectively [154,156]. The ¢rst search for L. brevis S-layer protein homologues from data bases revealed no genuinely related sequences [156]. The predicted amino acid sequences of the recently described L. acidophilus [154] and L. helveticus (EMBL: X91199 and X92752) slpA genes, however show 35.7% [154] and 28.8% similarity to that of the L. brevis S-layer protein [156], respectively. Hybridization of a L. brevis slpA speci¢c fragment with chromosomal DNA of L. buchneri, L. helveticus and L. acidophilus resulted in signals from clearly positive to weakly positive, respectively [174], indicating, according to phylogenetic relatednesses, that the slpA gene of L. buchneri is more closely related to that of L. brevis than the other two. In L. acidophilus, two slp genes with phase variation and in L. helveticus one functional slp gene and a truncated slp 3P end have been found [165]. In L. brevis, only one slp gene is present instead [174]. 9.2. In vivo expression of the slpA gene 9.2.1. mRNA analyses The size of the S-layer transcripts determined by Northern blot analysis is 1.5 kb, con¢rming that slpA is a monocistronic transcriptional unit [156]. Mapping of the transcription start site of slpA revealed two 5P ends located immediately downstream of the two 10 regions deduced from the DNA sequence, and thus con¢rming the functionality of the promoters [156]. Determination of the stability of the slpA mRNA showed that the half-life of the slpA transcripts was 3 Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 antiserum raised against the isolated protein (molecular mass 46 000) and analyzed by immunogold electron microscopy. The post embedding immunoelectron microscopy clearly showed that the protein (molecular mass 46 000) was heavily enriched in the outermost part of the cell wall of L. brevis cells [156]. The antiserum also recognized a major protein (molecular mass 55 000) of L. buchneri (DSM 20557) released from the cell similarly to that of L. brevis, thus indicating antigenic relatedness of these two surface proteins (molecular masses of 46 000 and 55 000) [174]. The N-terminal sequence of the intact S-layer protein was determined to be NH2 -Lys-Ser-Tyr-AlaThr-Ala-Gly-Ala-Tyr-Ser. N-terminal analyses were also performed for a few tryptic peptides of the S-layer protein and the amino acid sequence information was used to design degenerated oligonucleotides for the isolation of the slpA gene [156]. DNA sequencing of the slpA gene from L. brevis was performed with PCR fragments since the gene was unstably maintained in L. brevis gene libraries in E. coli and Bacillus subtilis [156]. Brie£y, the slpA gene is 1395 bp in size with a coding capacity for a protein with a molecular mass of 48 159. The ¢rst 90 nucleotides of the structural gene encode a signal peptide of 30 amino acid residues with features of typical Gram-positive type signal peptides [156,175]. The size of the mature polypeptide is 435 amino acid which is in good agreement with the molecular mass of 46 000 of the S-layer protein analyzed by SDSPAGE. The slpA gene is preceded by a well conserved ribosome binding site (RBS) and two putative promoter regions, P1 and P2. The 35 and 10 regions of P1 and P2 resemble the conserved prokaryotic 35 and 10 consensus sequence [176]. Furthermore, a transcription termination sequence is found downstream of the two translation stop codons of the slpA gene, indicating that the slpA gene is monocistronic [156]. Furthermore, in the coding region of slpA 10 partly overlapping direct repeats of 10^12 nucleotides are present [156] possibly partly explaining the instability of the gene in heterologous hosts [168,156]. Computer analyses of the predicted amino acid sequence of S-layer protein revealed that the codon usage was clearly biased [156] and most resembled that reported for highly expressed B. subtilis proteins H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 9.2.2. S-layer synthesis In vivo expression studies of L. brevis have shown that the kinetics of the accumulations of the slpA mRNA and protein correlates well up to the onset of the stationary phase followed by a sharp decrease at the level of slpA mRNA. The rate of mRNA decay is, however, slower than expected from the half-life of slpA transcripts suggesting that residual transcription continues even though the total amount of the S-layer protein does not further increase at the stationary phase [179]. In addition to the in vivo transcription studies of L. brevis slpA, growth dependent S-layer mRNA synthesis has been described only for A. salmonicida. The level of transcripts derived from the A. salmonicida S-layer protein gene, vapA, was found to be highest at the mid-exponential phase of growth, whereas a relatively sharp decline of vapA transcripts already occurred at the late exponential phase [180]. The L. brevis S-layer protein is not released into the supernatant fractions at any of the growth phases studied, con¢rming earlier observations [156] and suggesting a tight regulation of S-layer synthesis and assembly [179]. Breitwieser et al. [131] have demonstrated the presence of substantial amounts of S-layer subunits on the inner surface or within the peptidoglycan layer in B. stearothermophilus suggesting an intermediate phase between the synthesis and ¢nal location of the S-layer protein. This has also been quite commonly observed in S-layers of other Gram-positive eubacteria [131]. However, in L. brevis over 95% of the S-layer subunits could be released with the SDS-PAGE sample bu¡er from intact cells, as Western blot analyses of intact and disrupted cells indicated. Thus, it appears that essentially no accumulation of the L. brevis S-layer subunits took place inside the peptidoglycan layer prior to translocation to the outer surface. 9.3. Heterologous protein secretion with the L. brevis S-layer signals 9.3.1. Construction of a secretion vector based on the slpA signals A derivative (pKTH2095) of the shuttle vector pGK12 [182] has been utilized as the carrier of the secretion cassette constructed to contain the two promoters (P1, P2), signal sequence (SS) and transcription terminator (tslpA) of the L. brevis slpA and another terminator (t) upstream of splA. As reporter the L-lactamase gene (bla) of pUC19 was used. The secretion vector (pKTH2121, see Fig. 21) was constructed stepwise using PCR technology [183]. 9.3.2. Expression and secretion of Bla by the slpA secretion cassette (MG1614), transformed with pKTH2121, e¤ciently secreted L-lactamase into the culture medium and DNA sequencing of the cassette con¢rmed its stability and correctness. To test the utility of the cassette in lactobacilli, L. brevis (ATCC 8287), L. plantarum (NCDO 1193), L. gasseri (NCK 334) and L. casei (ATCC 393) hosts were also transformed with pKTH2121 and expression and secreL. lactis Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 14 min [179]. When compared to typical half-lives of prokaryotic mRNAs, the slpA transcripts are exceptionally stable. Recently, the half-life of the L. acidophilus slpA mRNA has been determined showing a value of 15 min [114]. Furthermore, the transcripts of the Aeromonas salmonicida vapA gene have also been shown to be very stable (11^22 min) [180]. The long half-lives of these three S-layer mRNAs from three di¡erent species may indicate that a high mRNA stability is a general feature of S-layer mRNAs. As they mediate the synthesis of a major structural component of the cell, the high stability of S-layer mRNAs is not unexpected. A study of the usage of the two L. brevis slpA promoters (P1, P2) in di¡erent stages of growth by Northern blot analysis has revealed that the P2 promoter, located closer to the start codon, is e¤ciently used during both the exponential and the early stationary phase whereas slpA mRNA derived from P1 was only weakly detectable [179]. Further quantitative analysis by dot blot hybridization showed that transcripts derived from both promoters are present throughout the entire growth phase but the level of transcripts derived from promoter P2 is 10 times higher than that of P1 [179]. Some other S-layer genes carrying multiple promoters have also been described [47,181]. The usage of three promoters has been described in the expression of the cwp operon of Bacillus brevis. Of these P2 is constitutively used and P3 preferentially at the exponential phase of growth [47]. 