Few diazotrophs have been found to belong to the family Cytophagaceae so far. In the present study, a Gram-negative, rod-shaped bacterium that forms red colonies, was isolated from sands of the Takalamakan desert. It was designated H4XT. Phylogenetic and biochemical analysis indicated that the isolate is a new species of the genus Pontibacter. The 16S rRNA gene of H4XT displays 94.2–96.8% sequence similarities to those of other strains in Pontibacter. The major respiratory quinone is menaquinone-7 (MK-7). The DNA G+C content is 46.6 mol%. The major cellular fatty acids are iso-C15∶0, C16∶1ω5c, summed feature 3 (containing C16∶1ω6c and/or C16∶1ω7c) and summed feature 4 (comprising anteiso-C17∶1B and/or iso-C17∶1I). The major polar lipids are phosphatidylethanolamine (PE), one aminophospholipid (APL) and some unknown phospholipids (PLs). It is interesting to see that this bacterium can grow very well in a nitrogen-free medium. PCR amplification suggested that the bacterium possesses at least one type of nitrogenase gene. Acetylene reduction assay showed that H4XT actually possesses nitrogen-fixing activity. Therefore, it can be concluded that H4XT is a new diazotroph. We thus referred it to as Pontibacter diazotrophicus sp. nov. The type strain is H4XT ( = CCTCC AB 2013049T = NRRL B-59974T).
Citation: Xu L, Zeng X-C, Nie Y, Luo X, Zhou E, Zhou L, et al. (2014) Pontibacter diazotrophicus sp. nov., a Novel Nitrogen-Fixing Bacterium of the Family Cytophagaceae. PLoS ONE 9(3): e92294. https://doi.org/10.1371/journal.pone.0092294
Editor: Paul Jaak Janssen, Belgian Nuclear Research Centre SCK/CEN, Belgium
Received: June 8, 2013; Accepted: February 20, 2014; Published: March 19, 2014
Copyright: © 2014 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (Grant no. 41272257 and no. 41072181), the China Postdoctoral Science Foundation (Grant no. 20110491233) and Research Program for BGEG lab staff (Grant no. GBL11208). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The Takalamakan desert is situated in the middle of the Tarim basin, Xinjiang province of China. It is the world's second largest shifting sand desert. Taklamakan has another name “the Sea of Death” due to its extremely rigorous climate. The highest temperature reached 65.6°C in summer and the lowest was below −20°C in winter. Diurnal temperature difference reaches over 40°C. It is very arid in the Takalamakan area. The annual precipitation is less than 100 mm, while evaporation reaches 2500–3400 mm. Moreover, there are only trace-level organic compounds in the sands and soils. Although the environmental conditions are extremely rigorous, some plants, such as Populus euphratica, still exist in Taklamakan Desert .
It was shown that the oligotrophic ecosystem is largely dependent on nitrogen input from biological nitrogen fixation. Nitrogen-fixing bacteria are the only organisms capable of converting molecular N2 into NH4+, a more readily assimilated form of dissolved nitrogen . Diazotrophic bacteria also play a vital role in stabilizing soil against erosion and altering the hydrological properties of crust-covered soils for the plants in the deserts of India, Israel, Morocco, Chile and China –. Nitrogen-fixing bacteria are thus important for maintaining the ecological equilibrium of deserts and improving the environment. However, few of diazotrophs have been isolated from the Takalamakan desert so far.
We described a novel nitrogen-fixing bacterium H4XT isolated from Taklamakan Desert. We showed that this bacterium is a new species of the genus Pontibacter. The bacterium is able to grow very well in a nitrogen-free medium. We also found that this bacterium contains a typical nitrogen-fixing gene nifH. Acetylene reduction assay showed that H4XT actually possesses nitrogen-fixing ability. Therefore, the isolate is a new diazotroph. The bacterium was thus referred to as Pontibacter diazotrophicus sp. nov. This is the first nitrogen-fixing bacterium isolated from Taklamakan Desert.
Materials and Methods
No specific permits were required for the described field studies. We would like to confirm that the location is not privately-owned or protected in any way, and the field studies did not involve endangered or protected species.
