International Journal of Systematic and Evolutionary Microbiology (2006), 56, 1899–1903 DOI 10.1099/ijs.0.

64101-0

Novel nitrogen-fixing Acetobacter nitrogenifigens

sp. nov., isolated from Kombucha tea
Debasree Dutta and Ratan Gachhui
Correspondence Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700032, West
Ratan Gachhui Bengal, India
[email protected]

The four nitrogen-fixing bacteria so far described in the family Acetobacteraceae belong to the
genera Gluconacetobacter and Acetobacter. Nitrogen-fixing bacterial strain RG1T was isolated
from Kombucha tea and, based on the phylogenetic analysis of 16S rRNA gene sequence which is
supported by a high bootstrap value, was found to belong to the genus Acetobacter. Strain RG1T
differed from Acetobacter aceti, the nearest member with a 16S rRNA gene sequence similarity of
98?2 %, and type strains of other Acetobacter species with regard to several characteristics of
growth features in culture media, growth in nitrogen-free medium, production of c-pyrone from
glucose and dihydroxyacetone from glycerol. Strain RG1T utilized maltose, glycerol, sorbitol,
fructose, galactose, arabinose and ethanol, but not methanol as a carbon source. These results,
along with electrophoretic mobility patterns of nine metabolic enzymes, suggest that strain RG1T
represents a novel nitrogen-fixing species. The ubiquinone present was Q-9 and DNA G+C
content was 64?1 mol%. Strain RG1T exhibited a low value of 2–24 % DNA–DNA relatedness
to the type strains of related acetobacters, which placed it as a separate taxon. On the basis of this
data, the name Acetobacter nitrogenifigens sp. nov. is proposed, with the type strain RG1T
(=MTCC 6912T=LMG 23498T).

Endophytic bacteria colonize the internal tissues of the et al., 1997), Asaia (Yamada et al., 2000), Kozakia (Lisdiyanti
host plant for mutual benefits. The nitrogen-fixing endo- et al., 2002), Saccharibacter (Jojima et al., 2004) and
phyte Gluconacetobacter diazotrophicus has been found to be Swaminathania (Loganathan & Nair, 2004). The genus
associated with sugarcane, pineapple, cameroon grass, Acetobacter comprises, at present, 15 validly described
sweet potato, mango and banana (Muthukumarasamy species that were delineated mainly on the basis of DNA–
et al., 2002), while Gluconacetobacter azotocaptans and DNA relatedness and phylogenetic relationships (Sokollek
Gluconacetobacter johannae inhabit coffee plants (Fuentes- et al., 1998; Lisdiyanti et al., 2000, 2001; Cleenwerck et al.,
Ramı́rez et al., 2001) and provide host plants with useful 2002; Silva et al., 2006). This report of the isolation of
fixed nitrogen and growth stimulants. The first report of the nitrogen-fixing strain RG1T from the novel source
occurrence and association of nitrogen-fixing Acetobacter Kombucha tea is only second in the genus Acetobacter
peroxydans along with G. diazotrophicus inhabiting culti- after A. peroxydans (Muthukumarasamy et al., 2005).
vated wetland rice varieties demonstrated the presence
We present morphological, biochemical and genetic evid-
of nitrogen-fixing property in the genus Acetobacter
ence which indicates that this isolate represents a novel
(Muthukumarasamy et al., 2005). Based on the striking
nitrogen-fixing species within the genus Acetobacter isolated
resemblance of the high sugar and low pH habitats of
from a novel source. We propose the name Acetobacter
sugarcane and Kombucha tea (Blanc, 1996), we explored nitrogenifigens for the RG1T isolate.
and isolated a nitrogen-fixing strain RG1T belonging to
the genus Acetobacter of the family Acetobacteraceae. This Aliquots of Kombucha mat suspension, after teasing the
family has been divided into eight genera; Acetobacter, mat apart in the soup, were spread on to LGI (0?06 %
Gluconacetobacter, Gluconobacter, Acidomonas, (Yamada KH2PO4, 0?02 % K2HPO4, 0?02 % MgSO4, 0?002 % CaCl2,
0?001 % FeCl3, 0?0002 % Na2MoO4, 10 % sucrose, pH 4?5;
Abbreviation: MLEE, multilocus enzyme electrophoresis. Cavalcante & Döbereiner, 1988) agar plates containing
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA 150 mg cycloheximide l21 (Jimenez-Salgado et al., 1997)
gene and nifH gene sequences of strain RG1T are AY669513 and and 150 mg nystatin l21. Plates were incubated at 30 uC for
AY952470, respectively. 5 days. The bacterial isolate was purified by repeated
A MLEE dendrogram showing the relationships between Acetobacter streaking on to LGI plates, which have no combined
nitrogenifigens sp. nov. and type strains of closely related Acetobacter nitrogen source. Gas tight vials of bacteria-inoculated LGI
species is available as a supplementary figure in IJSEM Online. medium (under microaerophilic environment, without