85 86 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 tion of L-lactamase was determined as the function of growth in £ask cultivations (Fig. 22). In each what slower. In all strains studied, degradation of L-lactamase due to proteolysis was observed. With strain carrying pKTH2121 all detectable Bla activity L. plantarum, L. gasseri and L. casei the Bla activity was in the growth medium. The highest yield (10 240 rapidly decreased already at the early stationary U/ml ; 50 mg Bla/l) in the culture supernatants was phase, suggesting higher protease activity in these obtained with strains [183]. L. lactis at the early stationary phase. The highest production levels of Bla in the early sta- L. brevis cells and in the exponential phase L. plantarum cells were 60% and 30%, respectively, of that in L. lactis [183]. However, the rate of Bla production was roughly equal in L. lactis and L. plantarum, whereas that of L. brevis was sometionary phase The comparison of the activity and amount of Bla protein by Western blots revealed a good correlation and lack of cell associated L-lactamase. The size of Bla secreted to the culture medium was equal to that of the mature Bla of E. coli, suggesting that the enzyme was correctly processed [183]. Small amounts Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Fig. 21. Secretion vector based on the a slpA expression and secretion signals. The L. brevis promoter signal sequence (PslpA-SSslpA) region, the transcription terminators (t and tslpA) and the E. coli L-lactamase (bla) gene were isolated byPCR ampli¢cations and stepwise joined to form the ¢nal t-PslpA-SSslpA-bla-tslpA cassette which was then ligated with pKTH2095 to result in pKTH2121. The nucleotide and the corresponding amino acid sequences of the Pslp-SSslpA-bla joint region of the secretion construct are shown and the signal peptide cleavage site is indicated by a vertical arrow. The reporter gene region is underlined. < H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 9.3.3. pH controlled cultivation ) L % L. lactis L. brevis ) *& +!, +-. L. lactis% & ++ L = >#)++ L. lactis lactis :? 8- * .% L. brevis #44 + < * .% L. plantarum (421 ;! * .% L. gasseri (4> !!- * .% L. casei #44 !;! * . & +! L ) L. lactis L. brevis 4 L. lactis L. brevis % % >#)++ 4 :< +6 *+ :<?. :' % # L. lactis L. brevis !/@4 !<@4 % * % : . *// . & L. lactis% = +6 ) A 55 ( ()3 & L. brevis% ) 8/ B ++ % $ U / 0 *& +!. % / 12600 3 % L % $ ) 4 L -% 5" -6 slp # slp # 7 L 856 + 12600 3 *& +!.% Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 slpbla L. brevis !" # L. brevis slp $ L. lactis% L. brevis L plantarum% L. gasseri% slp $ L. casei !" & % $ bla '( % L. casei% H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 88 U 105 mole- On the basis of the knowledge gathered, the future cules/cell/h. This value is comparable to or exceeds perspectives include further developments of lactoba- the best exoenzyme producing laboratory strains of cillar S-layers for di¡erent biotechnological applica- production hosts. However, lengthening of the e¡ec- protein is its development as a carrier vehicle for tive duration of the production phase, in order to foreign antigenic epitopes. Further elucidation of improve product yields, requires optimization of the adhesive properties of growth or alternatively the use of e.g. immobilized may also allow and extend its utilization as a carrier cell systems. Similarly to lactococcal Bla production, of oral vaccines and other substances for animal and the pH control resulted in the stabilization of human use. Further developments also include the secretion with a calculated value of 5 Bacillus [53], and thus suggests utility of lactococci as Bla production L. brevis than in L. brevis (Fig. 23). The rate of was, however, L. lactis. clearly lower in Thus, it is evident that extension of the use of L. brevis L. brevis S-layer S-layer protein L. brevis slpA signal both for secretion and for intracellular protein production to improve fermentation processes. the presumed competition caused by the translocation of S-layer protein has to be overcome before L. brevis can be e¤ciently utilized as a production host [183]. 9.4. Conclusions and perspectives 10. Biotechnological applications of recombinant S-layer proteins rSbsA and rSbsB from Bacillus stearothermophilus PV72 Michaela Truppe, Stefan Howorka, S-layers are common surface structures in lactobacilli and are present in many industrially used Lacto- bacillus species. The DNA sequences available for a few Lactobacillus S-layer protein genes suggest that Gerhard Schroll, Sonja Lechleitner, Beatrix Kuen, Stephanie Resch3 and Werner Lubitz As several S-layer sequences have been elucidated, lactobacillar S-proteins share common characteristics a great potential for the biotechnological use of re- di¡erent from other S-layers in possessing a large combinant S-layers exists [186]. The possibility to amount of basic amino acids and thus very high build two-dimensional crystalline arrays of identical predicted p values, even though the consequences protein or glycoprotein subunits on di¡erent surfaces of that property are unknown. Functions of lactoba- or interfaces opens appealing possibilities for func- cillar S-layers are unknown but recent observations tioning surfaces and to build supramolecular struc- of the adhesive properties of some S-proteins may tures in the third dimension. Most importantly, suggest that they may have an important role in in- S-layer lattices are highly anisotropic structures ex- volvement in di¡erent gastrointestinal ecosystems. hibiting remarkable di¡erences in the topography The gene is the ¢rst lactobacillar and physicochemical properties of their surfaces S-layer protein gene characterized. Also its in vivo [187^189]. In nature, the inner surface often interacts expression at the level of transcription and transla- either with peptidoglycan or with membrane surfaces tion is well studied. Furthermore, the utility of the [190]. As no detailed information on the arrange- I L. brevis slpA slpA expression and secretion signals in heterologous ment of single amino acids or atoms within the ter- protein secretion has been demonstrated. It is evident tiary structure of any S-layers with known sequences that the is available, recombinant modi¢cations by exchange L. brevis slpA signals can be e¤ciently used for protein secretion in a variety of lactic acid bac- or introduction of single amino acids, introduction teria even though the recognition of of slpA promoters is host dependent. At present, the slpA based secretion cassette functions most e¤ciently in Lactococcus, giving a yield of the secreted model protein, L-lactamase, at the highest level of heterologous pro- tein production described for lactic acid bacteria so far. epitopes within surface loops, weakening or enforcing intra- and intermolecular interactions remain so far the most practical tools to functionally describe the molecular architecture of speci¢c S-layers [137]. In this section examples of the latter approach will be given for the two di¡erent S-layer Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 tamase formation in L-lac- tions. An evident application of H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 /. 