Isolation of diazotrophic bacteria
About 1.0 gram of sands were taken from a dune ridge of Taklimakan Desert (84.173400W, 40.485143N). Scattered grass can be seen at the sampling site. The sands were suspended in 0.85% (w/v) NaCl solution. After removal of insoluble sands and large particles, supernatant containing bacteria was serially diluted and plated onto an agar plate containing 1 g K2HPO4, 0.2 g MgSO4, 1 g CaCO3, 0.2 g NaCl, 5 mg FeSO4, 10 g glucose per liter (pH 7.0). The plate was incubated at 30°C for 2 weeks.
16S rRNA gene sequence analysis
Genomic DNAs of bacteria were isolated using MiniBEST Bacterial Genomic DNA Extraction Kit Version 2.0 (TaKaRa Biotechnology Co., Tokyo, Japan). 16S rRNA gene was amplified by PCR using the primers 27F and 1492R as described previously . PCR products were gel purified and sequenced by Genscript (Nanjing, China). Pairwise sequence identities of 16S rRNA genes were calculated using the Eztaxon-e server (http://eztaxon-e.ezbiocloud.net/) . Multiple sequence alignment was performed using ClustalW . Phylogenetic trees were constructed using the maximum-likelihood and Bayesian method implemented in MEGA 5.0 and MrBayes v3.1, respectively , . The topology of the tree was evaluated using the bootstrap resampling method with 1000 replicates.
Bacterial morphology and motility were observed under a phase contrast microscope using the cells that were grown in the 0.3×Marine Broth 2216 (Difco) medium at 28°C into exponential phase. Gram staining was performed as described previously . Salt tolerance was determined by growing the bacteria in 0.3×Marine Broth 2216 containing different concentrations of NaCl (0–10%, w/v), respectively. Bacterial growth at different temperatures (4, 10, 20, 28, 30, 35, 37, 42°C) and different pH values (5.0–11.0) were also examined. Oxidase activity was determined from the oxidation of 1% p-aminodimethylaniline oxalate. Catalase activity was tested by measuring bubble production after the application of 3% (v/v) hydrogen peroxide solution. Capability to hydrolyze starch (1%, w/v), cellulose (0.1%, w/v), chitin from crab shells (1%, w/v), casein (1%, w/v) and tyrosine (0.5%, w/v) were also tested as described previously . Other enzyme activities and biochemical features were determined using the API kits (API 20NE, API 20E, API 50CH and API ZYM) according to the manufacturer's instruction (BioMerieux, France). DNA G+C content of the strain H4XT was determined using HPLC (UltiMate 3000, Dionex) , . Respiratory quinones were extracted and detected by HPLC as described previously . Polar lipids were isolated using a standard TLC technique . For analysis of fatty acid methyl esters (FAMEs), the isolate and closely related type strains from the genus Pontibacter were cultured on the 0.3×Marine Broth 2216 agar plate for appropriate time, respectively. FAMEs were further prepared and analyzed using Sherlock Microbial Identification System (MIDI, Inc., Newwark, USA).
Nitrogen-free growth assay
Bacteria were initially grown in the 0.3×Marine Broth 2216 medium into exponential phase. Cells were harvested by centrifugation (8000 rpm, 10 min, JA 20 rotor, Beckman). The pellets were washed twice with 0.85% (w/v) NaCl solution, and re-suspended in distilled water. The suspension was inoculated into a nitrogen-free agar plate containing 19.45 g NaCl, 8.8 g MgCl2, 3.24 g Na2SO4, 1.8 g CaCl2, 0.55 g KCl, 0.16 g NaHCO3, 0.1 g Ferric citrate, 0.08 g KBr, 0.034 g SrCl2, 0.022 g H3BO3, 8.0 mg Na2HPO4, 4.0 mg Na2SiO3, 2.4 mg NaF per liter (pH 7.4). Survived bacteria were passaged at least 20 times on the agar plate. The strains Azospirillum lipoferum Sp59T and Escherichia coli DH5α were included as positive and negative control, respectively.
