| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700032, West Bengal, India
Correspondence
Ratan Gachhui
ratangachhui{at}yahoo.com
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
-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).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and nifH gene sequences of strain RG1T are AY669513 and AY952470, respectively.
A MLEE dendrogram showing the relationships between Acetobacter nitrogenifigens sp. nov. and type strains of closely related Acetobacter species is available as a supplementary figure in IJSEM Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
), while Gluconacetobacter azotocaptans and Gluconacetobacter johannae inhabit coffee plants (Fuentes-Ramírez et al., 2001) and provide host plants with useful fixed nitrogen and growth stimulants. The first report of the occurrence and association of nitrogen-fixing Acetobacter peroxydans along with G. diazotrophicus inhabiting cultivated wetland rice varieties demonstrated the presence of nitrogen-fixing property in the genus Acetobacter (Muthukumarasamy et al., 2005). Based on the striking resemblance of the high sugar and low pH habitats of sugarcane and Kombucha tea (Blanc, 1996), we explored and isolated a nitrogen-fixing strain RG1T belonging to the genus Acetobacter of the family Acetobacteraceae. This family has been divided into eight genera; Acetobacter, Gluconacetobacter, Gluconobacter, Acidomonas, (Yamada et al., 1997), Asaia (Yamada et al., 2000), Kozakia (Lisdiyanti et al., 2002), Saccharibacter (Jojima et al., 2004) and Swaminathania (Loganathan & Nair, 2004). The genus Acetobacter comprises, at present, 15 validly described species that were delineated mainly on the basis of DNA–DNA relatedness and phylogenetic relationships (Sokollek et al., 1998; Lisdiyanti et al., 2000, 2001; Cleenwerck et al., 2002; Silva et al., 2006). This report of the isolation of nitrogen-fixing strain RG1T from the novel source Kombucha tea is only second in the genus Acetobacter after A. peroxydans (Muthukumarasamy et al., 2005). We present morphological, biochemical and genetic evidence which indicates that this isolate represents a novel nitrogen-fixing species within the genus Acetobacter isolated from a novel source. We propose the name Acetobacter nitrogenifigens for the RG1T isolate.
Aliquots of Kombucha mat suspension, after teasing the mat apart in the soup, were spread on to LGI (0.06 % KH2PO4, 0.02 % K2HPO4, 0.02 % MgSO4, 0.002 % CaCl2, 0.001 % FeCl3, 0.0002 % Na2MoO4, 10 % sucrose, pH 4.5; Cavalcante & Döbereiner, 1988) agar plates containing 150 mg cycloheximide l–1 (Jimenez-Salgado et al., 1997) and 150 mg nystatin l–1. Plates were incubated at 30 °C for 5 days. The bacterial isolate was purified by repeated streaking on to LGI plates, which have no combined nitrogen source. Gas tight vials of bacteria-inoculated LGI medium (under microaerophilic environment, without shaking) were assayed for acetylene reduction activity (Stal, 1988). Nitrogenase-positive isolates were selected for further characterization. Nitrogen-fixing bacterial strain RG1T was isolated. Other reference strains and the novel isolates were grown in mannitol broth for further study. Colony morphology was examined on LGI agar plates and on potato agar plates containing 10 % sucrose. Various phenotypic and morphological studies were performed using standard techniques described elsewhere (Cavalcante & Döbereiner, 1988; Caballero-Mellado et al., 1995; Jimenez-Salgado et al., 1997; Cleenwerck et al., 2002). Isoprenoid quinone of the isolate was extracted with chloroform/methanol [2 : 1, (v/v)], and then purified by TLC on silica gel 60 F254 (20x20 cm; Merck) by using benzene as the developing solvent. Quinones recovered from the TLC plates were dissolved in acetone and analysed by HPLC (Lu et al., 1999). The HPLC system was equipped with a reverse-phase column [Luna 5U C18 (2) 100A, 250x4.6 mm; Phenomenex] and a mixture of methanol/2-propanol [2 : 1, (v/v)] was used as the mobile phase at a flow rate of 1 ml min–1. Types of quinone were identified by absorption at 275 nm and compared with coenzymes Q-9 and Q-10 standards from Sigma-Aldrich. Ubiquinone Q-9 was present in strain RG1T 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 biochemically and morphologically from other species of the genus Acetobacter as shown in Table 1.