10.1. Heterologous expression of sbsA and sbsB in Escherichia coli 10.2. Heterologous expression of sbsA in Bacillus subtilis ) - sbs ! E. coli sbs sbs B. stearothermophilus ) 2 5 &.' .' '*# //2 :9 > > > > sbs sbs sbs - rSbs &'# &#& :9 rSbs & '! 0 2 9 rSbs rSbs Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 B. stearothermophilus ! " #$ %&#!&'#( ) # " $ &'* *** + , '* " $ - %&.( + ! " $ " $ ./ *** - " $ %&'#( &'* *** ./ ***! 01 %&.!&'/( ) sbs "'#/2 $ &/ - '* %&.( sbs "#* $ .* - '& - 3 %&'/( ) - VpL " 42$ ) sbs 42 E. coli V5/6 ") '$ 7 3 %&'*( E. coli sbs "&$ 1 sbs - E. coli 3 , 8 9 E. coli : sbs E. coli 4# ") '$ lacpo %&'/( ) ' = )3 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 10.3. Structural/functional analysis of SbsA and SbsB + sbs sbs , (#)-* Apa ' ' 8 5. & +;< & "% 10.4. Construction of SbsA/SbsB fusion proteins 10.4.1. Insertion of streptavidin in SbsA/SbsB 0 #)1& -)2& $3) 442 5 6+5 7 & $3) 442 "' 8% ' 9 9 ' # += #23 ' sbs sbs E. coli . "' /% 9 #)1 -)2 $3) 442 #$) #2/ > > >9> >9> >9> >9> 3 3 # #2# -)2 #$) +;<? -88 ##2- 8#3 > > > #2# 8-4 #2# >9> >9> >9> + @ " % 8)3 -)- >9 @ # "% + - @ $ / 0 & & +;<& Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 sbs B. subtilis ! "#$% & & '# "' $% sbs xyl ' B. subtilis sbs sbs (#)#* H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 10.4.2. Insertion of Bet v 1 in SbsA/SbsB 10.4.3. Insertion of pseudorabies virus antigens into SbsA/SbsB Insertion of the pseudorabies virus [195] gB epitope SmaBB (489^1224 nucleotide fragment coordinates, refer to EMBL HEHSSGP2) into sbsA/sbsB (Table 5) was performed to test gB speci¢c immune responses in experimental animals. Western blot analysis with a monoclonal antibody corresponding to the inserted sequence showed the accessibility of the virus polypeptide within the SbsA/rSbsB proteins. An extended study with other PCR fragments inserted into sbsA and/or sbsB is in progress. 10.4.4. Insertion of PHB synthase (PhbC) of Alcaligenes eutrophus H16 into SbsA A regular arrangement of polypeptide structures with enzymatic activities on the surface of S-layers is an ambitious goal for the construction of immobilized enzymes within a living cell and in the case of PHB synthase (590 aa) for the construction of a molecular machine for biopolymer synthesis. The phbC gene was ampli¢ed by PCR from plasmid p4A [196] and inserted into the 878 ApaI site (Table 4) in frame with sbsA giving rise to plasmid pSbsA-PhbC. For the functional test of the enzymatic activity of the SbsA-PhbC construct E. coli cells harboring the corresponding plasmid had to be cotransformed with plasmid pUMS harboring the L-ketothiolase (PhbA) and acetoacetyl-CoA reductase (PhbB) of A. eutrophus [197]. Poly-L-hydrox- ybutyrate (PHB) formation in E. coli (pSbsA-PhbC, pUMS) cells was indicated by staining with Sudan black, gas chromatography and electron microscopy. These ¢ndings indicate that the SbsA-PhbC construct is enzymatically active and could serve as an example for immobilizing enzymes on intracellular S-layer matrices. 10.5. Perspectives These few examples show that the architectural principles using S-layer structures as rigid matrices can be applied to modulate their surfaces, e.g. to build multiple, multifunctional recombinant S-layers with various functions. It was demonstrated that the expression of recombinant SbsA/SbsB constructs in various cell systems combined with future ultrastructural approaches is useful to develop new tools in biotechnology. For example, recent data obtained from the comparison of protein and prostaglandin mediators suggest that even in extremely high doses, S-layer proteins do not exhibit endotoxic properties. They enhance immune cell functions and are therefore a promising basis for the construction of new types of vaccines or diagnostic systems. In addition, multivaccine components can be derived from multiple, di¡erent rSbsA subunits, each carrying relevant immunodeterminants of pathogens, when mixed together. Molecular S-layer speci¢c machines can contribute to new ways of metabolic designs by S-layer immobilized enzymes. Acknowledgments Nicolas Bayan et al. thank J.C. Dedieu for his skilful technical assistance. Their article is dedicated to the memory of J.L. Peyret, who tragically died in 1993 and who initiated this work. Agneés Fouet et al. wish to thank Gervaise Mosser (Institut Curie, Paris) for scienti¢c advice, and Caroline Frolet for the Southern experiments. Everly Conway de Macario and Alberto J.L. Macario thank the members of their laboratory who over the years participated in the study of S-layer and ABC transporter genes, particularly Linda E. Mayerhofer, Rong Yao, Charles B. Dugan, Robert J. Jovell, and Wayne Decatur. They thank Javier Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Bet v 1 is the major pollen allergen of the birch and is responsible for atopic (IgE mediated) allergies in an increasing percentage of the population [193]. Sequencing and cloning of Bet v 1 by others [194] made it possible to insert the open reading frame of Bet v 1 into nucleotide position 878 (aa 296) of sbsA (Table 5). This Bet v 1-S-layer fusion protein is being studied to determine whether it is capable of converting a (TH2 directed) IgE antibody response into a TH1 mediated response against Bet v 1. If so, it would indicate an ability to suppress the manifestations of allergy in patients susceptible to pollen allergies. Furthermore, SbsA-Bet v 1 fusion proteins are investigated for the ability to assay anti-Bet v 1 antibody concentrations and/or to reduce high levels of anti-Bet v 1 IgE. 91 92 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 References [1] Aggerbeck, L.P. and Gulik-Krzywicki, T. (1986) Studies of lipoproteins by freeze-fracture and etching electron microscopy. Methods Enzymol. 128, 457^472. [2] Richter, W., Haënel, F. and Hilliger, M. (1985) Freeze-fracture observations of Corynebacterium glutamicum: the occurrence of an outer membrane-like structure and the in£uence of temperature on the cytoplasmic membrane. J. Basic Microbiol. 25, 527^536. [3] Beveridge, T.J. and Graham, L.L. (1991) Surface layers of bacteria. Microbiol. Rev. 55, 684^705. [4] Messner, P. and Sleytr, U.B. (1992) Crystalline bacterial cellsurface layers. Adv. Microbiol. Physiol. 33, 213^275. [5] Chami, M., Bayan, N., Dedieu, J.-C., Leblon, G., Shechter, E. and Gulik-Krzywicki, T. (1995) Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: a combined freeze-etch electron microscopy and biochemical study. Biol. Cell. 83, 219^229. [6] Luckevich, M.D. and Beveridge, T.J. (1989) Characterization of a dynamic S-layer on Bacillus thuringiensis. J. Bacteriol. 171, 6656^6667. [7] Yamada, H., Tsukagoshi, N. and Udaka, S. (1981) Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47. J. Bacteriol. 148, 322^332. [8] Kawata, T. and Masuda, K. (1972) Extracellular crystalline lattice material of Corynebacterium diphtheriae revealed by electron microscopy. Jap. J. Microbiol. 16, 515^523. [9] Stewart, M. and Murray, R.G.E. (1982) Structure of the regular surface layer of Aquaspirillum serpens MW5. J. Bacteriol. 150, 348^357. [10] Beveridge, T.J. and Murray, R.G.E. (1976) Super¢cial cellwall layers on Spirillum `ordal' and their in vitro reassembly. Can. J. Microbiol. 22, 567^582. [11] Remsen, C.C., Valois, F.W. and Watson, S.W. (1970) Fine structure of the cytomembranes of Nitrosocystis oceanus. J. Bacteriol. 94, 422^433. [12] Austin, J.W. and Murray, R.G.E. (1990) Isolation and in vitro assembly of the components of the outer S-layer of Lampropedia hyalina. J. Bacteriol. 172, 3681^3689. [13] Baumeister, W., Karrenberg, F., Rachel, R., Engel, A., Ten Heggeler, B. and Saxton, W.O. (1982) The major cell envelope protein of Micrococcus radiodurans (R1): structural and chemical characterization. Eur. J. Biochem. 