Pseudacidovorax Austerolens Sp. Nov., a Nifh Bacterium Isolated From

Ann Microbiol (2015) 65:217–223 DOI 10.1007/s13213-014-0852-9

ORIGINAL ARTICLE

Pseudacidovorax austerolens sp. nov., anifHbacterium isolated from Himalayan valley soil, India

Shivani Tyagi & Dileep Kumar Singh

Received: 12 June 2013 /Accepted: 28 February 2014 /Published online: 15 March 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Gram-negative, rod-shaped bacterial strain Introduction ptl-2T, was isolated from soil of the Himalayan valley, India. The bacterial strain ptl-2T has been characterized Bacterial strain ptl-2T was isolated from agricultural soil of using a polyphasic taxonomic approach including mor- Chamba valley, situated at latitude 31.4300° S and longitude phological characterization, fatty acid analysis, biochemi- 76.1297° E in Himachal Pradesh, India. It is situated in the cal tests, 16S rRNA and nifH gene sequence analysis. 16S Himalayan valley, with an average altitude of 996 m, subtrop- rRNA gene sequence analysis showed that the strain ptl- ical climate, natural vegetation and organic agricultural farm- 2T belonged to the genus Pseudacidovorax and is closely ing. The temperatures in summer vary between 38 and 15 °C related to Pseudacidovorax intermedius (99.3 % similari- and in winter between 15 and 0 °C. The average annual ty). It showed <97 % similarity to species of the genera rainfall in the valley is 786 mm. Rice, wheat and maize are Acidovorax, Alicycliphilus, Xylophilus, Giesbergeria and the main crops of this region. Here, biological nitrogen fixa- Simplicispira. The generic assignment has been confirmed tion is supposed to play an important role in maintaining the on the basis of chemotaxonomic data, which revealed the soil fertility. The process of biological nitrogen fixation has fatty acid profile, characteristic of the genus been found to be unique in bacteria and archaea, which play a

Pseudacidovorax,consistingofC16:0 (21.78) and significant role in nitrogen budgeting of the soil. Therefore, a C18:1w7c (19.78) as major fatty acids. Phylogenetic, che- study on nitrogen fixing in the microbial population of this motaxonomic and phenotypic analysis based on signature region becomes significant. So, studies on nifH gene express- sequences, DNA-DNA hybridization and physiological ing bacterial diversity of this region have been carried out. In characterizations, confirms that strain ptl-2T represents a this paper, polyphasic taxonomy of the strain ptl-2T was different species of the genus Pseudacidovorax for which described and it is placed as a member of the genus the name Pseudacidovorax austerolens sp. nov. is pro- Pseudacidovorax.GenusPseudacidovorax was first reported posed. The type strain ptl-2T (= CCUG 58759T,=DSM by Kämpfer et al. (2008) with a single known species 24877T) has been submitted to two culture collection P. intermedius. It was isolated from a soyabean-cultivated field centres. GenBank accession numbers for the 16S rRNA soil. Since then no new member has been reported in this and nifH sequence of strain Pseudacidovorax austerolens genera. Therefore, the present study describing the second ptl-2T are FJ581042 and GQ249664, respectively. known species of the genus Pseudacidovorax is significant.

Keywords Pseudacidovorax austerolens sp. nov. . 16S rRNA gene sequencing . nifH gene Materials and methods

Physicochemical analyses of the soil

: * S. Tyagi D. K. Singh ( ) Soil from Chamba valley was analyzed for its physico- Soil Microbial Ecology and Environment Toxicology Laboratory, Department of Zoology, University of Delhi, 110007 Delhi, India chemical properties by standard methods as described by e-mail: [email protected] Öhlinger (1996). 218 Ann Microbiol (2015) 65:217–223