Measurement of nitrogenase activity
Bacterial nitrogenase activity of the strain H4XT was examined using the acetylene reduction assay. The strains Azospirillum lipoferum Sp59T and Escherichia coli DH5α were included as positive and negative control, respectively. Other members of the genus Pontibacter, such as P. actinarum KMM 6156T, P. korlensis X14-1T and P. xinjiangensis 311-10T were also included as parallel comparison. Bacteria were grown in the 0.3×Marine Broth 2216 medium into exponential phase at 28°C with shaking. Cells were harvested by centrifugation (8000 rpm, 10 min, JA 20 rotor, Beckman), and washed twice with 0.85% (w/v) NaCl solution. The cells were re-suspended in distilled water. Aliquots of 0.2 ml were inoculated into vials (21 ml) containing 10 ml of semisolid NFb medium . Cultures were incubated, unshaken, at 28°C. After 48 hours, the vials were sealed with rubber stoppers. The gas phase in the headspace was replaced with acetylene (10% v/v). Ethylene content was measured at 13 h intervals. Measurement was performed using a gas chromatograph (GC-4000, GL Science inc., Tokyo, Japan) with a flame-ionization detector and a column (2.0 m×2.0 mm i.d., stainless steel) packed with GDX-502. Controls with medium and inoculated culture without acetylene gas were run in parallel to each strain for the full incubation time.
NifH gene sequence analysis
Genomic sequence of the nifH gene was amplified by direct PCR followed by nested PCR using two pairs of primers FGPH19 and PolR (for direct PCR), PolF and AQER (for nested PCR) as described previously , . The primers was designed to amplify the partial sequence of the nifH gene that codes for the amino acid sequence from residue 38 to 149 in nitrogenase H. The PCR products were gel purified and cloned into the pMD18-T® vector (TaKaRa Biotechnology Co., Tokyo, Japan). Positive clones were sequenced by Genscript (Nanjing, China). Multiple sequence alignment of the deduced amino acid sequences of the nifH genes from the strain H4XT and other closely related bacteria were performed using ClustalW . Phylogenetic tree was constructed using the maximum-likelihood or Bayesian method , . The topology of the tree was evaluated using the bootstrap resampling method with 1000 replicates.
Isolation of candidate diazotrophic bacteria from Taklamakan Desert
From 1.0 g of sands, we isolated twenty-six different bacteria that are capable of growing well in the nitrogen-free medium. Among them, twenty-five isolates formed white or whitish colonies, and the last one formed red colonies. Sequence analysis for the 16S rRNA genes of these bacteria showed that we discovered a new strain of bacteria with potential nitrogen-fixing activity, which was designated H4XT.
Phylogeny of 16S rRNA gene sequences
The 16S rRNA gene sequence of H4XT shows 96.8% and 95.5% identities to those of Pontibacter toksunensis and Pontibacter saemangeumensis, respectively. It also shows 94.2–95.4% identities to those of other species of the genus Pontibacter, such as P. korlensis, P. lucknowensis, P. actiniarum, P. ramchanderi, P. odishensis, P. roseus, P. xinjiangensis, P. akesuensis, P. niistensis, P. populi, P. rhizosphera, P. salisaro, P. indicus and P. jeungdoensis. Phylogenetic analysis indicated that the strain H4XT is most closely related with P. saemangeumensis GCM0142T and P. xinjiangensis 311-10T (Figure 1). Thus, it is likely that H4XT represents a new species of the genus Pontibacter.
Bootstrap values (expressed as percentages of 1000 replicates) that are >75% are shown at branch points. Asterisks indicate that the corresponding nodes were also recovered in the Bayesian tree. Bar, 0.01 substitutions per nucleotide position.
The G+C content of H4XT was 46.6 mol%, which falls within the range for the genus Pontibacter. Only menaquinone-7 (MK-7) was detectable as respiratory menaquinone. Phosphatidylethanolamine was found to be one of the major polar lipids in the cells. In addition, we found that there are several unknown phospholipids and an aminophospholipid (Figure 2). The major fatty acids include iso-C15∶0 (10.9%), C16∶1ω5c (14.3%), summed feature 3 (containing C16∶1ω6c and/or C16∶1ω7c) (21.6%) and summed feature 4 (comprising anteiso-C17∶1 B and/or iso-C17∶1 I) (31.9%) (Table 1). All these chemotaxonomic properties of the strain H4XT are consistent with those of other members of the genus Pontibacter described so far , .
