The molecular aspects of the type 37 pneumococcal capsular biosynthesis, a homopolysaccharide com... more The molecular aspects of the type 37 pneumococcal capsular biosynthesis, a homopolysaccharide composed of sophorosyl units (�-d-Glc-(1→2)-�-d-Glc) linked by �-1,3 bonds, have been studied. Remarkably, the biosynthesis of the type 37 capsule is driven by a single gene (tts) located far apart from the cap locus responsible for capsular formation in all of the types characterized to date in Streptococcus pneumoniae. However, a cap37 locus virtually identical to the cap33f cluster has been found in type 37 strains, although some of its genes are inactivated by mutations. The tts gene has been sequenced and its transcription start point determined. Tts shows sequence motifs characteristic of cellulose synthases and other �-glycosyltransferases. Insertion of the tts gene into the pneumococcal DNA causes a noticeable genome reorganization in such a way that genes normally separated by more than 350 kb in the chromosome are located together in clinical isolates of type 37. Encapsulated pneu...
El tipo 37 de Streptococcus pneumoniae sintetiza una capsula formada por un homopolisacarido de g... more El tipo 37 de Streptococcus pneumoniae sintetiza una capsula formada por un homopolisacarido de glucosa ramificado, siendo la estructura y composicion mas sencilla de las descritas hasta el momento entre los polisacaridos capsulares de neumococo (se han descrito 90 tipos capsulares diferentes en S.pneumoniae). La capsula es el principal factor de virulencia de este microorganismo, ya que las estirpes que han perdido la capacidad de sintetizarla son practicamente avirulentas ya que son fagocitadas rapidamente por el sistema inmunitario del huesped. Dada la importancia de la capsula, el estudio del tipo 37 parecia ser adecuado para aportar datos acerca de los procesos desconocidos de la sintesis capsular en este microorganismo. No obstante, el estudio del locus cap, responsable de la sintesis en los tipos capsulares estudiados hasta el momento, revelo que este serotipo es un caso especial, ya que la sintesis capsular no esta asociada a locus cap37, sino que este constituye el resto ge...
Many streptococci are human and/or animal pathogens and the frequent cause of life-threatening di... more Many streptococci are human and/or animal pathogens and the frequent cause of life-threatening diseases. Among various streptococcal virulence factors, capsular polysaccharides (CPs) are recognized as essential to prevent phagocytosis by macrophages and neutrophils. In the last decade, an impressive advance on the knowledge of the genetic bases underlying capsule formation has been achieved. The capsular gene cluster driving the formation of the CP of Streptococcus pyogenes and other hyaluronate-producing streptococci, represents one of the simplest cases of gene organization to synthesize a capsule. A more complex situation has been found in Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, and other streptococci. On the whole, there exists a direct relationship between the structural and chemical complexity of the repeating unit of the polysaccharide and the number of genes found in the corresponding capsular locus. Streptococcal vaccines, either polysaccharide or conjugate, are currently being tested in clinical trials to overcome the rise of worldwide antibiotic resistance, although, for different reasons, none of these vaccines are expected to provide the required full coverage in a near future. This concern has prompted to explore alternative possibilities with an improved therapeutic potential against streptococcal diseases.
Background: Protein folding in the envelope is a crucial limiting step of protein export and secr... more Background: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H 2 O 2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H 2 O 2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.
The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococ... more The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0?04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS " mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS " phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS " mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.
It has been recently reported that different type 37 clinical isolates of Streptococcus pneumonia... more It has been recently reported that different type 37 clinical isolates of Streptococcus pneumoniae have an identical tts gene directing the formation of type 37 capsular polysaccharide. Here we show that type 37 S. pneumoniae strains isolated in two different continents (Europe and America) some 60 years apart frequently gave rise to nontypable variants upon in vitro cultivation. The tts gene from three independent nontypable mutants was PCR amplified and sequenced showing different classes of inactivating mutations. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that the type 37 pneumococcal isolates studied so far constitute a highly related strain cluster (a clonal complex), and strongly suggested that every type 37 pneumococcus has spread globally from a single, old clone.
Journal of Molecular Microbiology and Biotechnology, 2008
using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox w... more using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox we are developing should contribute to enlarge the use of L. lactis as a protein cell factory.