Bacterial isolation Percent G + C content

Strain ptl-2T, was isolated from the soil of Chamba The G + C content of genomic DNA was determined as valley, Himachal Pradesh, India. Soil sampling was car- described by Gonzalez and Saiz-Jimenez (2002) using Ap- ried out from the rhizospheric region of maize plants. plied Biosystems 7500 Real Time PCR at the Department of Samples were transported to the laboratory and stored at Zoology, University of Delhi. 4 °C until further analyses. Soil samples were serially Percentage G + C was analysed using SYBR Green I diluted and inoculated on nitrogen free Jensen media (SYBR Green I, Applied Biosystems, USA). Genomic DNA T plates(2.0gsucrose,0.1gK2 HPO4 ,0.05g was isolated from the pure strain of the bacterium (ptl-2 ) T MgSO4·7H2O, 0.05 g NaCl, 0.01 g FeSO4·7H2O, 0.2 g isolated from soil. Nitrobacter winogradskyi (ATCC 25391 ) CaCO3, 1.6 g agar, 100 ml distilled water, pH 7.0). was used as a positive control for the analysis. Purity and Several bacterial strains were isolated from the soil, but quantification of isolated genomic DNA of both the pure only those showing good growth in nitrogen free medium strains was checked using a spectrophotometer (NanoDrop, were picked. After 48 h of incubation at 30 °C, single ND100, USA). The Tm value calculated was used for per- colonies were picked and re-streaked on agar plates, and centage G + C calculation. this was repeated five to seven times to ensure purity of the culture. Subculturing was also done on Luria-Bertani Nitrogen fixation (LB) medium (1 g tryptone, 0.5 g yeast extract, 0.5 g NaCl, 0.1 g glucose, 1.6 g agar, 100 ml distilled water, Acetylene reduction assay (Burris 1972) was performed to test pH 7.6) at 30 °C, for 48–72 h. the nitrogen fixing ability of the bacterium. Strain ptl-2T was grown in nitrogen free Jensen media for 72 h at 37 °C; it was then sealed with rubber septa. Acetylene was added to a final Phenotypic characterization concentration of 10 % (v/v) in the gas phase and the vials were reincubated at 37 °C for 20 h. The amount of ethylene pro- Cell morphology of the strain ptl-2T was observed using duced was measured using a Nucon Gas Chromatograph a scanning electron microscope (SEM). For sample prep- equipped with FID detector. Uninoculated tubes in triplicates aration, bacterial cells were fixed and stored in phosphate were used as control. buffer. The presence of flagella was determined by a transmission electron microscope (TEM). For TEM sam- 16S rRNA gene amplification ple preparation, bacterial cells were negatively stained with 0.5 % uranyl acetate on copper grids and, after air Amplification of the 16S rRNA gene sequence was carried out drying, the grids were examined with a TEM (Morgagni using a single colony from an overnight-grown culture of the model 268). bacterium. This colony was picked up with a sterile tip, boiled Cellular fatty acid profiles of isolate ptl-2T and in deionized water and centrifuged at 10,000 g for 10 min. The P. intermedius CCUG 54492T were determined with a gas supernatant was diluted 10-fold and used for PCR amplifica- chromatograph, using the Sherlock Microbial Identification tion. PCR amplification was carried out with thermocycler System (MIDI). Bacterial cultures were harvested by centri- (2720 Thermal Cycler, Applied Biosystems, USA) using a fugation and the pellets were subjected to saponification, universal primer set corresponding to positions 27 F (16S methylation and extraction using a standard protocol forward primer; 5′-AGAGTTTGATCCTGGCTCAG-3′)and (Paisley 1996). 1492R (16S reverse primer; 5′-TACGGTTACCTTGTTACG Susceptibility towards antibiotics was determined on L B ACTT-3′)ofEscherichia coli (Prakash et al. 2007). The PCR agar at 28 °C using the commercial susceptibility test discs product was purified using a gel/PCR DNA extraction kit (Hi (Antibiotic Icosa disc I and II, HiMedia, Maharashtra, India) Yield™, Real Biotech Corporation, Taiwan) according to the with varying amount of antibiotics, from 5 to 30 μg/disc. manufacturer’s instructions and sent for sequencing to Range of zone diameter used for determination of susceptibil- Macrogen, Seoul, Korea. ity was 4–8 mm for sensitive, 1–3 mm for intermediate and zero mm for resistant. nifH gene amplification Gram staining was done by modified Hucker’smethod (Alexander et al. 2003). Growth at different salt concentra- The nifH gene was amplified from genomic DNA of strain ptl- tions (1–10 %) and temperature (4–50°C)wasstudiedinL 2T by PCR using degenerate primers Pol-F and Pol-R (Poly B broth (Arden-Jones et al. 1979). Different biochemical et al. 2001). Reaction products were separated by agarose gel tests were carried out using a biochemical test kit (HiMedia, electrophoresis and stained with ethidium bromide. The band Mumbai, India). position was determined by comparing with the standard Ann Microbiol (2015) 65:217–223 219 having 100 bp ladder (Bangalore Genei, India). The purified was performed overnight at 65 °C. After hybridization, the nifH PCR product was directly sequenced for further analysis. filter was washed with SSC and SDS to remove unbound probe. The amount of probe bound to the DNA was estimated Phylogenetic tree construction using a scintillation counter (Perkin Elmer) and the hybridization values obtained were expressed as percentages of the The 16S rRNA gene sequence (1416 bases long) was subject- amount of probe bound relative to the homologous reaction. ed to a similarity search using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) and sequence ERIC-PCR matching tool of Ribosomal Database Project II (http://rdp. cme.nsu.edu/) and identify tool of EzTaxon (Chun et al. Both the bacteria were further studied by ERIC-PCR finger- 2007). Sequences of closely related strains were obtained printing. Primer pairs ERIC 1R (5′-ATGTAA GCT CCTGGG using the sequence match program of the Ez-taxon, and a GAT TCA C-3′) and ERIC 2 (5′-AAG TAA GTG ACT GGG phylogenetic tree was constructed using 1,416 bases long GTG AGCG-3′) were used for ERIC PCR (Alam et al. 1999). 16S rRNA gene sequence of strain ptl-2T and 23 most closely related neighbours of ptl-2T with authentically published Nucleotide sequence accession number names. Multiple alignments of all 24 sequences were performed using CLUSTAL_X (Thompson et al. 1997)and GenBank accession numbers for the 16S rRNA and nifH common gaps from all the selected sequences were removed; sequences of strain Pseudacidovorax austerolens ptl-2T sp. the alignment was checked manually for quality. Terminal nov. (= CCUG 58759T = DSM 24877T) are FJ581042 and nucleotides not common to all 24 sequences were removed. GQ249664, respectively. A phylogenetic tree was constructed using the neighbour- joining method (Saitou and Nei 1987) with 1,416 bases. The evolutionary distance matrices for the neighbour-joining Results and discussion method were calculated using the Kimura two-parameter method (Kimura 1980). Similarity values for the 16S rRNA Physicochemical analyses of the soil gene sequences were calculated using the FASTA program (Pearson and Lipman 1988). Topologies of the resultant tree The physicochemical analysis of soil showed that the soil was were evaluated using bootstrap analysis based on 1,000 re- sandy loam type, soil pH was 7.56±0.01, organic matter was sampling. 2.11±0.17 % in cultivated soil, dry matter 92±0.40 % and soil A phylogenetic tree based on 329 base sequences of ptl-2T water content was 6.75±0.40 % in cultivated soil. The con- nifH gene and partial nifH gene sequences of other 14 closely centration of exchangeable potassium and sodium in the soil related strains was also constructed using the neighbour- was in the range of 300±2.02 kg ha−1 and 9.40±0.06 ppm in joining method. cultivated soil. Soil physicochemical properties are important factors that govern the type and distinctiveness of the micro- DNA-DNA hybridization flora and micro-fauna found in a particular soil.