125, 535^544. [14] Mescher, M.F. and Strominger, J.L. (1976) Puri¢cation and characterization of a prokaryotic glycoprotein from the cell envelope of Halobacterium salinarium. J. Biol. Chem. 251, 2005^2014. [15] Kuëpcuë, Z., Maërz, L., Messner, P. and Sleytr, U.B. (1984) Evidence for the glycoprotein nature of the crystalline cell wall surface layer of Bacillus stearothermophilus strain NRS2004/3a. FEMS Lett. 173, 185^190. [16] Peyret, J.L., Bayan, N., Joli¡, G., Gulik-Krzywicki, T., Mathieu, L., Shechter, E. and Leblon, G. (1993) Characterization of the cspB gene encoding PS2, an ordered surface layer protein in Corynebacterium glutamicum. Mol. Microbiol. 9, 97^109. [17] Blaser, M.J. and Gotschlich, E.C. (1990) Surface array protein of Campylobacter fetus cloning and gene structure. J. Biol. Chem. 265, 14529^14535. [18] Evenberg, D. and Lugtenberg, B. (1982) Cell surface of the ¢sh pathogenic bacterium Aeromonas salmonicida: II. Puri¢cation and characterization of a major cell envelope protein related to autoagglutination, adhesion and virulence. Biochim. Biophys. Acta 684, 249^254. [19] Fujimoto, S., Takade, A., Amako, K. and Blaser, M.J. (1991) Correlation between molecular size of the surface array protein and morphology and antigenicity of the Campylobacter fetus S-layer. Infect. Immun. 59, 2017^2022. [20] Wang, E., Garcia, M.M., Blake, M.S., Pei, Z. and Blaser, M.J. (1993) Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus. J. Bacteriol. 175, 4979^4984. [21] Liebl, W. and Sinskey, A.J. (1988) Molecular cloning and nucleotide sequence of a gene involved in the production of extracellular DNase by Corynebacterium glutamicum. In: Genetics and Biotechnology of Bacilli (Ganesan, A.T. and Hoch, J.A., Eds.), pp. 383^388. Academic Press, New York. [22] Joli¡, G., Mathieu, L., Hahn, V., Bayan, N., Duchiron, F., Renaud, M., Shechter, E. and Leblon, G. (1992) Cloning and nucleotide sequence of the csp1 gene encoding PS1, one of the two major secreted proteins of Corynebacterium glutamicum: the deduced NH2 terminal region of PS1 is similar to the Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 Cordero and Rosemarie A. Jack for assistance, and Tracy L. Godfrey for word processing and the members of the Photo Unit for the artwork and illustrations. Work in their laboratory was partially supported by a grant from NYSERDA (706-RIERBEA). Luis A. Fernaèndez-Herrero et al. appreciate the excellent technical assistance of J. de la Rosa. Their work has been supported by Project BIO94-9789 from the CICYT and by an institutional grant from the `Fundacioèn Ramoèn Areces'. During the development of their work L.A. Fernaèndez Herrero and G. Olabarr|èa were the holders of fellowships from the Comunidad Autoènoma de Madrid and the Gobierno Vasco, respectively. The work of Michaela Truppe et al. was supported by the Fonds zur Foërderung der Wissenschaftlichen Forschung (FWF, Projects S72/02 and S72/08). Stefan Howorka holds a fellowship from the Austrian Academy of Sciences. We thank Monika Timm for editorial help. The work of Martin J. Blaser and colleagues was supported by RO1 24145 from the National Institutes of Health. H. Bahl et al./FEMS Microbiology Reviews 20 (1997) 47^98 Mycobacterium antigen 85 complex. Mol. Microbiol. 6, 2349^ [23] [24] Corynebacterium glutamicum [25] Acetoge- nium kiwi [26] [27] [28] [30] [31] [34] Caulobacter cres- [46] Haloferax volcanii Corynebacterium [36] [37] [38] [43] [45] Corynebacterium glutamicum. [35] [42] Campylobacter fetus Halobacterium volcanii [33] [41] [44] Rickettsia prowazekii Rickettsia typhi [32] [40] Thermus thermophilus centus [29] [39] [46a] ogy and surface anchoring of the tetragonal paracrystalline array of . J. Mol. Biol. 228, 652^661. Nikaido, H. (1994) Prevention of drug access to bacterial targets: permeability barriers and active e¥ux. Science 264, 382^388. Brennan, P.J. and Nikaido, H. (1995) The envelope of mycobacteria. Annu. Rev. Biochem. 64, 29^63. Mikesell, P., Ivins, B.E., Ristroph, J.D. and Dreier, T.M. (1983). Evidence for plasmid mediated toxin production in . Infect. Immun. 39, 371^376. Green, B.D., Battisti, L., Koehler, T.M., Thorne, C.B. and Ivins, B.E. (1985). Demonstration of a capsule plasmid in . Infect. Immun. 49, 291^297. Gerhardt, P. (1967) Cytology of . Fed. Proc. 26, 1504^1517. Holt, S.C. and Leadbetter, E.R. (1969) Comparative ultrastructure of selected aerobic spore-forming bacteria: a freezeetching study. Bacteriol. Rev. 33, 346^378. Sleytr, U.B., Messner, P., Pum, D. and Saèra, M. (1993) Crystalline bacterial cell surface layers. Mol. Microbiol. 10, 911^ 916. Etienne-Toumelin, I., Sirard, J.-C., Du£ot, E., Mock, M. and Fouet, A. (1995) Characterization of the S-layer: cloning and sequencing of the structural gene. J. Bacteriol. 177, 614^620. Mesnage, S., Tosi-Couture, E., Mock, M., Gounon, P. and Fouet, A. (1997) Molecular characterization of the main S-layer component; evidence that it is the major cell-associated antigen. Mol. Microbiol. 23, 1147^ 1155. Adachi, T., Yamagata, H., Tsukagoshi, N. and Udaka, S. (1989) Multiple and tandemly arranged promoters of the cell wall protein gene operon in 47. J. Bacteriol. 171, 1010^1016. Tummuru, M.K.R. and Blaser, M.J. (1993) Rearrangement of homologs with conserved and variable regions in . Proc. Natl. Acad. Sci. USA 90, 7265^ 7269. Blaser, M.J., Wang, E., Tummuru, M.K.R., Washburn, R., Fujimoto, S. and Labigne, A. (1994) High frequency S-layer protein variation in revealed by mutagenesis. Mol. Microbiol. 14, 521^532. Dworkin, J. and Blaser, M.J. (1996) Generation of S-layer protein diversity utilizes a single promoter on an invertible DNA segment. Mol. Microbiol. 19, 1241^1253. Moran, C.P.J. (1993) RNA polymerase and transcription factors. In: and other Gram-positive Bacteria (Sonenshein, A.L., Hoch, J.A. and Losick, R., Eds.), pp. 653^667. American Society for Microbiology, Washington, DC. Vellanoweth, R.L. and Rabinowitz, J.C. (1992) The in£uence of ribosome-binding-site elements on translational e¤ciency in and in vivo. Mol. Microbiol. 6. 1105^1114. Simonen, M. and Palva I. (1993) Protein secretion in species. Microbiol. Rev. 57, 109^137. Aeromonas hydrophila Bacillus anthracis Bacillus anthracis Bacillus anthracis Bacillus anthracis Bacillus anthracis [47] Bacillus brevis [48] [49] sapA Campylobacter fetus Campylobacter fetus [50] [51] [52] lobacter fetus Bacillus subtilis Bacillus subtilis Bacillus sphaericus [53] sapA Campy- Escherichia coli Bacillus Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 2362. Ould, L.K. (1995) Seècreètion de proteèines heèteèrologues chez les Coryneèbacteèries productrices d'acides amineès. Thesis, Universiteè de Paris XI, Paris. Chami, M., Bayan, N., Peyret, J.L., Gulik-Krzywicki, T., Leblon, G. and Shechter, E. (1997) The S-layer protein of is anchored to the cell wall by its C-terminal hydrophobic domain. Mol. Microbiol. 23, 483^ 492. Lupas, A., Engelhardt, H., Peters, J., Santarius, U., Volker, S. and Baumeister, W. (1994) Domain structure of the surface layer revealed by electron crystallography and sequence analysis. J. Bacteriol. 176, 1224^1233. Olabarr|èa, G., Carrascosa, J.L., De Pedro, M.A. and Berenguer, J. (1996) A conserved motif in S-layer proteins is involved in peptidoglycan binding in . J. Bacteriol. 178, 4765^4772. Yang, L., Pei, Z., Fujimoto, S. and Blaser, M.J. (1992) Reattachment of surface array proteins to cells. J. Bacteriol. 174, 1258^1267. Walker, S.G., Karunaratne, D.N., Ravenscroft, N. and Smit, J. (1994) Characterization of mutants of defective in surface attachment of the paracrystalline surface layer. J. Bacteriol. 176, 6312^6323. Lechner, J. and Sumper, M. (1987) The primary structure of a procaryotic glycoprotein. J. Biol. Chem. 262, 9724^9729. Sumper, M., Berg, E., Mengele, R. and Strobel, I. (1990) Primary structure and glycosylation of the S-layer protein of . J. Bacteriol. 