International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1551–1558 DOI : 10.1099/ijs.0.

02064-0

Re-examination of the genus Acetobacter, with

descriptions of Acetobacter cerevisiae sp. nov.
and Acetobacter malorum sp. nov.
BCCM/LMG Bacteria I. Cleenwerck, K. Vandemeulebroecke, D. Janssens and J. Swings
Collection, Laboratory of
Microbiology, Ghent
University, K.L.
Author for correspondence : I. Cleenwerck. Tel : j32 9 2645123. Fax :j32 9 2645346.
Ledeganckstraat 35,
e-mail : Ilse.Cleenwerck!rug.ac.be
B-9000 Ghent, Belgium

Thirty-four Acetobacter strains, representing Acetobacter aceti, Acetobacter

pasteurianus, Acetobacter pomorum, Acetobacter peroxydans, Acetobacter
lovaniensis, Acetobacter estunensis, Acetobacter orleanensis, Acetobacter
indonesiensis and Acetobacter tropicalis, were subjected to a polyphasic study
that included DNA–DNA hybridizations, DNA base ratio determinations, 16S
rDNA sequence analysis and phenotypic characterization. Two novel species
are proposed, Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp.
nov. The type strains of these species are respectively LMG 1625T (l DSM
14362T l NCIB 8894T l ATCC 23765T) and LMG 1746T (l DSM 14337T).

Keywords : Acetobacter cerevisiae sp. nov., Acetobacter malorum sp. nov., acetic acid
bacteria, taxonomy, classification

INTRODUCTION In the course of a larger study set up to improve the

identification of Acetobacteraceae, 16 strains from the
Acetic acid bacteria are classified into the genera heterogeneous species A. pasteurianus, as well as 17
Acetobacter, Gluconobacter, Gluconacetobacter, reference strains representing all known Acetobacter
Acidomonas and the recently described genus Asaia species, were analysed genotypically and pheno-
(Yamada et al., 2000). These genera join phylo- typically. DNA homology data demonstrate that five
genetically into a broad rRNA cluster within the strains currently allocated to A. pasteurianus could not
α-subclass of the Proteobacteria, the acetic acid bac- be assigned to any known Acetobacter species. Four of
teria lineage. these strains constituted a separate but homogeneous
taxon, for which the name Acetobacter cerevisiae sp.
The genus Acetobacter is differentiated from the other nov. is proposed. The name Acetobacter malorum
genera by its Q9 ubiquinone system and by the sp. nov. is proposed for strain LMG 1746T.
oxidation of acetate and lactate to CO and H O (De
#
Ley & Frateur, 1974 ; Yamada et al., 1997, #
2000). At METHODS
the time of writing, the genus consisted of nine species :
Acetobacter aceti, Acetobacter pasteurianus, Aceto- Bacterial strains. Strains used in this study are listed in Table
bacter pomorum, Acetobacter peroxydans, Acetobacter 1. They were checked for purity by plating on medium 13
lovaniensis, Acetobacter estunensis, Acetobacter orlean- from the Catalogue of Cultures of the BCCM\LMG Bacteria
ensis, Acetobacter indonesiensis and Acetobacter Collection (Janssens et al., 1998) containing 2n5 % -
mannitol, 0n5 % yeast extract, 0n3 % peptone and 1n5 % agar.
tropicalis. These species were delineated mainly on the
basis of DNA–DNA relatedness and phylogenetic DNA isolation. Total DNA for the determination of the
relationships (Sokollek et al., 1998 ; Lisdiyanti et al., DNA base composition and for DNA–DNA hybridizations
was prepared by the method of Wilson (1987), with minor
2000). modifications. Cells were washed with RS buffer (0n15 M
NaCl, 10 mM EDTA, pH 8n0) and then suspended and lysed
................................................................................................................................................. in a Tris\EDTA buffer (10 mM Tris\HCl with up to
Published online ahead of print on 18 January 2002 as DOI 200 mM EDTA, pH 8n0) containing RNase A (Sigma), SDS
10.1099/ijs.0.02064-0. (Serva) and proteinase K (Merck) to final concentrations of
The EMBL accession numbers for the 16S rDNA sequences of strains LMG 400 µg ml−", 2 % (w\v) and 200 µg ml−", respectively. NaCl
1629, LMG 18848T, LMG 1633, LMG 1617T, LMG 1626T, LMG 1572, LMG (5 M stock solution) and CTAB\NaCl solution [10 % (w\v)
1531, LMG 1588, LMG 1663, LMG 1625T, LMG 1746T and LMG 1583T are hexadecyltrimethylammonium bromide in 0n7 M NaCl] were
respectively AJ419834–AJ419845. added to final concentrations of 1 M and 13n3 % (v\v),