To further clarify the taxonomic status of strain ptl-2T,DNA– Phenotypic characterization DNA hybridization studies were carried out between strain ptl-2T and P. intermedius CCUG 54492T as described by Pal Bacterial colonies were circular and white in color in J H et al. (2005). The amount of bound probe DNA was estimated medium after 24 h. Colony size was less than 1 mm after using a scintillation counter (Triathler, Hidex, Finland) and 48 h of incubation in J H medium. Strain ptl-2T had Gram- hybridization values were expressed as percentages of bound negative, rod shaped cells. Cell size was 971±50 nm in length probe. Cells were disrupted using a French pressure cell and 450±50 nm in width. The cells were motile by means of a (Thermo Spectronic) and the DNA in the crude lysate was single, polar flagellum (Fig. 1) which was around 5,845± purified by chromatography on hydroxyapatite as described 200 nm in length. Optimum temperature for growth was by Cashion et al. (1977). 37 °C. Although the strain can grow in a temperature range Total genomic DNA of bacterial strains was extracted and 20–40 °C. The optimum salt concentration was found to be purified, and DNA (10 μg) from each strain was transferred 1 %, and can tolerate up to 3 % NaCl. Optimum pH for growth onto a positively charged nylon membrane (Hybond-N; was 7 although it could grow from a pH range 6 to 9. Strain Amersham) using a dot blot apparatus (Bio-Rad). The mem- ptl-2T is different from its nearest neighbour in various bio- brane was air-dried and cross-linked and the DNA probe for chemical tests such as utilization of citrate, glucose, arabinose, each strain was labeled with [α-32P] ATP (BRIT) using a rhamnose, saccharose and trehalose as shown in Table 1. nick-translation kit (Amersham Pharmacia). Hybridization Results of other tests such as lysine decarboxylase, ornithine 220 Ann Microbiol (2015) 65:217–223