PE, phosphatidylethanolamine; APL, aminophospholipid; PL1-4, unknown phospholipids.
The bacterial cells of the strain H4XT are Gram-staining negative, mobile by gliding. Typical cells are straight, slightly curved or curved rods. The bacteria form red colonies on the 0.3×Marine Broth 2216 agar plate. Colonies are convex and circular with entire margin. The cells are catalase-positive and oxidase-positive (Table 2). The strain grows at a wide range of temperatures from 4°C to 40°C, and the optimum is 30°C. Growth occurs at pH values of 6.0–8.0, and the optimum pH is 7.0. The strain tolerates high salt concentrations up to 8% (w/v) NaCl. We found that there are a lot of phenotypic features of the strain H4XT that make it distinguishable from the reference species (Table 2). These data suggest that the strain H4XT represents a novel species of the genus Pontibacter.
The cells of the strains H4XT, E. coli DH5α, A. lipoferum Sp59T, P. actinarum KMM 6156T, P. korlensis X14-1T and P. xinjiangensis 311-10T were inoculated onto the agar plates containing nitrogen-free medium, and passaged at least 20 times, respectively. We found that only H4XT and the positive control A. lipoferum Sp59T were capable of proliferating in the nitrogen-free medium even after multiple passages. Thus, it is likely that the isolate is a diazotroph.
The discovery that the nitrogenase enzyme responsible for nitrogen-fixation also reduced acetylene to ethylene provided a useful assay for the quantification of the nitrogen-fixation process . To further confirm that H4XT is a nitrogen-fixer, we performed acetylene reduction assay. As shown in Table 3, we found that, if the assay for the strain H4XT was performed without acetylene, ethylene was not detectable. This suggests that the strain H4XT does not produce detectable native ethylene. The strain H4XT was able to convert acetylene into ethylene at the rate of 7.13±1.2 nmol per hour per 108 cells at 28°C, whereas the positive control, A. lipoferum Sp59T can reduce ethylene at the rate of 97.85±1.6 nmol per hour per 108 cells. However, the negative control, E. coli DH5α, and other members of the genus Pontibacter, such as P. actinarum KMM 6156T, P. korlensis X14-1T and P. xinjiangensis 311-10T were totally unable to reduce acetylene.
Moreover, we detected the existence of a nitrogenase gene (nifH) in the stain H4XT. The nif genes are a family of genes encoding enzymes involved in the fixation of atmospheric nitrogen. PCR strategy was employed to amplify the nifH gene from the genomic DNAs of H4XT using two pairs of primers FGPH19 and PolR, PolF and AQER as described previously , . The PCR amplification using the primers FGPH19 and PolR yielded some non-specific bands. Nested PCR using the primers PolF and AQER was further employed to increase the specificity of DNA amplification. PCR products were gel purified and cloned into a T-vector for sequencing. The result showed that we successfully obtained the partial genomic sequence (298 bp) of the nifH gene from the strain H4XT, but failed to get it from other related species of the genus Pontibacter, including P. actiniarum KMM 6156T, P. korlensis X14-1T and P. xinjiangensis 311-10T. Phylogenetic analyses indicated that the nifH gene of the strain H4XT is most closely related to those of some species of the genus Azospirillum, including A. halopraeferens, A. picis and A. rugosum (Figure 3).
Bootstrap values (expressed as percentages of 1000 replicates) that are >75% are shown at branch points. Asterisks indicate that the corresponding nodes were also recovered in the Bayesian tree. Bar, 0.1 substitutions per nucleotide position.
Therefore, based on the phenotypic, genotypic and biochemical properties of the strain H4XT, it can be concluded that this bacterium represents a novel species of the genus Pontibacter. It was thus referred to as Pontibacter diazotrophicus sp. nov. It is noteworthy that P. diazotrophicus is the first nitrogen fixer described so far from the genus Pontibacter.