02064 # 2002 IUMS Printed in Great Britain 1551

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I. Cleenwerck and others

Table 1. List of strains studied

.................................................................................................................................................................................................................................................................................................................

Strains are listed under their proposed species designations. LMG, BCCM\LMG Bacteria Collection, Laboratorium
Microbiologie, Universiteit Gent, Belgium ; NCIB, National Collections of Industrial and Marine Bacteria, Aberdeen, UK ; LMD,
Laboratorium voor Microbiologie, Technische Universiteit, Delft, The Netherlands ; IFO, Institute for Fermentation, Osaka,
Japan ; NRIC, NODAI Research Institute Culture Collection, Tokyo University of Agriculture, Tokyo, Japan.

Strain As received Current species Source (if known)

designation

A. pasteurianus
LMG 1555 NCIB 8163 A. pasteurianus
LMG 1686 LMD 31.4 A. pasteurianus
LMG 1262Tt1* LMD 22.1Tt1* A. pasteurianus Beer
LMG 1630 EQ A. pasteurianus Sugar cane bagasse, Brazil
LMG 1629 A A. pasteurianus Fermented Agave sisalina juice, Brazil
LMG 1658 MM 80 A. pasteurianus Toddy palm, Burma
LMG 1659 MM 73 A. pasteurianus Toddy palm, Burma
A. pomorum
LMG 18848T LTH 2458T A. pomorum Submerged cider vinegar fermentation, Germany
A. peroxydans
LMG 1633 LMD 53.9 A. pasteurianus Ditch water
LMG 1635T NCIB 8618T A. peroxydans Ditch water, the Netherlands
A. lovaniensis
LMG 1617T NCIB 8620T A. lovaniensis Sewage on soil, Belgium
A. estunensis
LMG 1626T NCIB 8935T A. estunensis Cider, UK
LMG 1572 LMG E A. pasteurianus Cider, UK
LMG 1580 LMD 50.6 A. estunensis Beer, the Netherlands
A. aceti
LMG 1531 NCIB 8941 A. aceti Quick vinegar, the Netherlands
LMG 1535 LMG Ch31 A. aceti Vinegar plant, Belgium
LMG 1504T NCIB 8621T A. aceti Beech-wood shavings of vinegar plant
LMG 1496 LMG 24WR A. aceti
A. cerevisiae sp. nov.
LMG 1625T NCIB 8894 A. pasteurianus Beer (ale) in storage, Toronto, Canada
LMG 1599 NCIB 6425 A. pasteurianus
LMG 1699 MARTIN 2 A. pasteurianus Brewery, UK
LMG 1682 C101 A. pasteurianus Beer, Ireland
A. malorum sp. nov.
LMG 1746T LMG 76.10 A. pasteurianus Rotting apple, Ghent, Belgium
A. orleanensis
LMG 1583T NCIB 8622T A. orleanensis Beer, Belgium
LMG 1592 NCIB 2224 A. orleanensis
LMG 1608 NCIB 8088 A. orleanensis Beer
LMG 1545 IFO 3296 A. orleanensis Film in fermenter of rice vinegar, Japan
A. indonesiensis
LMG 19824T NRIC 0313T A. indonesiensis Fruit of Annona muricata, Indonesia
LMG 1588 LMD 39.6 A. indonesiensis
LMG 1571 LMD 39.2 A. indonesiensis
A. tropicalis
LMG 19825T NRIC 0312T A. tropicalis Coconut juice, Indonesia
LMG 19826 NRIC 0321 A. tropicalis Lime, Indonesia
LMG 1754 LMG 79.18 A. pasteurianus Fruit of Ficus capensis, Ivory Coast
LMG 1663 592 A. pasteurianus Fermenting putrefied meat sample, UK

* The type strain of A. pasteurianus shows two stable colonial variations, t1 and t2, that give identical protein profiles by SDS-PAGE.