64101 G 2006 IUMS Printed in Great Britain 1899

D. Dutta and R. Gachhui

Strains/species: 1, A. nitrogenifigens RG1T; 2, A. cibinongensis LMG 21418T; 3, A. orleanensis; 4, A. malorum LMG 1746T; 5, A. aceti; 6, A. tropicalis; 7, A. oeni B13T; 8, A. indonesiensis;
9, A. orientalis LMG 21417T; 10, A. estunensis; 11, A. cereviseae. W, Weak; V, variable; ND, not determined; +, positive; 2, negative. Data for species 1 and 5 are from this study. Motility
shaking) were assayed for acetylene reduction activity (Stal,

Non-motile

56?0–57?6
1988). Nitrogenase-positive isolates were selected for further

ND
11

2
2
2
2
characterization. Nitrogen-fixing bacterial strain RG1T was
isolated. Other reference strains and the novel isolates were
grown in mannitol broth for further study. Colony morphol-

59?2–60?2
ogy was examined on LGI agar plates and on potato agar plates

ND
+
10

2
2
2
V
containing 10 % sucrose. Various phenotypic and morpho-
logical studies were performed using standard techniques
described elsewhere (Cavalcante & Döbereiner, 1988;

52?0–52?8
Caballero-Mellado et al., 1995; Jimenez-Salgado et al., 1997;

ND
2
2

2
2
W
W
9
Cleenwerck et al., 2002). Isoprenoid quinone of the isolate was

Table 1. Differential characteristics between Acetobacter nitrogenifigens sp. nov. and closely related members of the genus Acetobacter
extracted with chloroform/methanol [2 : 1, (v/v)], and then
purified by TLC on silica gel 60 F254 (20620 cm; Merck) by

54?0–54?2
using benzene as the developing solvent. Quinones recovered

ND
+
2
2

2
2
2
8
from the TLC plates were dissolved in acetone and analysed by
HPLC (Lu et al., 1999). The HPLC system was equipped with a

data for species 4 and 11 are taken from Cleenwerck et al. (2002). All other data are from Silva et al. (2006) unless otherwise indicated.

Peritrichous
reverse-phase column [Luna 5U C18 (2) 100A, 25064?6 mm;

flagellation
Phenomenex] and a mixture of methanol/2-propanol [2 : 1, (v/

58?1
+
2

2
2
2
2
7
v)] was used as the mobile phase at a flow rate of 1 ml min21.
Types of quinone were identified by absorption at 275 nm and
compared with coenzymes Q-9 and Q-10 standards from

55?6–56?2
Sigma-Aldrich. Ubiquinone Q-9 was present in strain RG1T

ND
+
2
2

2
2
2
6
and fitted with the previous observations that showed the
presence of this ubiquinone type in the genus Acetobacter
(Cleenwerck et al., 2002). The type strain RG1T deviated

56?9–58?3
ND
biochemically and morphologically from other species of the

+
5*

2
2
2
V
genus Acetobacter as shown in Table 1.