Description of Pontibacterdiazotrophicus sp. nov
Pontibacterdiazotrophicus (di.a.zo.tro'phi.cus. Gr.prefix di, two, double; N.L.n. azotum, nitrogen; Gr.adj.trophikos, nursing, ending or feeding; M.L. masc. adj. diazotrophicus, one that feeds on dinitrogen).
Cells are Gram-staining negative, rod-shaped (0.4–0.6×1.2–2.0 μm) and mobile by sliding. They form circular, convex, and red colonies with entire margin on the 0.3×Marine Broth 2216 agar plate. Growth occurs at temperatures from 4 to 40°C (optimum 30°C), at pH 6.0–8.0. The isolate grows in 0–8% (w/v) NaCl. The isolate is oxidase positive and catalase positive. It possesses the nifH gene, and is capable of fixing nitrogen. It can hydrolyse starch, casein, aesculin and ONPG, but not gelatin, tyrosine, chitin and cellulose. It is negative for nitrate reduction, H2S production, V-P test, indole production and glucose acidification. It assimilates D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, gluconate, trisodium citrate, D-ribose, D-saccharose, lactate, capric acid and 3-hydroxybutyric acid, but not L-rhamnose, suberic acid, malonate, D-melibiose, L-histidine, 2-ketogluconate, itaconic acid, acetate, 3-hydroxybenzoic acid, L-serine, salicin, L-fucose, D-sorbitol, propionate, valeric acid, 4-hydroxybenzoic acid and L-proline in the API 20NE and API 32GN system. It also has the activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucinearylamidase, valine arylamidase, cystinearylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase, but negative for those of lipase (C14), α-chymotrypsin, α-mannosidase, α-fucosidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, arginine dihydrolase, lysine decarboxilase, ornithine decarboxilase, tryptophane deaminase and urease. The major fatty acids are iso-C15∶0, C16∶1ω5c, summed feature 3 (containing C16∶1ω6c and/or C16∶1ω7c) and summed feature 4 (comprising anteiso-C17∶1 B and/or iso-C17∶1 I). MK-7 is the predominant menaquinone. The major polar lipids are composed of PE, APL and unknown phospholipids. The G+C content of the genomic DNA of the type strain is 46.6 mol%.
The type strain, H4XT ( = CCTCC AB 2013049T = NRRL B-59974T), was isolated from the sands of the Takalamakan desert.
The genus Pontibacter, first described by Nedashkovskaya et al., is a member of the family Cytophagacea . Until now, at least fifteen species of this genus have been isolated from different habitats, including P. actiniarum and P. saemangeumensis from sea water , , P. roseus from occasional drainage system , P. xinjiangensis, P. korlensis, P. toksunensis and P. akesuensis from desert soils , , , , P. niistensis and P. populi from forest soil , , P. rhizosphera from the rhizosphere soil of Nerium indicum , P. salisaro, P. jeungdoensis and P. odishensis from solar saltern –, and P. lucknowensis and P. ramchanderi from the hexachlorocyclohexane contaminated soil , . Among all members of the family Cytophagacea, none has been found to have nitrogen-fixing activity so far. Our study showed that the strain H4XT is capable of growing well in a nitrogen-free medium. We also found that it possesses the nifH gene potentially encoding nitrogenase. Acetylene reduction assay suggested that H4XT possesses the nitrogenase activity. Therefore, H4XT is actually a diazotroph. This is the first report of a nitrogen-fixing bacterium belonging to the genus Pontibacter. Until now, only a few of the bacterial strains belonging to Cytophaga-Flavobacterium-Bacteroides (CFB) group have been found to be diazotrophs . Our study expands the knowledge of nitrogen-fixing bacteria in this evolutionary lineage.
It was shown that genes involved in nitrogen fixation may be transferred between distantly related species belonging to different phyla of bacteria , . Lateral gene transfer plays a major role in the genome evolution of Pontibacter sp. . Here, we found that the nucleotide sequence of the nifH gene of the strain H4XT is closely related to those from Azospirillum sp., affiliated with α-Proteobacteria. Therefore, it is interesting to further explore whether the nifH gene of the strain H4XT was acquired by horizontal gene transfer.