|
) 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 Acetobacter estunensis, 97.4 % with Acetobacter indonesiensis, 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, 96.9 % with Acetobacter orleansis, 96.6 % with Acetobacter orientalis and 97.0 % with Acetobacter syzygii after performing a similarity search with FASTA (ungapped). The phylogenetic tree (Fig. 1) was deduced using MEGA version 3.1 (Kumar et al., 2004) software after multiple alignment of 16S rRNA gene sequences of other acetic acid bacteria with CLUSTAL W (Thompson et al., 1994). Distances (distance options according to the Kimura two-parameter model) and clustering with the neighbour-joining method was determined by using bootstrap values (Felsenstein, 1985) based on 100 replications. Two subclusters were evident in the genus Acetobacter: one composed of Acetobacter pomorum, 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 confirmed by the formation of dihydroxyacetone from glycerol by both strains RG1T and A. aceti ATCC 15973T.
|
; Caballero-Mellado & Martínez-Romero, 1994). A. cibinongensis JCM 11196T, A. orleansis LMG 1583T, A. malorum LMG 1746T, A. aceti ATCC 15973 T, A. tropicalis LMG 19825T, A. oeni B13T, A. orientalis JCM 11195T, A. indonesiensis LMG 19824T, A. estunensis LMG 1626T and A. cerevisiae LMG 1625T were included as reference strains. The relationships among non-nitrogen-fixing species of Acetobacter and RG1T are illustrated by the dendrogram (Supplementary Fig. S1 available in IJSEM Online) derived by program ETMEGA (T. S. Whittam; http://foodsafe.msu.edu/whittam/programs/index.html) and MEGA version 3.1 software based on the electrophoretic mobility of nine metabolic enzymes, indophenol oxidase, alcohol dehydrogenase, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine dehydrogenase, leucine dehydrogenase, lysine dehydrogenase, hexokinase and esterase. In MLEE, strain RG1T exhibited highest similarity to A. aceti ATCC 15973T, which is in agreement with its phylogenetic position (Fig. 1
). Analysis of the dendrogram revealed that strain RG1T formed a unique branch with A. aceti ATCC 15973T that deviated at a genetic distance of more than 0.5. This value has been used as a criterion to suggest species limits (Selander et al., 1985; Musser et al., 1987). Thus, MLEE results strongly support the existence of strain RG1T as a novel Acetobacter species.
The nitrogenase enzyme complex, responsible for nitrogen-fixation, is composed of nifHDK and other gene fragments. A 336 bp region encoding dinitrogenase reductase, nifH, was amplified from strain RG1T using degenerate primers 19F and 407R (Franke et al., 1998) and sequenced. Presence of nifH gene further confirmed the nitrogen-fixing ability of strain RG1T.
To determine genomic relatedness of the novel isolate, dot-blot hybridization experiments were carried out with DIG-labelled DNA as described previously (Labrenz et al., 2000) using the detection kit from Roche Applied Sciences and following the manufacturer's instructions. Colorimetric quantification of dot intensities was done using the Molecular Analyst software (Bio-Rad) by determining mean pixel densities in equal sized circles. A genomic DNA probe was prepared from the novel strain RG1T, digested with EcoRI and run on 0.7 % agarose gel. Total DNA digests were transferred from gels to nylon membrane by Southern blotting. Hybridization was performed at a temperature of 75 °C for 16 h and the membrane was washed under high stringency conditions (twice with 2x SSC/0.1 % SDS at room temperature for 10 min; once with 0.1x SSC/0.1 % SDS at 75 °C for 15 min). A low level of genomic DNA relatedness (DNA–DNA hybridization values less than 50 %) was observed between the phylogenetically closest species and genera. Strain RG1T showed low DNA–DNA relatedness with the type strains of A. oeni (24 %), A. aceti (22 %), A. cerevisiae (19 %), A. cibinongensis (18.91 %), A. orientalis (18.9 %), A. estunensis (11 %), A. orleansis (10.37 %), A. malorum (7.31 %), A. indonesiensis (3.71 %) and A. tropicalis (2 %).
Although the limitations of 16S rRNA gene sequencing to differentiate closely related species have been documented (Fox et al., 1992), it is suggested that strains sharing greater than 97 % similarity might belong to the same species but 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, these considerations were consistent with both the 16S rRNA gene sequence similarity levels and the low levels of DNA–DNA relatedness exhibited within the Acetobacter species. The genetic distance of different closely related acetobacters involving strain RG1T was found to be greater 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.
In view of low physiological, biochemical, phylogenetic and genetic similarities among different closely related members of the genus Acetobacter, we recommend that nitrogen-fixing strain RG1T described here should be assigned to a novel species of the genus Acetobacter, for which the name Acetobacter nitrogenifigens sp. nov. is proposed.