respectively. To obtain DNA solutions free of RNA, a 100 µg ml−" and incubated at 37 mC for 1 h. Finally, the
second RNase treatment was performed. RNase A was degraded RNA was removed by chloroform extraction. The
added to the DNA solutions to a final concentration of DNA was dissolved in 0n1i SSC (0n15 M NaCl, 0n015 M

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 Description of two novel Acetobacter species

citric acid, 0n4 M NaOH, pH 7n0) to obtain a concentration Phenotypic characterization. Cell shape and size were de-
of 0n3–0n8 mg ml−". DNA quantity and quality were de- termined for cells grown aerobically at 28 mC for 2–4 days on
termined by measuring the absorption at 260, 280 and medium 13. For the type strain of A. pomorum, cell shape
234 nm. Only high-quality DNA with A \A and and size were also determined for cells grown aerobically at
A \A ratios of 1n8–2n0 and 0n40–0n60 was#'!selected #)! for 28 mC for 2 days on RAE agar (Sokollek & Hammes, 1997).
#$% #'!
further use. The size of the DNA was checked by agarose gel Gram staining was carried out by the method of Hucker &
electrophoresis. Only high-molecular-mass DNA was used. Conn (1923). Oxidase activity was tested using 1 %
N,N,Nh,Nh-tetramethyl p-phenylenediamine (Kovacs,
DNA–DNA hybridizations. DNA–DNA hybridizations were
1956). Catalase activity was tested by adding young cells to
performed using a modification of the microplate method
a drop of a 10 % H O solution and observing production of
described by Ezaki et al. (1989) and Goris et al. (1998).
O . The production # of
# 2- and 5-keto--gluconic acid was
Briefly, biotinylated probe DNA was sheared by ultra- #
determined by the method described by Gossele! et al. (1980).
sonication, denatured and then hybridized with single-
The utilization of ammonium as the sole nitrogen source in
stranded unlabelled DNA, non-covalently bound to micro-
the presence of ethanol as carbon source was tested using
plate wells. Hybridizations were performed at 50 mC in a
Frateur’s modified Hoyer ethanol\vitamins medium (De
hybridization solution containing 50 % formamide (2i
Ley et al., 1984) containing 2n5 % agar. Growth was checked
SSC, 5i Denhardt’s solution, 50 % formamide, 2n5 %
after 7 days incubation at 28 mC. Growth on the carbon
dextran sulfate, low-molecular-mass denatured salmon
sources glycerol, maltose and methanol was tested in basal
sperm DNA to a final concentration of 100 µg ml−", 1n25 µg
medium (0n05 % yeast extract, 0n3 % vitamin-free Casamino
biotinylated probe DNA ml−"). After 3 h, the hybridization
acids, 2n5 % agar) with the carbon source to be tested added
solution was removed and streptavidin\β--galactosidase
at a final concentration of 0n3 %. Medium without the
(Sigma) was added to the wells. After 10 min of incubation
carbon source was used as a control. Growth was evaluated
at 37 mC, the wells were washed and 4-methylumbelliferyl β-
after 7 days incubation at 28 mC. Growth in 30 % -glucose
-galactoside (Sigma) was added. The fluorescence intensity
was tested in a medium containing 0n5 % yeast extract and
was measured with an HTS7000 Bio Assay reader (Applied
30 % -glucose. Growth was checked after 7 days incubation
Biosystems). Salmon sperm DNA (Sigma) was used as a
at 28 mC under stationary conditions. Growth with n-
negative control in all experiments. The DNA relatedness
propanol as carbon source was examined as described by
percentages presented are means, based on at least two
Sokollek et al. (1998).
independent hybridization experiments. Reciprocal