It was shown that bacteria inhabiting the oligotrophic Taklamakan desert could largely depend on the nitrogen input from biological nitrogen fixation. Thus, the nifH gene encoding the nitrogenase that is capable of converting molecular N2 into NH4+, could undergo high selective pressure. This would lead to high degree of sequence homology between the nifH gene of the strain H4XT and those of other bacterial species from the desert.
Conceived and designed the experiments: XCZ XL LX. Performed the experiments: LX YN EZ LZ YP. Analyzed the data: XCZ XL LX. Contributed reagents/materials/analysis tools: XCZ WL. Wrote the paper: XCZ.
- 1. Westermann J, Zerbe S, Eckstein D (2008) Age structure and growth of degraded Populus euphratica floodplain forests in north-west China and perspectives for their recovery. J Integr Plant Biol 50: 536–546.
- 2. Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5: 539–554.
- 3. Chowdhury SP, Schmid M, Hartmann A, Tripathi AK (2007) Identification of diazotrophs in the culturable bacterial community associated with roots of Lasiurus sindicus, a perennial grass of Thar Desert, India. Microb Ecol 54: 82–90.
- 4. Russow R, Veste M, Böhme F (2005) A natural 15N approach to determine the biological fixation of atmospheric nitrogen by biological soil crusts of the Negev Desert. Rapid Commun Mass Spectrom 19: 3451–3456.
- 5. Benata H, Mohammed O, Noureddine B, Abdehnoumen H, Abdelbasset B, et al. (2008) Diversity of bacteria that nodulate Prosopis juliflora in the eastern area of Morocco. Syst Appl Microbiol 31: 378–386.
- 6. Lacap DC, Warren-Rhodes KA, McKay CP, Pointing SB (2011) Cyanobacteria and chloroflexi-dominated hypolithic colonization of quartz at the hyper-arid core of the Atacama Desert, Chile. Extremophiles 15: 31–38.
- 7. Dai J, Liu X, Wang Y (2012) Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China. Genet Mol Res 11: 2683–2693.
- 8. Wang Y, Zhang K, Cai F, Zhang L, Tang Y, et al. (2010) Pontibacter xinjiangensis sp. nov., in the phylum ‘Bacteroidetes', and reclassification of [Effluviibacter] roseus as Pontibacter roseus comb. nov. Int J Syst Evol Microbiol 60: 99–103.
- 9. Lane DJ (1991) 16S/23S rRNA sequencing. In: E Stackebrandt and M Goodfellow, editors. Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, Chichester. pp. 115–175.
- 10. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, et al. (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62: 716–721.
- 11. Thompson JD, Higgins DG, Gibson TJ (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.
- 12. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
- 13. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
- 14. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: P Gerhart, R. G. E Murray, W. A Wood and N. R Krieg, editors. Methods for General and Molecular Bacteriology.Washington, DC: American Society for Microbiology. pp. 607–654.
- 15. Ludwig W (2007) Nucleic acid techniques in bacterial systematics and identification. Int J Food Microbiol 120: 225–236.
- 16. Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39: 159–167.
- 17. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye M, et al. (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2: 233–241.
- 18. Komagata K, Suzuki KI (1987) Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19: 1–207.
- 19. Han SO, New PB (1998) Variation in nitrogen fixing ability among natural isolates of Azospirillum. Microb Ecol 36: 193–201.
- 20. Gaby JC, Buckley DH (2012) A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase. PLoS ONE 7: e42149.
- 21. Langlois RJ, LaRoche J, Raab PA (2005) Diazotrophic diversity and distribution in the tropical and subtropical Atlantic ocean. Appl Environ Microbiol 71: 7910–7919.
- 22. Nedashkovskaya OI, Kim SB, Suzuki M, Shevchenko LS, Lee MS, et al. (2005) Pontibacter actiniarum gen. nov., sp. nov., a novel member of the phylum 'Bacteroidetes', and proposal of Reichenbachiella gen. nov. as a replacement for the illegitimate prokaryotic generic name Reichenbachia Nedashkovskaya, et al. 2003. Int J Syst Evol Microbiol 55: 2583–2588.