A 1451 bp fragment of 16S rRNA gene was PCR amplified

Non-motile
with bacteria-specific primers fD1 and rD1 (Weisburg et al.,

57?2
2
2

2
2
2
2
4

1991) using Taq polymerase and genomic DNA from strain

RG1T as template. The nucleotide sequence was similar to the
extent of 98?2 % with Acetobacter aceti, 97?7 % with

55?7–58?1
Acetobacter estunensis, 97?4 % with Acetobacter indonesiensis,

ND
2
2

2
2
2
V
3

97?2 % with Acetobacter tropicalis, 97?2 % with Acetobacter

oeni, 97?1 % with Acetobacter cibinongensis, 97?0 % with
Acetobacter cerevisiae, 97?0 % with Acetobacter malorum,

53?8–54?5
96?9 % with Acetobacter orleansis, 96?6 % with Acetobacter
ND
+
2

2
2

2
W
2

orientalis and 97?0 % with Acetobacter syzygii after performing

a similarity search with FASTA (ungapped). The phylogenetic
Polar-flagellation

tree (Fig. 1) was deduced using MEGA version 3.1 (Kumar et

al., 2004) software after multiple alignment of 16S rRNA gene
64?1

sequences of other acetic acid bacteria with CLUSTAL W

+
+

+
+
1*

2
2

(Thompson et al., 1994). Distances (distance options

according to the Kimura two-parameter model) and
clustering with the neighbour-joining method was deter-
Growth in presence of 10 % ethanol
Growth in ammonium with ethanol

mined by using bootstrap values (Felsenstein, 1985) based on

Growth on YE +30 % D-glucose

100 replications. Two subclusters were evident in the genus

Growth on N-free LGI plate*

Acetobacter: one composed of Acetobacter pomorum,

DNA G+C content (mol%)
Growth on carbon sources

Acetobacter pasteurianus, A. syzygii, Acetobacter lovaliensis

and A. peroxydans and the other included Acetobacter aceti and
other acetobacters along with the novel nitrogen-fixing strain
RG1T. The clustering of strain RG1T with A. aceti was further
Characteristic

confirmed by the formation of dihydroxyacetone from

Methanol
Maltose

*This work.

glycerol by both strains RG1T and A. aceti ATCC 15973T.

Motility

Cell extracts were prepared for multilocus enzyme electro-

phoresis (MLEE) and run on a starch gel as described

1900 International Journal of Systematic and Evolutionary Microbiology 56

 Nitrogen-fixing Acetobacter from Kombucha tea

Fig. 1. Phylogenetic relationships of acetic

acid bacteria based on 16S rRNA gene
sequences involving Acetobacter nitrogenifi-
gens sp. nov. and other related species of
the family Acetobacteraceae. Numbers at
nodes indicate bootstrap values derived from
100 replications. GenBank accession num-
bers of 16S rRNA gene sequences are indi-
cated in parentheses. Bar, 2 substitutions
per 100 nucleotides.

previously (Selander et al., 1986; Caballero-Mellado & 19F and 407R (Franke et al., 1998) and sequenced. Presence
Martı́nez-Romero, 1994). A. cibinongensis JCM 11196T, A. of nifH gene further confirmed the nitrogen-fixing ability of
orleansis LMG 1583T, A. malorum LMG 1746T, A. aceti strain RG1T.
ATCC 15973 T, A. tropicalis LMG 19825T, A. oeni B13T,
A. orientalis JCM 11195T, A. indonesiensis LMG 19824T, To determine genomic relatedness of the novel isolate,
A. estunensis LMG 1626T and A. cerevisiae LMG 1625T were dot-blot hybridization experiments were carried out with
included as reference strains. The relationships among DIG-labelled DNA as described previously (Labrenz et al.,
non-nitrogen-fixing species of Acetobacter and RG1T are 2000) using the detection kit from Roche Applied Sciences
illustrated by the dendrogram (Supplementary Fig. S1 and following the manufacturer’s instructions. Colorimetric
available in IJSEM Online) derived by program ETMEGA quantification of dot intensities was done using the Mole-
(T. S. Whittam; http://foodsafe.msu.edu/whittam/programs/ cular Analyst software (Bio-Rad) by determining mean pixel
index.html) and MEGA version 3.1 software based on the densities in equal sized circles. A genomic DNA probe was
electrophoretic mobility of nine metabolic enzymes, indo- prepared from the novel strain RG1T, digested with EcoRI
phenol oxidase, alcohol dehydrogenase, isocitrate dehydro- and run on 0?7 % agarose gel. Total DNA digests were
genase, glucose-6-phosphate dehydrogenase, xanthine transferred from gels to nylon membrane by Southern
dehydrogenase, leucine dehydrogenase, lysine dehydrogen- blotting. Hybridization was performed at a temperature of
ase, hexokinase and esterase. In MLEE, strain RG1T 75 uC for 16 h and the membrane was washed under high
exhibited highest similarity to A. aceti ATCC 15973T, stringency conditions (twice with 26 SSC/0?1 % SDS at
which is in agreement with its phylogenetic position room temperature for 10 min; once with 0?16 SSC/0?1 %
(Fig. 1). Analysis of the dendrogram revealed that strain SDS at 75 uC for 15 min). A low level of genomic DNA
RG1T formed a unique branch with A. aceti ATCC 15973T relatedness (DNA–DNA hybridization values less than
that deviated at a genetic distance of more than 0?5. This 50 %) was observed between the phylogenetically closest
value has been used as a criterion to suggest species limits species and genera. Strain RG1T showed low DNA–DNA
(Selander et al., 1985; Musser et al., 1987). Thus, MLEE relatedness with the type strains of A. oeni (24 %), A. aceti
results strongly support the existence of strain RG1T as a (22 %), A. cerevisiae (19 %), A. cibinongensis (18?91 %),
novel Acetobacter species. A. orientalis (18?9 %), A. estunensis (11 %), A. orleansis
(10?37 %), A. malorum (7?31 %), A. indonesiensis (3?71 %)
The nitrogenase enzyme complex, responsible for nitrogen- and A. tropicalis (2 %).
fixation, is composed of nifHDK and other gene fragments.
A 336 bp region encoding dinitrogenase reductase, nifH, Although the limitations of 16S rRNA gene sequencing to
was amplified from strain RG1T using degenerate primers differentiate closely related species have been documented

