Pontibacter diazotrophicus sp. nov., a Novel Nitrogen-Fixing Bacterium of the Family Cytophagaceae

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).


Introduction
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 65.6uC in summer and the lowest was below 220uC in winter. Diurnal temperature difference reaches over 40uC. 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 [1].
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 N 2 into NH 4 + , a more readily assimilated form of dissolved nitrogen [2]. 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 [3][4][5][6][7][8]. 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 H4X T 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 H4X T 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.

Ethics statement
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 K 2 HPO 4 , 0.2 g MgSO 4 , 1 g CaCO 3 , 0.2 g NaCl, 5 mg FeSO 4 , 10 g glucose per liter (pH 7.0). The plate was incubated at 30uC 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 [9]. 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/) [10]. Multiple sequence alignment was performed using ClustalW [11]. Phylogenetic trees were constructed using the maximum-likelihood and Bayesian method implemented in MEGA 5.0 and MrBayes v3.1, respectively [12,13]. The topology of the tree was evaluated using the bootstrap resampling method with 1000 replicates.

Phenotypic analysis
Bacterial morphology and motility were observed under a phase contrast microscope using the cells that were grown in the 0.36Marine Broth 2216 (Difco) medium at 28uC into exponential phase. Gram staining was performed as described previously [14]. Salt tolerance was determined by growing the bacteria in 0.36Marine Broth 2216 containing different concentrations of NaCl (0-10%, w/v), respectively. Bacterial growth at different temperatures (4, 10, 20, 28, 30, 35, 37, 42uC) 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 [14]. 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 H4X T was determined using HPLC (UltiMate 3000, Dionex) [15,16]. Respiratory quinones were extracted and detected by HPLC as described previously [17]. Polar lipids were isolated using a standard TLC technique [18]. For analysis of fatty acid methyl esters (FAMEs), the isolate and closely related type strains from the genus Pontibacter were cultured on the 0.36Marine Broth 2216 agar plate for appropriate time, respectively. FAMEs were further prepared and analyzed using Sherlock Microbial Identification System (MIDI, Inc., Newwark, USA).

Measurement of nitrogenase activity
Bacterial nitrogenase activity of the strain H4X T was examined using the acetylene reduction assay. The strains Azospirillum lipoferum Sp59 T and Escherichia coli DH5a were included as positive and negative control, respectively. Other members of the genus Pontibacter, such as P. actinarum KMM 6156 T , P. korlensis X14-1 T and P. xinjiangensis 311-10 T were also included as parallel comparison. Bacteria were grown in the 0.36Marine Broth 2216 medium into exponential phase at 28uC 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 [19]. Cultures were incubated, unshaken, at 28uC. 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 m62.0 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 [20,21]. 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 H 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 H4X T and other closely related bacteria were performed using ClustalW [11]. Phylogenetic tree was constructed using the maximum-likelihood or Bayesian method [12,13]. The topology of the tree was evaluated using the bootstrap resampling method with 1000 replicates.

Results
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 H4X T .

Chemotaxonomic characterization
The G+C content of H4X T 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-C 15:0 (10.9%), C 16:1 v5c (14.3%), summed feature 3 (containing C 16:1 v6c and/or C 16:1 v7c) (21.6%) and summed feature 4 (comprising anteiso-C 17:1 B and/or iso-C 17:1 I) (31.9%) ( Table 1). All these chemotaxonomic properties of the strain H4X T are consistent with those of other members of the genus Pontibacter described so far [22,23].

Phenotypic features
The bacterial cells of the strain H4X T are Gram-staining negative, mobile by gliding. Typical cells are straight, slightly curved or curved rods. The bacteria form red colonies on the 0.36Marine 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 4uC to 40uC, and the optimum is 30uC. 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 H4X T that make it distinguishable from the reference species (Table 2). These data suggest that the strain H4X T represents a novel species of the genus Pontibacter.

Nitrogen-fixing properties
The cells of the strains H4X T , E. coli DH5a, A. lipoferum Sp59 T , P. actinarum KMM 6156 T , P. korlensis X14-1 T and P. xinjiangensis 311-10 T were inoculated onto the agar plates containing nitrogenfree medium, and passaged at least 20 times, respectively. We found that only H4X T and the positive control A. lipoferum Sp59 T  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 [24]. To further confirm that H4X T is a nitrogen-fixer, we performed acetylene reduction assay. As shown in Table 3, we found that, if the assay for the strain H4X T was performed without acetylene, ethylene was not detectable. This suggests that the strain H4X T does not produce detectable native ethylene. The strain H4X T was able to convert acetylene into ethylene at the rate of 7.1361.2 nmol per hour per 10 8 cells at 28uC, whereas the positive control, A. lipoferum Sp59 T can reduce ethylene at the rate of 97.8561.6 nmol per hour per 10 8 cells. However, the negative control, E. coli DH5a, and other members of the genus Pontibacter, such as P. actinarum KMM 6156 T , P. korlensis X14-1 T and P. xinjiangensis 311-10 T were totally unable to reduce acetylene.
Moreover, we detected the existence of a nitrogenase gene (nifH) in the stain H4X T . 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 H4X T using two pairs of primers FGPH19 and PolR, PolF and AQER as described previously [20,21]. 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 H4X T , but failed to get it from other related species of the genus Pontibacter, including P. actiniarum KMM 6156 T , P. korlensis X14-1 T and P. xinjiangensis 311-10 T . Phylogenetic analyses indicated that the nifH gene of the strain H4X T is most closely related to those of some species of the genus Azospirillum, including A. halopraeferens, A. picis and A. rugosum ( Figure 3).
Therefore, based on the phenotypic, genotypic and biochemical properties of the strain H4X T , 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.

Discussion
The genus Pontibacter, first described by Nedashkovskaya et al., is a member of the family Cytophagacea [22]. Until now, at least fifteen species of this genus have been isolated from different habitats, including P. actiniarum and P. saemangeumensis from sea water [22,25], P. roseus from occasional drainage system [26], P. xinjiangensis, P. korlensis, P. toksunensis and P. akesuensis from desert soils [8,23,27,28], P. niistensis and P. populi from forest soil [29,30], P. rhizosphera from the rhizosphere soil of Nerium indicum [31], P. salisaro, P. jeungdoensis and P. odishensis from solar saltern [32][33][34], and P. lucknowensis and P. ramchanderi from the hexachlorocyclohexane contaminated soil [35,36]. Among all members of the family Cytophagacea, none has been found to have nitrogen-fixing activity so far. Our study showed that the strain H4X T 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 H4X T possesses the nitrogenase activity. Therefore, H4X T 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 [37]. 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 [2,38]. Lateral gene transfer plays a major role in the genome evolution of Pontibacter sp. [39]. Here, we found that the nucleotide sequence of the nifH gene of the strain H4X T is closely related to those from Azospirillum sp., affiliated with a-Proteobacteria. Therefore, it is interesting to further explore whether the nifH gene of the strain H4X T 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 N 2 into NH 4 + , could undergo high selective pressure. This would lead to high   Escherichia coli DH5a 0 0 Pontibacter actinarum KMM 6156 T 0 0 Pontibacter xinjiangensis 311-10 T 0 0 Pontibacter korlensis X14-1 T 0 0 *Data were obtained from three independent experiments. doi:10.1371/journal.pone.0092294.t003