reactions (e.g. AiB and BiA) were performed and also
considered as independent hybridization experiments. RESULTS AND DISCUSSION
Determination of the DNA GjC content. The GjC content DNA relatedness
of DNA was determined by HPLC according to the method
of Mesbah et al. (1989) using a Waters Symmetry Shield RP The results of DNA–DNA hybridizations of all strains
column thermostatted at 37 mC. The solvent was 0n02 M) examined are shown in Table 2. DNA–DNA
NH H PO with 1n5 % acetonitrile (pH 4n0). Non-meth- hybridization data revealed that four strains, LMG
% # phage
ylated % lambda DNA (Sigma) was used as the 1625T, LMG 1599, LMG 1699 and LMG 1682,
calibration reference. displayed a high level of DNA relatedness (66–85 %)
Sequencing of 16S rDNA. 16S rDNA was amplified using and low levels of relatedness to the known Acetobacter
oligonucleotide primers complementary to highly conserved species. The name Acetobacter cerevisiae sp. nov. is
regions of bacterial 16S rRNA genes. The forward primer proposed for this taxon. Strain LMG 1746T showed
was 5h-AGAGTTTGATCCTGGCTCAG-3h (hybridizing at DNA relatedness at an intermediate level (50–53 %) to
positions 8–27, according to the Escherichia coli numbering the A. cerevisiae strains and at low levels to the known
system) and the reverse primer was 5h-AAGGAGGTGA-
TCCAGCCGCA-3h (hybridizing at positions 1541–1522).
Acetobacter species. The name Acetobacter malorum
PCR products were purified using a QIAquick PCR puri- sp. nov. is proposed for this strain. The DNA–DNA
fication kit (Qiagen), according to the manufacturer’s hybridization data also revealed that the type strain of
instructions. Purified PCR products were sequenced by A. pomorum, LMG 18848T, is related to strains of A.
using the ABI Prism Big Dye Terminator cycle sequencing pasteurianus at an intermediate level (51–58 %). The
ready reaction kit and an Applied Biosystems 377 DNA latter result clearly differs from the results obtained by
sequencer, using the protocols of the manufacturer (Applied Sokollek et al. (1998), who found only 17 % DNA
Biosystems). The eight sequencing primers used are listed in relatedness between the type strain of A. pomorum,
Coenye et al. (1999). Sequence assembly was performed LTH 2458T, and the type strain of A. pasteurianus,
using the program AutoAssembler (Applied Biosystems). DSM 3509T. The discrepancy between these data could
Phylogenetic analysis. The 16S rRNA gene sequences de- be explained by the fact that Sokollek et al. (1998),
termined and sequences of strains belonging to the same who used the membrane method, did not perform
phylogenetic group, retrieved from EMBL, were aligned and reciprocal reactions, which are important to obtain
a phylogenetic tree was constructed by the neighbour-joining unequivocal results. The hybridization data also
method using the Bionumerics 1.01 software package (Ap- showed that four strains, LMG 1633, LMG 1572,
plied Maths). Unknown bases were discarded from the LMG 1754 and LMG 1663, currently classified as A.
calculations. Bootstrapping analysis was undertaken to test
the statistical reliability of the topology of the neighbour- pasteurianus, have DNA–DNA binding values of less
joining tree using 500 bootstrap resamplings of the data. The than 22 % to the type strain of A. pasteurianus and
strain numbers, species names and accession numbers of 16S should be allocated to A. peroxydans (LMG 1633), A.
rDNA sequences retrieved from EMBL for use in the estunensis (LMG 1572) and A. tropicalis (LMG 1754,
phylogenetic analysis are presented in Fig. 1. LMG 1663).