172, 7111^7118. Carl, M., Dobson, M.E., Ching, W.-M. and Dasch, G.A. (1990) Characterization of the gene encoding the protective paracrystalline surface layer protein of : presence of a truncated identical homolog in . Proc. Natl. Acad. Sci. USA 87, 8237^8241. Kessel, M., Wildhaber, I., Cohen, S. and Baumeister, W. (1988) Three-dimensional structure of the regular surface glycoprotein layer of from the dead sea. EMBO J. 7, 1549^1554. Trias, J., Jarlier, V. and Benz, R. (1992) Porins in the cell wall of mycobacteria. Science 258, 1479^1481. Niederweis, M., Maier, E., Lichtinger, T., Benz, R. and Kraëmer, R. (1995) Identi¢cation of channel-forming activity in the cell wall of J. Bacteriol. 177, 5716^5718. De Briel, D., Couderc, F., Riegel, P., Jehl, F. and Minck, R. (1992) High-performance liquid chromatography of coronomycolic acids as a tool in identi¢cation of species and related organisms. J. Clin. Microbiol. 30, 1407^1417. Kornberg, R.D. and Darst, S.A. (1991) Two-dimensional crystals of proteins on lipid layers. Curr. Opin. Struct. Biol. 1, 642^646. Hastie, A.T. and Brinton, C.C. Jr. (1979) Speci¢c interaction of the tetragonally arrayed protein layer of with its peptidoglycan sacculus. J. Bacteriol. 138, 1010^1021. Thomas, S., Austin, J.W., McCubbin, W.D., Kay, C.M. and Trust, T.J. (1992) Roles of structural domains in the morphol- 93 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 94 [54] Lemaire, M., Ohayon, H., Gounon, P., Fujino, P. and Beguin, P. (1995) OlpB, a new outer layer protein of dium thermocellum components of Clostri- , and binding of its S-layer-like domains to the cell envelope. J. Bacteriol. 177, 2451^ [54a] Zhu, X., McVeigh, R.R., Malathi, P. and Ghosh, B.K. (1996) Bacillus lichenifor- The complete nucleotide sequence of the from intestinal tracts of animals. Syst. Appl. Microbiol. 9, 210^213. Ghosh, B.K. (1989) Cloning of the crystalline cell wall protein gene of Bacillus licheniformis NM 105. J. Bacteriol. 171, 6637^6648. from and Daniels, L. (1991) Characterization of a Methanosarcina strain isolated from goat feces, and that grows on H2 -CO2 only after adaptation. Curr. Mirobiol. 23, 165^173. capsule membrane Bacillus licheniformis in . mediated biosynthetic Properties reaction. J. Biol. [57] Kist, M.L. and Murray, R.G.E. (1984) Components of the array dental plaque. Appl. Environ. Microbiol. 54, 600^603. [71] Belay, N., Mukhopadhyay, B., Conway de Macario, E., Galask, R. and Daniels, L. (1990) Methanogenic bacteria in Chem. 248, 305^315. surface E. and Daniels, L. (1988) Methanogenic bacteria from human Aquaspirillum serpens of MW5 and E. and Paris, J.M. (1991) Psychrophilic ecosystems of interest their assembly in vitro. J. Bacteriol. 157, 599^606. [58] Tsuboi, A., Uchihi, R., Tabata, R., Takahashi, Y., Hashiba, H., Sasaki, T., Yamagata, H., Tsukagoshi, N. and Udaka, S. (1986) Characterization of the genes coding for two major Bacillus brevis cell wall proteins from protein-producing human vaginal samples. J. Clin. Microbiol. 28, 1666^1668. è , J., Macario, A.J.L., Bardulet, M., Conway de Macario, [72] Cairo 47 : for wastewater treatment : Microbiologic and immunologic elucidation of the methanogenic £ora. Syst. Appl. Microbiol. 14, 85^92. [73] Franzmann, P.D., Springer, N., Ludwig, W., Conway de complete nucleotide sequence of the outer wall protein gene. Macario, E. and Rohde, M. (1992) A methanogenic archaeon J. Bacteriol. 168, 365^373. from [59] Boot, H.J., Kolen, C.P.A.M. and Pouwels, P.H. (1996) In- Ace Lake, Antarctica : Methanococcoides burtonii sp. nov. Syst. Appl. Microbiol. 15, 573^581. terchange of the active and silent S-layer protein genes of [74] Sieburth, J. McN., Johnson, P.W., Macario, A.J.L. and Con- by inversion of the chromosomal way de Macario, E. (1993) C1 bacteria in the water column of Lactobacillus acidophilus Chesapeake segment. Mol. Microbiol. 21, 799^809. èa, [60] Olabarr| and G., Fernandez-Herrero, Berenguer, J. (1996) SlpM , L.A., gene Carrascosa, coding for layer-like array' overexpressed in S-layer mutants of thermophilus an Bay. II. The dominant O2 and H2 S-tolerant J.L. methylotrophic methanogens, coenriched with their oxidative `S- and sulphate reducing bacterial consorts, are all new immu- Thermus HB8. J. Bacteriol. 178, 357^365. notypes and probably include new taxa. Marine Ecol. Prog. Ser. 95, 81^89. M è ra, M., Kuen, B., Mayer, H.F., Mandl, F., Schuster, K.C. [61] Sa [75] Blotevogel, K.-H., Gahl-Jan en, R., Jannsen, S., Fischer, U., and Sleytr, U.B. (1996) Dynamics in oxygen-induced changes Pilz, F., Auling, G., Macario, A.J.L. and Tindall, B.J. (1991) in S-layer protein synthesis from Isolation and characterization of a novel mesophilic, fresh- Bacillus stearothermophilus PV72 and the S-layer-de¢cient variant T5 in continuous culture and studies of the cell wall composition. J. Bacteriol. 178, 2108^2117. water methanogen from river sediment burgensis [76] Kotelnikova, [62] Takumi, K., Koga, T., Oka, T. and Endo, Y. (1991) Selfassembly, adhesion, and chemical properties of tetragonally arrayed S-layer proteins of Clostridium . J. Gen. Appl. Micro- biol. 37, 455^465. Methanoculleus olden- sp. nov. Arch. Microbiol. 157, 54^59. S., Pedersen, K. Methanobacterium subterranium and Macario, A.J.L. (1997) sp. nov., a new alcalophilic, eurythermic, and halotolerant methanogen isolated from deep granitic groundwater (submitted). [77] Jain, M.K., Thompson, T.E., Conway de Macario, E. and [63] Ezzell, J.W. and Abshire, T.G. (1988) Immunological analysis of cell-associated antigens Bacillus anthracis . Infect. Im- mun. 56, 349^356. Zeikus, J.G. ivanov as (1987) Speciation of Methanobacterium ivanovii Methanobacterium strain , sp. nov. Syst. Appl. Mi- crobiol. 9, 77^82. [64] Macario, A.J.L. and Conway de Macario, E. (1985) Mono- [78] Kim, B.-Y., Conway de Macario, E., No ë lling, J. and Daniels, clonal antibodies of prede¢ned molecular speci¢city for iden- L. (1996) Isolation and characterization of a copper-resistant ti¢cation and classi¢cation of methanogens and for probing methanogen from a copper-mining soil sample. Appl. Envi- their ecologic Bacteria niches. (Macario, In : Monoclonal A.J.L. and Antibodies Conway de Against Macario, E., Eds.), Vol. II, pp. 213^247. Academic Press, Orlando, FL. [65] Beveridge, T.J. (1994) Bacterial S-layers. Curr. Opin. Struct. Biol. 4, 204^212. (1994) Identi¢cation Conway de Macario, E. and Winter, J. (1989) Characterization of a new mesophilic, secondary alcohol-utilizing methanogen, [66] Konisky, J., Lynn, D., Hoppert, M., Mayer, F. and Haney, P. ron. Microbiol. 62, 2629^2635. [79] Zellner, G., Bleicher, K., Braun, E., Kneifel, H., Tindall, B.J., of Methanococcus voltae structural gene. J. Bacteriol. 176, 1790^1792. S-layer Methanobacterium palustre spec. nov. from a peat bog. Arch. Microbiol. 151, 1^9. [80] Fielding, E.R., Archer, D.B., Conway de Macario, E. and Macario, A.J.L. (1988) Isolation and characterization of Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 (Q-D-glutamyl) regular Methanobrevibacter smithii [70] Belay, N., Johnson, R., Rajagopal, B.S., Conway de Macario, [56] Troy, F.A. (1973) Chemistry and biosynthesis of the poly the cario, A.J.L. (1982) Isolation human feces. Appl. Environ. Microbiol. 43, 227^232. [69] Mukhopadhyay, B., Purwantini, E., Conway de Macario, E. NM105 S-layer-encoding gene. Gene 173, 189^194. [55] Tang, M., Owens, K., Pietri, R., Zhu, X., McVeigh, R. and of lin, M.J. (1987) Antigenic diversity of methanogenic bacteria [68] Miller, T.L., Wolin, M.J., Conway de Macario, E. and Ma- 2459. mis [67] Conway de Macario, E., Macario, A.J.L., Miller, T. and Wo- H. Bahl et al./FEMS Microbiology Reviews 20 (1997) 47^98 [81] [82] [83] [85] [86] [87] [88] [89] [90] [91] [92] [93] Borst, P., Bitter, W., McCulloch, R., Van Leeuwen, F. and Rudenko, G. (1995) Antigenic variation in malaria. Cell 82, 1^4. [94] Dybvig, K. (1993) DNA rearrangements and phenotypic switching in prokaryotes. Mol. Microbiol. 