http://ijs.sgmjournals.org 1901
D. Dutta and R. Gachhui

(Fox et al., 1992), it is suggested that strains sharing greater The type strain is strain RG1T (=MTCC 6912T=LMG
than 97 % similarity might belong to the same species but 23498T), isolated from Kombucha tea.
a lower similarity value could form the basis of delineation
of bacterial species (Stackebrandt & Goebel, 1994). Also a
DNA–DNA relatedness level below 70 % indicates a distinct
species (Stackebrandt & Goebel, 1994). In the present study, Acknowledgements
these considerations were consistent with both the 16S We thank Dr Encarna Velázquez for promptly sending us A. oeni B13T
rRNA gene sequence similarity levels and the low levels of as a gift and Dr Dhritiman Ghosh for help in the phylogenetic analysis.
DNA–DNA relatedness exhibited within the Acetobacter Work was partially supported from funding agencies of the
Government of India: CSIR (no. 38/1097/04/EMR-II) and DBT
species. The genetic distance of different closely related (no. BT/PR5111/BCE/08/340/2004). Financial support was also
acetobacters involving strain RG1T was found to be greater received from Jadavpur University to D. D. and R. G.
than 0?5 in all comparisons and such a distance indicates a
novel species; hence strain RG1T represent a novel species of
the genus Acetobacter.
References
In view of low physiological, biochemical, phylogenetic and
Blanc, P. J. (1996). Characterization of the tea fungus metabolites.
genetic similarities among different closely related members Biotechnol Lett 18, 139–142.
of the genus Acetobacter, we recommend that nitrogen-
Caballero-Mellado, J. & Martı́nez-Romero, E. (1994). Limited genetic
fixing strain RG1T described here should be assigned to a diversity in the endophytic sugarcane bacterium Acetobacter
novel species of the genus Acetobacter, for which the name diazotrphicus. Appl Environ Micrbiol 60, 1532–1537.
Acetobacter nitrogenifigens sp. nov. is proposed. Caballero-Mellado, J., Fuentes-Ramı́rez, L. E., Reis, V. M. &
Martı́nez-Romero, E. (1995). Genetic structure of Acetobacter diazo-
Description of Acetobacter nitrogenifigens trophicus populations and identification of a new genetically distant
group. Appl Environ Microbiol 61, 3008–3013.
sp. nov.
Cavalcante, V. & Döbereiner, J. (1988). A new acid tolerant
Acetobacter nitrogenifigens (ni.tro.gen.i9fi.gens. N.L. n. nitrogen-fixing bacterium associated with the sugarcane. Plant Soil
nitrogenium nitrogen; L. part. adj. figens fixing; N.L. part. 108, 23–31.
adj. nitrogenifigens nitrogen-fixing). Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J.
(2002). Re-examination of the genus Acetobacter, with descriptions
Cells are straight rods with rounded ends, approximately of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov.
1?5–2?0 mm in length and 0?1–0?2 mm in width and occur Int J Syst Evol Microbiol 52, 1551–1558.
singly or in chains. These Gram-negative bacteria are motile Felsenstein, J. (1985). Confidence limits on phylogenies: an
with polar flagellation, catalase-positive and oxidase- approach using the bootstrap. Evolution 39, 783–791.