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 Table 2. DNA–DNA binding values and GjC content of Acetobacter strains studied
1554

I. Cleenwerck and others

............................................................................................................................................................................................................................................................................................................................................................................................................................................
Mean standard deviation of DNA–DNA hybridization is ±7% (see Goris et al., 1998).

Strain G+C content (mol%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

A. pasteurianus
1. LMG 1555 54.3 100
2. LMG 1686 53.5 100
3. LMG 1262T t1 53.4 78 82 100
4. LMG 1630 53.7 79 71 100
5. LMG 1629 53.6 90 70 100
6. LMG 1658 53.2 66 62 59 100
7. LMG 1659 53.7 69 66 100
A. pomorum
8. LMG 18848T 52.1 51 58 53 51 55 55 100
A. peroxydans
9. LMG 1633 60.7 6 9 100
10. LMG 1635T 59.7 13 23 73 100
A. lovaniensis
11. LMG 1617T 57.4 21 14 18 20 17 16 100
A. estunensis
12. LMG 1626T 59.2 20 13 9 10 100
13. LMG 1572 60.2 19 21 21 19 3 12 7 9 98 100
14. LMG 1580 59.5 92 92 100
International Journal of Systematic and Evolutionary Microbiology 52

A. aceti
15. LMG 1531 58.3 16 18 6 12 16 100
16. LMG 1535 57.0 14 10 8 8 14 59 100
17. LMG 1504T 56.9 7 8 7 9 6 5 6 6 10 13 17 17 53 100 100
18. LMG 1496 56.9 6 9 7 7 12 55 104 95 100
A. cerevisiae sp. nov.
19. LMG 1625T 57.6 37 32 19 27 10 11 15 17 18 16 12 15 100
20. LMG 1599 56.8 30 23 25 11 77 100
21. LMG 1699 56.0 35 24 27 8 74 76 100
22. LMG 1682 57.4 22 32 13 19 18 15 18 10 9 9 85 80 66 100
A. malorum sp. nov.
23. LMG 1746T 57.2 10 11 7 8 6 9 7 9 5 5 3 53 50 100
A. orleanensis
24. LMG 1583T 55.7 12 12 11 10 6 7 4 33 37 37 40 28 100
25. LMG 1592 58.0* 90* 100
26. LMG 1608 58.1* 98* 100
27. LMG 1545 57.3* 77* 89* 85* 100
A. indonesiensis
28. LMG 19824T 54.0 18 100
29. LMG 1588 54.2 17 16 7 15 13 18 9 20 26 32 20 10 12 88* 100
30. LMG 1571 54.1 85* 83 100
A. tropicalis
31. LMG 19825T 56.0 34 18 100
32. LMG 19826 55.6 19 81 100
33. LMG 1754 56.2 18 11 12 7 100
34. LMG 1663 55.9 22 20 14 6 14 10 6 14 13 13 10 10 9 8 27 24 12 14 20 17 86 93 76 100

* Data taken from Lisdiyanti et al. (2000).

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Table 3. Characteristics that differentiate the species of the genus Acetobacter

......................................................................................................................................................................................................................................................................................................................................................................................................................

Taxa are listed as : 1, A. cerevisiae sp. nov. (n l 4) ; 2, A. malorum sp. nov. (n l 1) ; 3, A. pasteurianus (n l 7) ; 4, A. pomorum (n l 1) ; 5, A. peroxydans (n l 2) ; 6, A.
lovaniensis (n l 1) ; 7, A. orleanensis (n l 4) ; 8, A. indonesiensis (n l 2 ; strains LMG 1588 and LMG 1571) ; 9, A. tropicalis (n l 2 ; strains LMG 1754 and LMG 1663) ; 10,
A. estunensis (n l 3) ; 11, A. aceti (n l 4). Characters are scored as : j, positive ; v, variable ; k, negative. Abbreviation : YE, yeast extract.

Feature 1 2 3 4 5 6 7 8 9 10 11

Formation from -glucose :

5-Keto--gluconic acid k k k k* k k k k k k j
2-Keto--gluconic acid j j  (2j, k)† k* k j j j j j j
Growth on ammonium with ethanol k k k k j j k k k j j
Growth on carbon sources :
Glycerol j j  (2j, k) j k j j j j  (1j, j) j
Maltose k k  (2j, k) k j k  (1j, k) j j k  (1j, k)
Methanol k j k k k j k k k k k
Growth on YEj30 % -glucose k j  (1j, k) k k k k k k k k

Description of two novel Acetobacter species

Catalase j j j j k j j j j j j
GjC content of DNA (mol %) 56n0–57n6 57n2 53n2–54n3 52n1 59n7–60n7 57n1–58n9‡ 55n7–58n1 54n0–54n2 55n6–56n2 59n2–60n2 56n9–58n3

* Data taken from Sokollek et al. (1998).

† For variable characters, the number of strains testing positive and the result for the type strain are given in parentheses.
‡ Data taken from Lisdiyanti et al. (2000).
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I. Cleenwerck and others

.................................................................................................................................................................................................................................................................................................................
Fig. 1. Neighbour-joining tree reflecting the phylogenetic position of A. cerevisiae sp. nov. LMG 1625T and A. malorum
sp. nov. LMG 1746T within the acetic acid bacteria based on almost complete 16S rRNA gene sequences. Rhodopila
globiformis DSM 161T was used as an outgroup in this analysis. Bar, 1 % sequence dissimilarity. Numbers at branching
points indicate bootstrap percentages derived from 500 samples.