10, 465^471. [95] Jovell, R.J., Macario, A.J.L. and Conway de Macario, E. (1996) ABC transporters in Archaea: Two genes encoding homologs of the nucleotide-binding components in the methanogen Methanosarcina mazei S-6. Gene 174, 281^284. [96] Jovell, R.J., Macario, A.J.L. and Conway de Macario, E. (1997) An ABC-transporter system homolog in an organism of the phylogenetic domain Archaea. DNA Sequence 7, 193^ 198. [97] Castoèn, J., Carrascosa, J., de Pedro, M.A. and Berenguer, J. (1988) Identi¢cation of a crystalline layer on the cell envelope of the thermophilic eubacterium Thermus thermophilus. FEMS Lett. 51, 225^230. [98] Faraldo, M.L.M., de Pedro, M.A. and Berenguer, J. (1988) Puri¢cation, composition and Ca2 -binding properties of the monomeric protein of the S-layer of Thermus thermophilus. FEBS Lett. 235, 117^121. [99] Faraldo, M.L.M., de Pedro, M.A. and Berenguer, J. (1991) Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S-layer of Thermus thermophilus HB8. J. Bacteriol. 173, 5346^5351. [100] Faraldo, M.L.M., de Pedro, M.A. and Berenguer, J. (1992) Sequence of the S-layer gene of Thermus thermophilus HB8 and functionality of its promoter in Escherichia coli. J. Bacteriol. 174, 7458^7462. [101] Castoèn, J.R., Berenguer, J., de Pedro, M.A. and Carrascosa, J.L. (1993) The S-layer protein from Thermus thermophilus HB8 assembles into porin-like structures. Mol. Microbiol. 9, 65^75. [102] Castoèn, J.R., Berenguer, J., Kocsics, E. and Carrascosa, J.L. (1994) Three-dimensional structure of di¡erent aggregates built up by the S-layer protein of Thermus thermophilus. J. Struct. Biol. 113, 164^176. [103] Sleytr, U.B. (1978) Regular arrays of macromolecules on bacterial cell walls: structure, chemistry, assemble, and function. Int. Rev. Cytol. 53, 1^64. [104] Lasa, I., de Grado, M., de Pedro, M.A. and Berenguer, J. (1992) Development of Thermus-Escherichia shuttle vectors and their use for the expression in Thermus thermophilus of the celA gene from Clostridium thermocellum. J. Bacteriol. 174, 6424^6431. [105] Plumbridge, J.A., Cochet, O., Souza, J.M., Altamirano, M.M., Calgagno, M.L. and Badet, B. (1993) Coordinated regulation of animo sugar-synthesizing and -degrading enzymes in Escherichia coli K12. J. Bacteriol. 175, 4951^4956. [106] Fernaèndez-Herrero, L.A., Badet-Denisot, M.A., Badet, B. and Berenguer, J. (1995) GlmS of Thermus thermophilus HB8: An essential gene for cell wall synthesis identi¢ed immediately upstream the S-layer gene. Mol. Microbiol. 17, 1^12. [107] Beck, C.F. and Warren, R.A.J. (1988) Divergent promoters, a common form of gene organization. Microbiol. Rev. 52, 318^326. [108] Lasa, I., Castoèn, J.R., Fernandez-Herrero, L.A., Pedro, Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 [84] methanogenic bacteria from land¢lls. Appl. Environ. Microbiol. 54, 835^836. Macario, A.J.L. and Conway de Macario, E. (1988) Quantitative immunologic analysis of the methanogenic £ora of digestors reveals a considerable diversity. Appl. Environ. Microbiol. 54, 79^86. Macario, A.J.L., Earle, J.F.K., Chynoweth, D.P. and Conway de Macario, E. (1989) Distinctive patterns of methanogenic £ora determined with antibody probes in anaerobic digestors of di¡erent characteristics operated under controlled conditions. Syst. Appl. Microbiol. 12, 216^222. Zellner, G., Alten, C., Stackebrandt, E., Conway de Macario, E. and Winter, J. (1987) Isolation and characterization of Methanocorpusculum parvum, gen. nov., spec. nov., a new tungsten requiring, coccoid methanogen. Arch. Microbiol. 147, 13^20. Zellner, G., Stackebrandt, E., Messner, P., Tindall, B.J., Conway de Macario, E. Kneifel, H., Sleytr, U.B. and Winter, J. (1989) Methanocorpusculaceae fam. nov., represented by Methanocorpusculum parvum, Methanocorpusculum sinense spec. nov. and Methanocorpusculum bavaricum spec. nov. Arch. Microbiol. 151, 381^390. Conway de Macario, E., Macario, A.J.L. and Pastini, A. (1985) The super¢cial antigenic mosaic of Methanobrevibacter smithii : Identi¢cation of determinants and isolates by monoclonal antibodies. Arch. Microbiol. 142, 311^316. Conway de Macario, E., Macario, A.J.L., and Magarinìos, M.C., Koënig, H. and Kandler, O. (1983) Dissecting the antigenic mosaic of the archaebacterium Methanobacterium thermoautotrophicum by monoclonal antibodies of de¢ned molecular speci¢city. Proc. Natl. Acad. Sci. USA 80, 6346^6350. Conway de Macario, E. and Macario, A.J.L. (1986) Immunology of archaebacteria: Identi¢cation, antigenic relationships and immunochemistry of surface structures. Syst. Appl. Microbiol. 7, 320^324. Mayerhofer, L.E., Macario, A.J.L. and Conway de Macario, E. (1992) Lamina, a novel multicellular form of Methanosarcina mazei S-6. J. Bacteriol. 174, 309^314. Clarens, M., Cairoè, J.J., Par|ès, J.M., Macario, A.J.L. and Conway de Macario, E. (1993) Characterization and forms of JC3, a new Methanosarcina isolate: Comparison with Methanosarcina mazei strains S-6T, MC3, and LYC. Curr. Microbiol. 26, 167^174. Yao, R., Macario, A.J.L. and Conway de Macario, E. (1994) An archaeal S-layer gene homolog with repetitive subunits. Biochim. Biophys. Acta 1219, 697^700. Broëckl, G., Behr, M., Fabry, S., Hensel, R., Kaudewitz, H., Biendl, E. and Koënig, H. (1991) Analysis and nucleotide sequence of the genes encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis. Eur. J. Biochem. 199, 147^ 152. Mayerhofer, L.E., Conway de Macario, E. and Macario, A.J.L. (1995) Conservation and variability in Archaea: Protein antigens with tandem repeats encoded by a cluster of genes with common motifs in Methanosarcina mazei S-6. Gene 165, 87^91. 95 96 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 [123] Grogono-Thomas, R., Newell, D.G., Woodland, R.M. and Blaser, M.J. (1997) The role of S-layer proteins in ovine campylobacter abortion. FEMS Microbiol. Rev. (this issue). [124] Hollaus, F. (1969) Charakterisierung hochthermophiler Bacillus-Staëmme aus Extraktionsanlagen von Ru«benzuckerfabriken. Thesis, University of Agricultural Sciences, Vienna. [125] Messner, P., Hollaus, F. and Sleytr, U.B. (1984) Paracrystalline cell wall surface layers on di¡erent Bacillus stearothermophilus strains. Int. J. Syst. Bacteriol. 34, 202^210. [126] Sleytr, U.B., Saèra, M., Kuëpcuë, Z. and Messner, P. (1986) Structural and chemical characterization of S-layers of selected strains of Bacillus stearothermophilus and Desulfotomaculum nigri¢cans. Arch. Microbiol. 146, 19^24. [127] Bartelmus, W. and Perschak, F. (1957) Schnellmethode zur Keimzahlbestimmung in der Zuckerindustrie. Z. Zuckerind. 7, 276^281. [128] Schuster, K.C., Mayer, H.F., Kieweg, R., Hampel, W.A. and Saèra, M. (1995) A synthetic medium for continuous culture of the S-layer carrying Bacillus stearothermophilus PV72 and studies on the in£uence of growth conditions on cell wall properties. Biotechnol. Bioeng. 48, 66^77. [129] Kuen, B., Sleytr, U.B. and Lubitz, W. (1994) Sequence analysis of the sbsA gene encoding the 130 kDA surface layer protein of Bacillus stearothermophilus PV72. Gene 145, 115^ 120. [130] Kuen, B., Saèra, M. and Lubitz, W. (1996) Heterologous expression and self-assembly of the S-layer protein SbsA of Bacillus stearothermophilus in Escherichia coli. Mol. Microbiol. 19, 495^503. [131] Breitwieser, A., Gruber, K. and Sleytr, U.B. (1992) Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus. J. Bacteriol. 174, 8008^8015. [132] Sleytr, U.B. and Glauert, A.M. (1975) Analysis of regular arrays of subunits on bacterial surfaces: evidence for a dynamic process of assembly. J. Ultrastruct. Res. 50, 103^116. [133] Pink, T., Langer, K., Hotzy, C. and Saèra, M. (1996) Regulation of S-layer protein synthesis of Bacillus stearothermophilus PV72 through variation of continuous cultivation conditions. J. Biotechnol. 