The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and ... more The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.
The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating u... more The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating units of [-D-Glcp-(132)]--D-Glcp-(133). The elements governing the expression of the tts gene, coding for the glucosyltransferase involved in the synthesis of the type 37 pneumococcal capsular polysaccharide, have been studied. Primer extension analysis and functional tests demonstrated the presence of four new transcriptional start points upstream of the previously reported tts promoter (ttsp). Most interesting, three of these transcriptional start points are located in a RUP element thought to be involved in recombinational events (Oggioni, M. R., and Claverys, J. P. (1999) Microbiology 145, 2647-2653). Transformation experiments using either a recombinant plasmid containing the whole transcriptional unit of tts or chromosomal DNA from a type 37 pneumococcus showed that tts is the only gene required to drive the biosynthesis of a type 37 capsule in S. pneumoniae and other Gram-positive bacteria, namely Streptococcus oralis, Streptococcus gordonii, and Bacillus subtilis. The Tts synthase was overproduced in S. pneumoniae and purified as a membrane-associated enzyme. These membrane preparations used UDP-Glc as substrate to catalyze the synthesis of a high molecular weight polysaccharide immunologically identical to the type 37 capsule. In addition, UDP-Gal was also a substrate to produce type 37 polysaccharide since a strong UDP-Glc-4-epimerase activity is associated to the membrane fraction of S. pneumoniae. These results indicated that Tts has a dual biochemical activity that leads to the synthesis of the branched type 37 polysaccharide.
The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a c... more The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-i...
The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been... more The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 degrees C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.
A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galact... more A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution o...
Here we developed the new expression system PZn zitR, based on the regulatory signals (PZn promot... more Here we developed the new expression system PZn zitR, based on the regulatory signals (PZn promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn2+ high-affinity uptake and regulation. A PZn zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic β-galactosidase. Nuclease and β-galactosidase activities of L. lactis MG1363 cells expressing either uspnuc or lacLM under the control of PZn zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn2+, availability in the environment. Our results demonstrate that PZn zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the pr...
The molecular aspects of the type 37 pneumococcal capsular biosynthesis, a homopolysaccharide com... more The molecular aspects of the type 37 pneumococcal capsular biosynthesis, a homopolysaccharide composed of sophorosyl units (�-d-Glc-(1→2)-�-d-Glc) linked by �-1,3 bonds, have been studied. Remarkably, the biosynthesis of the type 37 capsule is driven by a single gene (tts) located far apart from the cap locus responsible for capsular formation in all of the types characterized to date in Streptococcus pneumoniae. However, a cap37 locus virtually identical to the cap33f cluster has been found in type 37 strains, although some of its genes are inactivated by mutations. The tts gene has been sequenced and its transcription start point determined. Tts shows sequence motifs characteristic of cellulose synthases and other �-glycosyltransferases. Insertion of the tts gene into the pneumococcal DNA causes a noticeable genome reorganization in such a way that genes normally separated by more than 350 kb in the chromosome are located together in clinical isolates of type 37. Encapsulated pneu...
El tipo 37 de Streptococcus pneumoniae sintetiza una capsula formada por un homopolisacarido de g... more El tipo 37 de Streptococcus pneumoniae sintetiza una capsula formada por un homopolisacarido de glucosa ramificado, siendo la estructura y composicion mas sencilla de las descritas hasta el momento entre los polisacaridos capsulares de neumococo (se han descrito 90 tipos capsulares diferentes en S.pneumoniae). La capsula es el principal factor de virulencia de este microorganismo, ya que las estirpes que han perdido la capacidad de sintetizarla son practicamente avirulentas ya que son fagocitadas rapidamente por el sistema inmunitario del huesped. Dada la importancia de la capsula, el estudio del tipo 37 parecia ser adecuado para aportar datos acerca de los procesos desconocidos de la sintesis capsular en este microorganismo. No obstante, el estudio del locus cap, responsable de la sintesis en los tipos capsulares estudiados hasta el momento, revelo que este serotipo es un caso especial, ya que la sintesis capsular no esta asociada a locus cap37, sino que este constituye el resto ge...