DNA GjC content known Acetobacter species were determined and com-
pared with deposited 16S rRNA gene sequences of
The GjC contents of the Acetobacter strains studied
strains of the known Acetobacter species and of the
are given in Table 2 and ranged from 52n1 to
type species of the other genera in the family Aceto-
60n7 mol %, whereas the GjC range of a species was
bacteraceae. A phylogenetic tree, reflecting the
limited to 2n4 mol %. The latter result shows that the
positions of these strains within the acetic acid bacteria
GjC content of Acetobacter species is no longer too
lineage, was generated using the neighbour-joining
broad for a species, as was previously the case,
method and is shown in Fig. 1. Bootstrap values
especially for A. pasteurianus, which had a GjC range
supported the topology of the tree. The tree showed
of 9n7 mol % (Gossele! et al., 1983b ; Swings et al.,
that the genus Acetobacter formed two major rRNA
1992). Analogous results were obtained by Lisdiyanti
groups, one containing the species A. pasteurianus, A.
et al. (2000). The GjC content of the strains belonging
pomorum, A. peroxydans and A. lovaniensis and the
to A. cerevisiae varied from 56n0 to 57n6 mol %. The A.
second containing A. cerevisiae LMG 1625T, A. mal-
malorum strain LMG 1746T had a GjC content of
orum LMG 1746T and the species A. estunensis, A.
57n2 mol %. The distribution of GjC content for each
aceti, A. indonesiensis, A. tropicalis and A. orleanensis.
Acetobacter species is given in Table 3.
It is noteworthy that the Acetobacter species within
Phylogenetic analysis based on 16S rRNA gene
each rRNA group showed more than 97n2 % 16S
sequences
rDNA sequence similarity and that strains belonging
to the same Acetobacter species showed more than
The nearly complete 16S rRNA gene sequences (1436– 99n6 % 16S rDNA sequence similarity. A. cerevisiae
1444 nucleotides) of A. cerevisiae LMG 1625T, A. LMG 1625T and A. malorum LMG 1746T showed
malorum LMG 1746T and ten strains representing the 99n9 % 16S rDNA sequence similarity, indicating that

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 Description of two novel Acetobacter species