50, 189^200. [134] Eder, J. (1983) Versuche zur Aufklaërung der Funktion parakristalliner Proteinmembranen Bacillus stearothermophilus. Thesis, University of Agricultural Sciences, Vienna. [135] Schuster, K.C., Pink, T., Mayer, H.F., Hampel, W.A. and Saèra, M. (1997) Oxygen-triggered synchronized variant formation of the S-layer carrying Bacillus stearothermophilus PV72 during continuous cultivation. J. Biotechnol. (in press). [136] Saèra, M. and Sleytr, U.B. (1994) Comparative studies of S-layer proteins from Bacillus stearothermophilus strains expressed during growth in continuous culture under oxygenlimited and non oxygen-limited conditions. J. Bacteriol. 176, 7182^7189. [137] Kuen, B. and Lubitz, W. (1996) Analysis of S-layer proteins and genes. In: Crystalline Bacterial Cell Surface Proteins (Sleytr, U.B., Messner, P., Pum, D. and Saèra, M., Eds.), pp. 77^102. Landes, Academic Press, Austin, TX. [138] Kuen, B., Koch, A., Asenbauer, E., Saèra, M. and Lubitz, W. (1997) Molecular characterization of the Bacillus stearother- Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 M.A. and Berenguer, J. (1992) Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus. Mol. Microbiol. 11, 1555^1564. [109] Henning, U. and Koebnik, R. (1994) Outer membrane proteins of Escherichia coli : mechanism of sorting and regulation of synthesis. In: Bacterial Cell Wall (Ghuysen, J.M. and Hakenbeck, R., Eds.), Vol. 27, pp. 381^395. Elsevier, London. [110] Fernaèndez-Herrero, L.A. (1995) Caracterizacioèn de genes implicados en la regulacioèn y la s|èntesis de la envoltura celular de T. thermophilus HB8. Universidad Autoènoma de Madrid. [111] Fernaèndez-Herrero, L.A., Olabarr|èa, G. and Berenguer, J. (1997) Surface proteins and a novel transcription factor regulate the expression of the S-layer gene in Thermus thermophilus HB8. Mol. Microbiol. 24, 61^72. [112] Travers, A.A. (1991) DNA bending and kinking: sequence dependence and function. Curr. Opin. Struct. Biol. 1, 114^ 122. [113] Boot, H.J. and Pouwels, P.H. (1996) Expression, secretion and antigenic variation of bacterial S-layer proteins. Mol. Microbiol. 21, 1117^1123. [114] Boot, H.J., Kolen, C.P.A.M., Andreadaki, F.J., Leer, R.J. and Pouwels, P.H. (1996) The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA. J. Bacteriol. 178, 5388^5394. [115] Blaser, M.J. (1993) Role of the S-layer proteins of Campylobacter fetus in serum resistance and antigenic variation: a model of bacterial pathogenesis. Am. J. Med. Sci. 306, 325^329. [116] Blaser, M.J. and Pei, Z. (1993) Pathogenesis of Campylobacter fetus infections. Critical role of high molecular mass S-layer proteins in virulence. J. Infect. Dis. 167, 372^377. [117] Neuzil, K.M., Wang, E., Haas, D. and Blaser, M.J. (1994) Persistence of Campylobacter fetus bacteremia associated with absence of opsonizing antibodies. J. Clin. Microbiol. 32, 1718^1720. [118] Garcia, M.M., Lutze-Wallace, C.L., Azucena, S.D., Eaglesome, M.D., Holst, E. and Blaser, M.J. (1995) Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs. J. Bacteriol. 177, 1976^1980. [119] Dworkin, J., Tummuru, M.K.R. and Blaser M.J. (1995) Segmental conservation of sapA sequences in type B Campylobacter fetus cells. J. Biol. Chem. 270, 15093^15101. [120] Tummuru, M.K.R. and Blaser, M.J. (1992) Characteristics of the Campylobacter fetus sapA promoter: evidence that the sapA promoter is deleted in spontaneous mutant strains. J. Bacteriol. 174, 5916^5922. [121] Dworkin, J., Tummuru, M.K.R. and Blaser, M.J. (1995) The lipopolysaccharide binding domain of the Campylobacter fetus S-layer protein resides within the conserved N-terminus of a family of silent and divergent homologs. J. Bacteriol. 177, 1734^1741. [122] Dworkin, J. and Blaser, M.J. (1997) Nested DNA inversion as a paradigm of programmed gene rearrangement. Proc. Natl. Acad. Sci. USA 94, 985^990. H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 mophilus PV72 S-layer gene sbsB induced by oxidative stress. [139] [140] [141] [143] [144] [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] [163] [164] [165] [166] [167] [168] P.H. (1993) S-layer protein of Lactobacillus acidophilus ATCC 4356: puri¢cation, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J. Bacteriol. 175, 6089^6096. Boot, H.J., Kolen, C.P.A.M. and Pouwels, P.H. (1995) Identi¢cation, cloning and nucleotide sequence of a silent S-layer protein gene of Lactobacillus acidophilus 4356 which has extensive similarity with the S-layer protein gene of this species. J. Bacteriol. 177, 7222^7230. Vidgren, G., Palva, I., Pakkanen, R., Lounatmaa, K. and Palva, A. (1992) S-layer protein gene of Lactobacillus brevis : cloning by polymerase chain reaction and determination of the nucleotide sequence. J. Bacteriol. 174, 7419^7427. Pouwels, P.H. and Leunissen, J.A.M. (1994) Divergence in codon usage of Lactobacillus species. Nucleic Acids Res. 22, 929^936. Boot, H.J., Kolen, C.P.A.M., Pot, B., Kersters, K. and Pouwels, P.H. (1996) The presence of two S-layer protein encoding genes is conserved among species related to Lactobacillus acidophilus. Microbiology 142, 2375^2384. Hellmann, C., Schweitzer, O., Gerke, C., Vanittanakom, N., Mack, D. and Gotz, F. (1996) Molecular basis of intercellular adhesion in the bio¢lm-forming Staphylococcus epidermidis. Mol. Microbiol. 20, 1083^1091. Pendrak, M.L. and Perry, R.D. (1993) Proteins essential for expression of the Hms phenotype of Yersinia pestis. Mol. Microbiol. 8, 857^864. Hinnebusch, B.J., Perry, R.D. and Schwan, T.G. (1996) Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by £eas. Science 273, 367^370. Noonan, B. and Trust, T.J. (1995) The leucine zipper of Aeromonas salmonicida AbcA is required for the transcriptional activation of the P2 promoter of the surface-layer structural gene, vapA, in Escherichia coli. Mol. Microbiol. 17, 379^386. Bowditch, R.D., Baumann, P. and Yousten, A.A. (1989) Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene. J. Bacteriol. 171, 4178^4188. Matern, H.T., Klein, J.R., Henrich, B. and Plapp, R. (1994) Determination and comparison of Lactobacillus delbrueckii ssp. lactis DSM7290 promoter sequences. FEMS Microbiol. Lett. 122, 121^128. Boot, H.J. (1996) The surface layer protein genes of Lactobacillus acidophilus. Thesis, University of Amsterdam, Amsterdam. Masuda, K. and Kawata, T. (1980) Reassembly of the regularly arranged subunits in the cell wall of Lactobacillus brevis and their reattachment to cell walls. Microbiol. Immunol. 24, 299^308. Feng, J.-A., Johnson, R.C. and Dickerson, R.E. (1994) Hin recombinase bound to DNA: the origin of speci¢city in major and minor groove interactions. Science 263, 348^355. Toba, T., Virkola, R., Westerlund, B., Bjorkman, Y., Sillanpaa, J., Vartio, T., Kalkkinen, N. and Korhonen, T.K. (1995) A collagen-binding S-layer protein in Lactobacillus crispatus. Appl. Environ. Microbiol. 61, 2467^2471. Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 [142] J. Bacteriol. 179, 1664^1670. Blaser, M. and Joel, D. (1996) Generation of Campylobacter fetus S-layer protein diversity utilizes a single promoter on an invertible DNA segment. Mol. Microbiol. 19, 1241^1253. Plasterk, R.H.A., Simon, M.I. and Barbour, A.G. (1985) Transposition of structural genes to an expression sequence on a linear plasmid causes antigenic variation in the bacterium Borrelia hermsii. Nature 318, 257^263. Rosa, P.A., Schwan, T. and Hogan, D. (1992) Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi. Mol. Microbiol. 6, 3031^3040. McKay, L.L. and Baldwin, K.A., (1990) Applications for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol. Rev. 87, 3^14. Perdigon, G., Medici, M., Bibas Bonet de Jorrat, M.E., Valverde de Budeguer, M. and Pesce de Ruiz Holgado, A. (1993) Immunomodulating e¡ects of lactic acid bacteria on mucosal and tumoral immunity. Int. J. Immunother. 