negative. Growth occurs on nitrogen-free LGI plates at Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P., Jr (1992). How close is
30 uC, but not at 37 uC and in LGI broth under close: 16S rRNA sequence identity may not be sufficient to guarantee
microaerophilic conditions. After incubation for 5 days, species identity. Int J Syst Bacteriol 42, 166–170.
colonies grown on LGI plates are smooth, round, glistening, Franke, I. J., Fegan, M., Hayward, A. C. & Sly, L. I. (1998). Nucleotide
transparent and 2–3 mm in diameter, while dark-yellow sequence of the nifH gene coding for nitrogen reductase in the acetic
acid bacterium Acetobacter diazotrophicus. Lett Appl Microbiol 26,
colonies are formed on LGI agar supplemented with
12–16.
0?001 % bromothymol blue. Colonies on potato agar are
Fuentes-Ramı́rez, L. E., Bustillos-Cristales, R., Tapia-Herńandez,
light brown after 5 days of incubation, but the intensity
A., Jimenez-Salgado, T., Wang, E. T., Martı́nez-Romero, E. &
increases after 10 days. Strains are aerobic and fix atmo- Caballero-Mellado, J. (2001). Novel nitrogen-fixing acetic acid
spheric nitrogen microaerobically. Growth occurs well in bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter
the presence of ammonium, but nitrate is not reduced. In azotocaptans sp. nov., associated with coffee plants. Int J Syst Evol
absence of yeast extract, strains can utilize different carbon Microbiol 51, 1305–1314.
sources such as D-galactose, D-xylose, D-fructose, D- Jimenez-Salgado, T., Fuentes-Ramı́rez, L. E., Tapia-Herńandez, A.,
arabinose, D-mannitol, D-sorbitol, and grow on 30 % Mascarúa-Esparza, M. A., Martı́nez-Romero, E. & Caballero-
sucrose and 30 % glucose. Dihydroxyacetone is produced Mellado, J. (1997). Coffea arabica L., a new host plant for Aceto-
from glycerol and c-pyrone from D-glucose. Ethanol is bacter diazotrophicus, and isolation of other nitrogen-fixing
Acetobacteria. Appl Environ Microbiol 63, 3676–3683.
oxidized to acetic acid, and acetate and lactate over oxidize
to CO2 and water. Methanol is not utilized. Single amino Jojima, Y., Mihara, Y., Suzuki, S., Yokozeki, K., Yamanaka, S. &
Fudou, R. (2004). Saccharibacter floricola gen. nov., sp. nov., a novel
acids like L-alanine, L-cysteine and L-phenylalanine can be osmophilic acetic acid bacterium isolated from pollen. Int J Syst Evol
used as a sole source of carbon and nitrogen. L-Threonine is Microbiol 54, 2263–2267.
not utilized as the sole source of carbon and nitrogen. The Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software
ubiquinone present is of the type Q-9 and DNA G+C for molecular evolutionary genetics analysis and sequence alignment.
content is 64?1 mol%. Based on data from the MLEE assay Brief Bioinform 5, 150–163.
along with DNA–DNA relatedness and nifH gene sequence, Labrenz, M., Tindall, B. J., Lawson, P. A., Collins, M. D., Schumann,
the type strain RG1T can be differentiated from other P. & Hirsch, P. (2000). Staleya guttiformis gen. nov., sp. nov., and
Acetobacter species. Sulfitobacter brevis sp. nov., a-3-Proteobacteria from hypersaline,