Many streptococci are human and/or animal pathogens and the frequent cause of life-threatening di... more Many streptococci are human and/or animal pathogens and the frequent cause of life-threatening diseases. Among various streptococcal virulence factors, capsular polysaccharides (CPs) are recognized as essential to prevent phagocytosis by macrophages and neutrophils. In the last decade, an impressive advance on the knowledge of the genetic bases underlying capsule formation has been achieved. The capsular gene cluster driving the formation of the CP of Streptococcus pyogenes and other hyaluronate-producing streptococci, represents one of the simplest cases of gene organization to synthesize a capsule. A more complex situation has been found in Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, and other streptococci. On the whole, there exists a direct relationship between the structural and chemical complexity of the repeating unit of the polysaccharide and the number of genes found in the corresponding capsular locus. Streptococcal vaccines, either polysaccharide or conjugate, are currently being tested in clinical trials to overcome the rise of worldwide antibiotic resistance, although, for different reasons, none of these vaccines are expected to provide the required full coverage in a near future. This concern has prompted to explore alternative possibilities with an improved therapeutic potential against streptococcal diseases.
Background: Protein folding in the envelope is a crucial limiting step of protein export and secr... more Background: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H 2 O 2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H 2 O 2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.
The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococ... more The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0?04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS " mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS " phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS " mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.
It has been recently reported that different type 37 clinical isolates of Streptococcus pneumonia... more It has been recently reported that different type 37 clinical isolates of Streptococcus pneumoniae have an identical tts gene directing the formation of type 37 capsular polysaccharide. Here we show that type 37 S. pneumoniae strains isolated in two different continents (Europe and America) some 60 years apart frequently gave rise to nontypable variants upon in vitro cultivation. The tts gene from three independent nontypable mutants was PCR amplified and sequenced showing different classes of inactivating mutations. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that the type 37 pneumococcal isolates studied so far constitute a highly related strain cluster (a clonal complex), and strongly suggested that every type 37 pneumococcus has spread globally from a single, old clone.
Journal of Molecular Microbiology and Biotechnology, 2008
using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox w... more using NICE or P170 expression systems under similar laboratory conditions. Finally, the toolbox we are developing should contribute to enlarge the use of L. lactis as a protein cell factory.
The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and ... more The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.
The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating u... more The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating units of [-D-Glcp-(132)]--D-Glcp-(133). The elements governing the expression of the tts gene, coding for the glucosyltransferase involved in the synthesis of the type 37 pneumococcal capsular polysaccharide, have been studied. Primer extension analysis and functional tests demonstrated the presence of four new transcriptional start points upstream of the previously reported tts promoter (ttsp). Most interesting, three of these transcriptional start points are located in a RUP element thought to be involved in recombinational events (Oggioni, M. R., and Claverys, J. P. (1999) Microbiology 145, 2647-2653). Transformation experiments using either a recombinant plasmid containing the whole transcriptional unit of tts or chromosomal DNA from a type 37 pneumococcus showed that tts is the only gene required to drive the biosynthesis of a type 37 capsule in S. pneumoniae and other Gram-positive bacteria, namely Streptococcus oralis, Streptococcus gordonii, and Bacillus subtilis. The Tts synthase was overproduced in S. pneumoniae and purified as a membrane-associated enzyme. These membrane preparations used UDP-Glc as substrate to catalyze the synthesis of a high molecular weight polysaccharide immunologically identical to the type 37 capsule. In addition, UDP-Gal was also a substrate to produce type 37 polysaccharide since a strong UDP-Glc-4-epimerase activity is associated to the membrane fraction of S. pneumoniae. These results indicated that Tts has a dual biochemical activity that leads to the synthesis of the branched type 37 polysaccharide.
The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a c... more The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-i...
The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been... more The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 degrees C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.
A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galact... more A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution o...
Here we developed the new expression system PZn zitR, based on the regulatory signals (PZn promot... more Here we developed the new expression system PZn zitR, based on the regulatory signals (PZn promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn2+ high-affinity uptake and regulation. A PZn zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic β-galactosidase. Nuclease and β-galactosidase activities of L. lactis MG1363 cells expressing either uspnuc or lacLM under the control of PZn zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn2+, availability in the environment. Our results demonstrate that PZn zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the pr...
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Papers by Daniel Llull