9, 29^ 52. Kitazawa, H., Matsumura, K., Itoh, T. and Yamaguchi, T. (1992) Interferon induction in murine peritoneal macrophage by stimulation with Lactobacillus acidophilus. Microbiol. Immunol. 36, 311^315. Fernandes, C.F., Shahani, K.M. and Amer, M.A. (1987) Therapeutic role of dietary lactobacilli and lactobacillic fermented dairy products. FEMS Microbiol. Rev. 46, 343^356. Coconnier, M.-H., Bernet, M.-F., Kerneis, S., Chauviere, G., Fourniat, J. and Servin, A.L. (1993) Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiol. Lett. 110, 299^306. Beachey, E.H. (1981) Bacterial adherence: Adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143, 325^345. Masuda, K. and Kawata, T. (1983) Distribution and chemical characterization of regular arrays in the cell walls of strains of the genus Lactobacillus. FEMS Microbiol. Lett. 20, 145^150. Ray, B. and Johnson, M.C. (1986) Freeze-drying injury of surface layer protein and its protection in Lactobacillus acidophilus. Cryo-Letters 7, 210^217. Lortal, S. (1993) Crystalline surface layers of the genus Lactobacillus. In: Advances in Paracrystalline Bacterial Surface Layers (Beveridge, T.J. and Koval, S.F., Eds.), pp. 57^65. Plenum, New York. Mukai, T. and Arihara, K. (1994) Presence of intestinal lectin-binding glycoproteins on the cell surface of Lactobacillus acidophilus. Biosci. Biotech. Biochem. 58, 1851^1854. Schneitz, C., Nuotio, L. and Lounatma, K. (1993) Adhesion of Lactobacillus acidophilus to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer (S-layer) J. Appl. Bacteriol. 74, 290^294. Greene, J.D. and Klaenhammer, T.R. (1994) Factors involved in adherence of lactobacilli to human Caco-2 cells. Appl. Environ. Microbiol. 60, 4487^4494. Boot, H.J., Kolen, C.P.A.M., van Noort, J.M. and Pouwels, 97 H. Bahl et al. / FEMS Microbiology Reviews 20 (1997) 47^98 98 [169] Pouwels, P.H., Leer, R.J. and Boersma, W.J.A. (1996) The potential of Lactobacillus as a carrier for oral immunization : isolated from the chromosome of Lactococcus lactis lactis. Appl. Environ. Microbiol. 57, 341^348. subsp. development and preliminary characterization of vector sys- è ra, M., Ku [186] Sa ë pcu ë , S. and Sleytr, U.B. (1996) Biotechnological tems for targeted delivery of antigens. J. Biotechnol. 44, 183^ applications of S-layers. In : Crystalline Bacterial Cell Surface è ra, M., Proteins (Sleytr, U.B., Messner, P., Pum, D. and Sa 192. [170] Sleytr, U.B. and Messner, P. (1988) Crystalline surface layers è ra, M. and Sleytr, U.B. (1989) Structure, surface [187] Pum, D., Sa in prokaryotes. J. Bacteriol. 170, 2891^2897. [171] Board, R.G., Jones, D. and Jarvis, B. (1995) Microbial Fermentations : Beverages, Foods, and Feeds. Society for Applied Bacteriology Symposium Series Number 24. Suppl. to [172] Wood, J.B. (Ed.) (1992) The Lactic Acid Bacteria in Health coagulans E38-66. J. Bacteriol. 171, 5296^303. Bacillus è ra, M. and Sleytr, U.B. (1987) Charge distribution on the [188] Sa Bacillus stearothermophilus NRS 1536/3c and the importance of charged groups for morphogenesis and func- tial characterization of regular array in the cell wall of tobacillus brevis. Microbiol. Immunol. 23, 941^953. Lac- [174] Palva, A., Vidgren, G., Palva, I., Pakkanen, R. and Lounatmaa, K. (1992) Characterization of expression and secretion for use in food and feed è ra, [189] Sa M. and Sleytr, U.B. (1993) Relevance of charged groups for the integrity of the layer from Bacillus coagulans E38^66 J. and for molecular interactions. Bacteriol. 175, 2248^2254. è ra, M. (1996) Oc[190] Sleytr, U.B., Messner, P., Pum, D. and Sa currence, location, Crystalline and Bacterial morphogenesis Cell Surface of S-layers. technology. Med. Fac. Landbouwwetensch. Univ. Gent, 57/ è ra, M., Eds.), (Sleytr, U.B., Messner, P., Pum, D. and Sa Proteins pp. 5^34. Landes, Academic Press, Austin, TX. 4b, 1891^1898. [175] Von Heijne, G. (1985) Signal sequences : The limits of varia- re£ects both translational selection Bacillus subtilis integration vector and its application. Gene 181, 71^76. [192] Chen, ogy. Academic Press, New York. [177] Shields, D.C. and Sharp, P.M. (1987) Synonymous codon Bacillus subtilis [191] Kim, L., Mogk, A. and Schumann, W. (1996) A xylose-inducible tion. J. Mol. Biol. 184, 99^105. [176] Von Heijne, G. (1987) Sequence Analysis in Molecular Biol- usage in In : ultrastructure industry. In : Proceedings of Sixth Forum for Applied Bio- W.J., [178] Smit, J. (1986) Protein surface layers of bacteria. In Bacterial L., Joho, K.E. and McAllister, W.T. two-codon insertion mutagenesis. Gene 111, 143^144. [193] Breiteneder, and mutational biases. Nucleic Acids Res. 15, 8023-8040. Gross, (1992) A modi¢ed kanamycin-resistance cassette to facilitate H., Pettenburger, Kraft, D., Rumpold, H. and K., Bito A., Breitenbach, Valenta, M. (1989) R., The Bet v 1, Outer Membranes as Model Systems (Inouye, M., Ed.), pp. gene coding for the major birch pollen allergen 343^376. John Wiley and Sons, New York. highly homologous to a pea disease response gene. EMBO J. [179] Kahala, M., Savijoki, K. and Palva, A. (1997) In vivo expression of the Lactobacillus brevis S-layer gene. J. Bacteriol. S., Gustafson, C.E., Feutrier, J., Cavaignac, Trust, T.J. (1993) Transcriptional analysis of the salmonidica S-layer protein gene vapA. J. S. and Aeromonas Bacteriol. 175, [181] Ebisu, S., Tsuboi, A., Takagi, H., Naruse, Y., Yamagata, H., Tsukagoshi, N. and Udaka, S. (1990) Conserved structures of cell wall protein genes among protein-producing brevis strains. J. Bacteriol. 172, 1312^1320. Bacillus Ho¡mann-Sommergruber, K., Breiteneder, R., Bohle, B., Sperr, W.R., Valent, P., Kungl, A.J., Breitenbach, M., Kraft, D. and Schreiner, O. (1993) Puri¢cation and characterization of recombinant Bet v 1, the major birch Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals. Gene 186, 255^262. Lactococcus and [184] Koivula, T., Sibakov, M. and Palva, I. (1991) Isolation and characterization of Lactococcus lactis subsp. lactis pro- moters. Appl. Environ. Microbiol. 57, 333^340. Koivula, T., (1991) Secretion of TEM von Wright, L-lactamase virus : T.C. Pseudorabies (1994) (Aujesky's disease) state of the art, August 1993. Acta Vet. Hung. 42, [196] Janes, B., Hollar, J. and Dennis, D. (1990) Molecular characterisation of the poly- L-hydroxy-butyrate Alcaligenes eutrophus H16. biosynthesis in In : Novel Biodegradable Micro- bial Polymers (Dawes, E.A., Ed.), pp. 175^190. Kluwer, Dor- Appl. Environ. Microbiol. 48, 726^731. [183] Savijoki, K., Kahala, M. and Palva, A. (1997) High level heterologous protein production in 1. J. Biol. Chem. 268, 19574^19580. [195] Mettenleiter, 153^177. [182] Kok, J., van der Vossen, M.B.M. and Venema, G. (1984) M., F.D., pollen allergen. Immunological equivalence to natural Bet v 7968^7975. [185] Sibakov, 8, 1935^1938. [194] Ferreira, H., Pettenburger, K., Ebner, C., Sommergruber, W., Steiner, 179, 284^286. [180] Chu, is A. and Palva, I. with signal sequences drecht. [197] Kalousek, S., Dennis, D. and Lubitz, W. (1993) Genetic engineering of PHB-synthase from Alcaligenes eutrophus H16. In : Proceedings of the International Symposium on Bacterial Polyhydroxy-alkanoates. (Schlegel, H.G. A., Eds.), pp. 426^427. Goltze, Go ë ttingen. and Steinbu ë chel, Downloaded from https://academic.oup.com/femsre/article/20/1-2/47/512411 by guest on 01 January 2023 tion. J. Bacteriol. 169, 2804^2809. and Disease. Elsevier, Amsterdam. [173] Masuda, K. and Kawata, T. (1979) Ultrastructure and par- Lactobacillus brevis charge and self-assembly of the S-layer lattice from S-layer of J. Appl. Bacteriol. Vol. 79. Blackwell Science, Oxford. signals from Eds.), pp. 133^160. Landes, Academic Press, Austin, TX.

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