1902 International Journal of Systematic and Evolutionary Microbiology 56

 Nitrogen-fixing Acetobacter from Kombucha tea

heliothermal and meromictic Antarctic Ekho Lake. Int J Syst Evol Selander, R. K., McKinney, R. M., Whittam, T. S., Bibb, W. F.,
Microbiol 50, 303–313. Brenner, D. J., Nolte, F. S. & Pattison, P. E. (1985). Genetic
Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & structures of populations of Legionella pneumophila. J Bacteriol 163,
Komagata, K. (2000). Systematic study of the genus Acetobacter 1021–1037.
with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M., Gilmour,
tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) M. N. & Whittam, T. S. (1986). Methods in multilocus enzyme
comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and electrophoresis for bacterial population genetics and systematics.
Acetobacter estunensis (Carr 1958) comb. nov. J Gen Appl Microbiol Appl Environ Microbiol 51, 873–884.
46, 147–165. Silva, L. R., Cleenwerck, I., Rivas, R., Swings, J., Trujillo, M. E.,
Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & Willems, A. & Velázquez, E. (2006). Acetobacter oeni sp. nov.,
Komagata, K. (2001). Identification of Acetobacter strains isolated isolated from spoiled red wine. Int J Syst Evol Microbiol 56, 21–24.
from Indonesian sources, and proposals of Acetobacter syzygii Sokollek, S. J., Hertel, C. & Hammes, W. P. (1998). Description of
sp. nov., Acetobacter cibinongensis sp. nov., and Acetobacter orientalis Acetobacter oboediens sp nov. and Acetobacter pomorum sp nov., two
sp. nov. J Gen Appl Microbiol 47, 119–131. new species isolated from industrial vinegar fermentations. Int J Syst
Lisdiyanti, P., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., Bacteriol 48, 935–940.
Yamada, Y., Uchimura, T. & Komagata, K. (2002). Kozakia Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a
baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the place for DNA-DNA reassociation and 16S rRNA sequence analysis
a-Proteobacteria. Int J Syst Evol Microbiol 52, 813–818. in the present species definition in bacteriology. Int J Syst Bacteriol
Loganathan, P. & Nair, S. (2004). Swaminathania salitolerans 44, 846–849.
gen. nov., sp. nov., a salt-tolerant, nitrogen-fixing and phosphate- Stal, L. J. (1988). Nitrogen fixation in cyanobacterial mats. Methods
solubilizing bacterium from wild rice (Porteresia coarctata Tateoka). Enzymology 167, 475–484.
Int J Syst Evol Microbiol 54, 1185–1190. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W:
Lu, S.-F., Lee, F.-L. & Chen, H.-K. (1999). A thermotolerant and high improving the sensitivity of progressive multiple sequence alignment
acetic-acid producing bacterium Acetobacter sp. 114-2. J Appl through sequence weighting, position specific gap penalties and
Microbiol 86, 55–62. weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Musser, J. M., Bemis, D. A., Ishikawa, H. & Selander, R. K. (1987). Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991).
Clonal diversity and host distribution in Bordetella bronchiseptica. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol
J Bacteriol 169, 2793–2803. 173, 697–703.
Muthukumarasamy, R., Revathi, G., Seshadri, S. & Yamada, Y., Hoshino, K. & Ishikawa, T. (1997). The phylogeny of
Lakshminarsimhan, C. (2002). Gluconacetobacter diazotrophicus acetic acid bacteria based on the partial sequences of 16S ribosomal
(syn. Acetobacter diazotrophicus), a promising diazotrophic endo- RNA: the elevation of the subgenus Gluconacetobacter to the generic
phyte in tropics. Current Science 83, 137–145. level. Biosci Biotechnol Biochem 61, 1244–1251.
Muthukumarasamy, R., Cleenwerck, I., Revathi, G. & 8 other Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S.,
authors (2005). Natural association of Gluconacetobacter diazotro- Seki, T., Uchimura, T. & Komagata, K. (2000). Asaia bogorensis
phicus and diazotrophic Acetobacter peroxydans with wetland rice. gen. nov., sp. nov., an unusual acetic acid bacterium in the
Syst Appl Microbiol 28, 277–286. a-Proteobacteria. Int J Syst Evol Microbiol 50, 823–829.

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