- 23. Zhang L, Zhu L, Wei L, Li C, Wang Y, et al. (2013) Pontibacter toksunensis sp. nov., isolated from soil, and emended descriptions of Pontibacter roseus and Pontibacter akesuensis. Int J Syst Evol Microbiol 63: 4462–4468.
- 24. Dilworth MJ (1966) Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochim Biophys Acta 127: 285–294.
- 25. Kang JY, Joung Y, Chun J, Kim H, Joh K, et al. (2013) Pontibacter saemangeumensis sp. nov., isolated from seawater. Int J Syst Evol Microbiol 63: 565–569.
- 26. Suresh K, Mayilraj S, Chakrabarti T (2006) Effluviibacter roseus gen. nov., sp. nov., isolated from muddy water, belonging to the family "Flexibacteraceae". Int J Syst Evol Microbiol 56: 1703–1707.
- 27. Zhang L, Zhang Q, Luo X, Tang Y, Dai J, et al. (2008) Pontibacter korlensis sp. nov., isolated from the desert of Xinjiang, China. Int J Syst Evol Microbiol 58: 1210–1214.
- 28. Zhou Y, Wang X, Liu H, Zhang KY, Zhang YQ, et al. (2007) Pontibacter akesuensis sp. nov., isolated from a desert soil in China. Int J Syst Evol Microbiol 57: 321–325.
- 29. Dastager SG, Raziuddin QS, Deepa CK, Li WJ, Pandey A (2010) Pontibacter niistensis sp. nov., isolated from forest soil. Int J Syst Evol Microbiol 60: 2867–2870.
- 30. Xu M, Wang Y, Dai J, Jiang F, Rahman E, et al. (2012) Pontibacter populi sp. nov., isolated from the soil of a Euphrates poplar (Populus euphratica) forest. Int J Syst Evol Microbiol 62: 665–670.
- 31. Raichand R, Kaur I, Singh NK, Mayilraj S (2011) Pontibacter rhizosphera sp. nov., isolated from rhizosphere soil of an Indian medicinal plant Nerium indicum. Antonie Van Leeuwenhoek 100: 129–135.
- 32. Joung Y, Kim H, Ahn TS, Jon K (2011) Pontibacter salisaro sp. nov., isolated from a clay tablet solar saltern in Korea. J Microbiol 49: 290–293.
- 33. Joung Y, Kim H, Lee BI, Kang H, Jang TY, et al. (2013) Pontibacter jeungdoensis sp. nov., isolated from a solar saltern in Korea. J Microbiol 51: 531–535.
- 34. Subhash Y, Tushar L, Sasikala C, Ramana CV (2013) Erythrobacter odishensis sp. nov. and Pontibacter odishensis sp. nov. isolated from a dry soil of a solar saltern. Int J Syst Evol Microbiol. 63: 4524–4532.
- 35. Dwivedi V, Niharika N, Lal R (2013) Pontibacter lucknowensis sp. nov., isolated from a hexachlorocyclohexane dump site. Int J Syst Evol Microbiol 63: 309–313.
- 36. Singh AK, Garg N, Sangwan N, Negi V, Kumar R, et al. (2013) Pontibacter ramchanderi sp. nov., isolated from hexachlorocyclohexane contaminated pond sediment. Int J Syst Evol Microbiol 63: 2829–2834.
- 37. Yeager CM, Kornosky JL, Housman DC, Grote EE, Belnap J, et al. (2004) Diazotrophic community structure and function in two successional stages of biological soil crusts from the Colorado Plateau and Chihuahuan Desert. Appl Environ Microbiol 70: 973–983.
- 38. Fernandez C, Farias L, Ulloa O (2011) Nitrogen fixation in denitrified marine waters. PLoS ONE 6: e20539.
- 39. Joshi MN, Sharma AC, Pandya RV, Patel RP, Saiyed ZM, et al. (2012) Draft genome sequence of Pontibacter sp. nov. BAB1700, a halotolerant, industrially important bacterium. J Bacteriol 194: 6329–6330.