they are phylogenetically very closely related. Both In conclusion, on the basis of DNA–DNA
strains shared 99n6 % 16S rDNA sequence similarity to reassociation values, DNA base compositions, phylo-
A. orleanensis. Somewhat lower values were observed genetic relationships and phenotypic characteristics,
to members of A. estunensis, A. aceti, A. indonesiensis this study revealed the existence of two novel
and A. tropicalis (97n6–98n9 %). The 16S rDNA se- Acetobacter species, A. cerevisiae (type strain LMG
quence of A. pomorum LMG 18848T was compared to 1625T) and A. malorum (type strain LMG 1746T). It
the sequence deposited for A. pomorum LTH 2458T also showed that four A. pasteurianus strains should be
(EMBL accession no. AJ001632). The two sequences renamed : LMG 1633 should be allocated to A.
were 100 % identical over 1440 bases, reinforcing the peroxydans, LMG 1572 should be allocated to A.
assumption that we used the same strain as Sokollek et estunensis and LMG 1754 and LMG 1663 should be
al. (1998). assigned to A. tropicalis.
Furthermore, this study confirms that Acetobacter
Phenotypic characteristics strains should be classified and identified mainly on the
Table 3 gives characteristics useful in the differen- basis of genotypic characteristics, as identification on
tiation of the species of the genus Acetobacter. A. the basis of phenotypic tests does not always lead to
cerevisiae and A. malorum could not be differentiated clear-cut results. The results above show that DNA–
from the known Acetobacter species by an exclusive DNA hybridization is a recommended technique for
phenotypic characteristic. However, the combination accurate identification of Acetobacter strains.
of growth on methanol as a carbon source, the inability
to grow on ammonium as a nitrogen source with Description of Acetobacter cerevisiae sp. nov.
ethanol as the carbon source and the ability to grow
on 30 % -glucose allowed the differentiation of Acetobacter cerevisiae (ce.re.vihsi.a.e. L. fem. gen. n.
A. malorum from the other Acetobacter species. A. cerevisiae of beer, referring to the source from which
cerevisiae could be differentiated from the species most strains have been isolated).
A. pomorum, A. peroxydans, A. lovaniensis, A. Cells are Gram-negative, ellipsoidal to rod-shaped,
indonesiensis, A. tropicalis, A. estunensis and A. aceti approximately 1n0i2n0–4n0 µm in size, occurring
by the combination of the following phenotypic singly, in pairs or occasionally in short chains. In-
characteristics : production of 2-keto--gluconic acid volution forms like swollen cells occur in some strains.
(but not 5-keto--gluconic acid) from -glucose, the Cells are non-motile. Endospores are not detected.
inability to grow on ammonium as a nitrogen source Colonies are beige to brown, round, regular to wavy,
with ethanol as the carbon source and the inability to raised and smooth with a diameter of 0n3–0n5 mm on
grow on maltose as a carbon source. It is important YPM agar. Obligately aerobic. Oxidase-negative.
to mention that some strains of A. pasteurianus, Catalase-positive. Characterized by the combination
A. pomorum, A. orleanensis, A. indonesiensis and A. of the following phenotypic features : 2-keto--glu-
tropicalis, even without the addition of A. cerevisiae, conic acid is produced from -glucose, 5-keto--
have phenotypic characteristics that are similar to one gluconic acid is not produced from -glucose, no
another. For identification of these strains to the growth with ammonium as the sole nitrogen source on
species level, genotypic characterization (as deter- ethanol as carbon source, no growth on maltose or
mining DNA similarity) is required. methanol as carbon source (Table 3). The range of
The type strains of A. pomorum and A. pasteurianus GjC content of the DNA is 56n0–57n6 mol %. The
were evaluated for growth in the presence of 30 % - type strain is LMG 1625T (l DSM 14362T l NCIB
glucose, growth on methanol as carbon source and 8894T l ATCC 23765T), which has a GjC content of
growth on n-propanol with ammonium as the sole 57n6 mol % and was isolated from beer (ale) in storage
nitrogen source, three features useful in the differen- at Toronto, Canada (Kozulis & Parsons, 1958).
tiation of the two species according to Sokollek et al.
(1998). In our hands, however, strain LMG 18848T Description of Acetobacter malorum sp. nov.
could not be distinguished from the type strain of A.
pasteurianus by any of these features. Acetobacter malorum (ma.lohrum. L. neut. gen. pl. n.
malorum of apples, referring to the isolation of the type
Some controversial reports exist on the growth of strain from a rotting apple).
Acetobacter strains on mannitol agar (Gossele! et al.,
1983b ; Franke et al., 1999 ; Boesch et al., 1998 ; Cells are Gram-negative, ellipsoidal, approximately
Yamada et al., 1997 ; Yamada, 2000 ; Lisdiyanti et al., 0n9i1n1–1n3 µm in size, occurring singly or in pairs.
2000). In our hands, all Acetobacter strains grew on Cells are non-motile. Endospores are not detected.
medium 13 (Janssens et al., 1998), also known as YPM Colonies are beige, round, regular to wavy, convex
agar (0n5 % yeast extract, 0n3 % peptone, 2n5 % - and smooth with a diameter of 0n5 mm on YPM
mannitol and 1n5 % agar), although in most cases this agar. Obligately aerobic. Oxidase-negative. Catalase-
growth was not abundant. Medium 13 is, however, positive. Characterized by the combination of the
very useful to maintain Acetobacter strains viable on following phenotypic features : 2-keto--gluconic acid
plates for a more extended period. is produced from -glucose, 5-keto--gluconic acid is

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I. Cleenwerck and others

not produced from -glucose, no growth with am- Gossele! , F., Swings, J., Kersters, K. & De Ley, J. (1983a). Numerical
monium as the sole nitrogen source on ethanol as analysis of phenotypic features and protein gel electropherograms of
Gluconobacter Asai 1935 emend. mut. char. Asai, Iizuka, and Komagata
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a GjC content of 57n2 mol % and was isolated from a electropherograms of a wide variety of Acetobacter strains. Proposal for
rotting apple in Ghent, Belgium (Gossele! et al., the improvement of the taxonomy of the genus Acetobacter Beijerinck
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