Description of Acetobacter nitrogenifigens sp. nov.
Acetobacter nitrogenifigens (ni.tro.gen.i'fi.gens. N.L. n. nitrogenium nitrogen; L. part. adj. figens fixing; N.L. part. adj. nitrogenifigens nitrogen-fixing).
Cells are straight rods with rounded ends, approximately 1.5–2.0 µm in length and 0.1–0.2 µm in width and occur singly or in chains. These Gram-negative bacteria are motile with polar flagellation, catalase-positive and oxidase-negative. Growth occurs on nitrogen-free LGI plates at 30 °C, but not at 37 °C and in LGI broth under microaerophilic conditions. After incubation for 5 days, colonies grown on LGI plates are smooth, round, glistening, transparent and 2–3 mm in diameter, while dark-yellow colonies are formed on LGI agar supplemented with 0.001 % bromothymol blue. Colonies on potato agar are light brown after 5 days of incubation, but the intensity increases after 10 days. Strains are aerobic and fix atmospheric nitrogen microaerobically. Growth occurs well in the presence of ammonium, but nitrate is not reduced. In absence of yeast extract, strains can utilize different carbon sources such as D-galactose, D-xylose, D-fructose, D-arabinose, D-mannitol, D-sorbitol, and grow on 30 % sucrose and 30 % glucose. Dihydroxyacetone is produced from glycerol and -pyrone from D-glucose. Ethanol is oxidized to acetic acid, and acetate and lactate over oxidize to CO2 and water. Methanol is not utilized. Single amino acids like L-alanine, L-cysteine and L-phenylalanine can be used as a sole source of carbon and nitrogen. L-Threonine is not utilized as the sole source of carbon and nitrogen. The ubiquinone present is of the type Q-9 and DNA G+C content is 64.1 mol%. Based on data from the MLEE assay along with DNA–DNA relatedness and nifH gene sequence, the type strain RG1T can be differentiated from other Acetobacter species.
The type strain is strain RG1T (=MTCC 6912T=LMG 23498T), isolated from Kombucha tea.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Caballero-Mellado, J. & Martínez-Romero, E. (1994). Limited genetic diversity in the endophytic sugarcane bacterium Acetobacter diazotrphicus. Appl Environ Micrbiol 60, 1532–1537.
Caballero-Mellado, J., Fuentes-Ramírez, L. E., Reis, V. M. & Martínez-Romero, E. (1995). Genetic structure of Acetobacter diazotrophicus populations and identification of a new genetically distant group. Appl Environ Microbiol 61, 3008–3013.[Abstract]
Cavalcante, V. & Döbereiner, J. (1988). A new acid tolerant nitrogen-fixing bacterium associated with the sugarcane. Plant Soil 108, 23–31.
Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. (2002). Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J Syst Evol Microbiol 52, 1551–1558.[Abstract]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P., Jr (1992). How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42, 166–170.
Franke, I. J., Fegan, M., Hayward, A. C. & Sly, L. I. (1998). Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus. Lett Appl Microbiol 26, 12–16.
Fuentes-Ramírez, L. E., Bustillos-Cristales, R., Tapia-Her
andez, A., Jimenez-Salgado, T., Wang, E. T., Martínez-Romero, E. & Caballero-Mellado, J. (2001). Novel nitrogen-fixing acetic acid bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter azotocaptans sp. nov., associated with coffee plants. Int J Syst Evol Microbiol 51, 1305–1314.[Abstract]
Jimenez-Salgado, T., Fuentes-Ramírez, L. E., Tapia-Herandez, A., Mascarúa-Esparza, M. A., Martínez-Romero, E. & Caballero-Mellado, J. (1997). Coffea arabica L., a new host plant for Acetobacter diazotrophicus, and isolation of other nitrogen-fixing Acetobacteria. Appl Environ Microbiol 63, 3676–3683.[Abstract]
Jojima, Y., Mihara, Y., Suzuki, S., Yokozeki, K., Yamanaka, S. & Fudou, R. (2004). Saccharibacter floricola gen. nov., sp. nov., a novel osmophilic acetic acid bacterium isolated from pollen. Int J Syst Evol Microbiol 54, 2263–2267.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Labrenz, M., Tindall, B. J., Lawson, P. A., Collins, M. D., Schumann, P. & Hirsch, P. (2000). Staleya guttiformis gen. nov., sp. nov., and Sulfitobacter brevis sp. nov., 
-3-Proteobacteria from hypersaline, heliothermal and meromictic Antarctic Ekho Lake. Int J Syst Evol Microbiol 50, 303–313.[Abstract]
Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & Komagata, K. (2000). Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov. J Gen Appl Microbiol 46, 147–165.
Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & Komagata, K. (2001). Identification of Acetobacter strains isolated from Indonesian sources, and proposals of Acetobacter syzygii sp. nov., Acetobacter cibinongensis sp. nov., and Acetobacter orientalis sp. nov. J Gen Appl Microbiol 47, 119–131.
Lisdiyanti, P., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., Yamada, Y., Uchimura, T. & Komagata, K. (2002). Kozakia baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the -Proteobacteria. Int J Syst Evol Microbiol 52, 813–818.[Abstract]
Loganathan, P. & Nair, S. (2004). Swaminathania salitolerans gen. nov., sp. nov., a salt-tolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice (Porteresia coarctata Tateoka). Int J Syst Evol Microbiol 54, 1185–1190.
Lu, S.-F., Lee, F.-L. & Chen, H.-K. (1999). A thermotolerant and high acetic-acid producing bacterium Acetobacter sp. 114-2. J Appl Microbiol 86, 55–62.[CrossRef]
Musser, J. M., Bemis, D. A., Ishikawa, H. & Selander, R. K. (1987). Clonal diversity and host distribution in Bordetella bronchiseptica. J Bacteriol 169, 2793–2803.
Muthukumarasamy, R., Revathi, G., Seshadri, S. & Lakshminarsimhan, C. (2002). Gluconacetobacter diazotrophicus (syn. Acetobacter diazotrophicus), a promising diazotrophic endophyte in tropics. Current Science 83, 137–145.
Muthukumarasamy, R., Cleenwerck, I., Revathi, G. & 8 other authors (2005). Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice. Syst Appl Microbiol 28, 277–286.
Selander, R. K., McKinney, R. M., Whittam, T. S., Bibb, W. F., Brenner, D. J., Nolte, F. S. & Pattison, P. E. (1985). Genetic structures of populations of Legionella pneumophila. J Bacteriol 163, 1021–1037.
Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M., Gilmour, M. N. & Whittam, T. S. (1986). Methods in multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol 51, 873–884.
Silva, L. R., Cleenwerck, I., Rivas, R., Swings, J., Trujillo, M. E., Willems, A. & Velázquez, E. (2006). Acetobacter oeni sp. nov., isolated from spoiled red wine. Int J Syst Evol Microbiol 56, 21–24.
Sokollek, S. J., Hertel, C. & Hammes, W. P. (1998). Description of Acetobacter oboediens sp nov. and Acetobacter pomorum sp nov., two new species isolated from industrial vinegar fermentations. Int J Syst Bacteriol 48, 935–940.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Stal, L. J. (1988). Nitrogen fixation in cyanobacterial mats. Methods Enzymology 167, 475–484.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.
Yamada, Y., Hoshino, K. & Ishikawa, T. (1997). The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconacetobacter to the generic level. Biosci Biotechnol Biochem 61, 1244–1251.[Medline]
Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., Uchimura, T. & Komagata, K. (2000). Asaia bogorensis gen. nov., sp. nov., an unusual acetic acid bacterium in the -Proteobacteria. Int J Syst Evol Microbiol 50, 823–829.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  I. Cleenwerck, A. Gonzalez, N. Camu, K. Engelbeen, P. De Vos, and L. De Vuyst Acetobacter fabarum sp. nov., an acetic acid bacterium from a Ghanaian cocoa bean heap fermentation Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2180 - 2185. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Ndoye, I. Cleenwerck, K. Engelbeen, R. Dubois-Dauphin, A. T. Guiro, S. Van Trappen, A. Willems, and P. Thonart Acetobacter senegalensis sp. nov., a thermotolerant acetic acid bacterium isolated in Senegal (sub-Saharan Africa) from mango fruit (Mangifera indica L.) Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1576 - 1581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Cleenwerck, N. Camu, K. Engelbeen, T. De Winter, K. Vandemeulebroecke, P. De Vos, and L. De Vuyst Acetobacter ghanensis sp. nov., a novel acetic acid bacterium isolated from traditional heap fermentations of Ghanaian cocoa beans Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1647 - 1652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Dutta and R. Gachhui Nitrogen-fixing and cellulose-producing Gluconacetobacter kombuchae sp. nov., isolated from Kombucha tea Int J Syst Evol Microbiol, February 1, 2007; 57(2): 353 - 357. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