Table 2 Percentage cellular fatty acid composition of strain ptl-2T and P. intermedius CCUG 54492T

Fatty acid ptl-2T P. intermedius CCUG 54492T

8:0 3OH 3.29 1.1 10:0 3OH 6.52 4.9 12:00 6.68 N.A 14:00 4.87 5.2 16:1 w7c/16:1w6c 15.53 21.2 16:00 21.78 23.4 17:0 cyclo 10.36 8.2 16:1 2OH 2.44 N.A 18:1 w7c 19.78 24.2 18:1 w5c 2.34 1.3 18:00 2.59 1.8 18:1 2OH 3.82 2.1

T Fig. 1 Morphology of strain ptl-2 observed showing the presence of a N.A. not available single, polar flagellum observed under transmission electron microscopy Both the neighbours were further compared on the basis of decarboxylase, urease, nitrate reduction and oxidase were also sensitivity to different antibiotics, and out of 26 different found to be different from those of P. intermedius CCUG sensitivity tests performed, they showed differences in the T 54492 . following 16 antibiotics: norfloxacin, gentamicin, chloram- Cellular fatty acid profile consists of fatty acids C8:0 3OH phenicol, cefuroxime, ciprofloxacin, cefaperazone, (3.29 % of total fatty acids), C10:0 3OH (6.52) C12:00 (6.68), roxithromycin, co-trimoxazole, cefaclor, cephotaxime, C14:00 (4.87), C16:1w7c/16:1w6c (15.53), C16:00 (21.78), C17:0 ampicillin/cloxacillin, penicillin, ampicillin/sulbactam, cyclo (10.36), C16:1 2OH (2.44), C18:1 w7c (19.78), C18:1 w5c nalidixic acid, tobramycin and amoxycillin (Table 3). (2.34), C18:00 (2.59) and C18:1 2OH (3.82). The fatty acid profile of strain ptl-2T was similar to P. intermedius but also Table 3 Antibacterial susceptibility test for strains ptl-2T and showed significant differences. Both the bacteria showed high P. intermedius CCUG 54492T amounts of C16:00,C18:1 w7c,andC16:1w7c/16:1w6c.Thisisin agreement with Kämpfer et al. (2008). But the presence of Antibiotics Quantity ptl-2T P. intermedius T CCUG 54492T fatty acid C12:0 and C16:1OH distinguished strain ptl-2 from P. intermedius (Table 2). Norfloxacin 10 μg± − Gentamicin 10 μg± + Table 1 Differential biochemical characteristics of strains ptl-2T and Chloramphenicol 30 μg+ ± T P. intermedius CCUG 54492 Cefuroxime 30 μg+ − Characteristic ptl-2T P. intermedius Ciprofloxacin 5 μg±− CCUG 54492T Cefaperazone 75 μg± − Roxithromycin 30 μg+ − Citrate − + Co-Trimoxazole 25 μg± + Glucose − + Cefaclor 30 μg+ − Arabinose ± + Cephotaxime 30 μg± ± Rhamnose − ± Ampicillin/Cloxacillin 10 μg+ − Saccharose − + Penicillin 10 Units ± − Trehalose − + Ampicillin/Sulbactam 10/10 μg − ± Lysine decarboxylase − + Nalidixic acid 10 μg± − Ornithine decarboxylase − + Tobramycin 10 μg± − Urease − + Amoxycillin 10 μg+ − Nitrate reduction − + ONPG test − + +Sensitive Oxidase ± + ±Intermediate −Resistant Ann Microbiol (2015) 65:217–223 221

Phylogenetic relationship In both phylogenetic trees (Figs. 2 and 3), strain ptl-2T is grouped together with P. intermedius CCUG 54492T but nei- The 16S rRNA sequence of strain ptl-2T was a continuous ther of them cluster with other related genera. stretch of 1,416 base pairs. This complete sequence was used for similarity search and for the construction of a phylogenetic Nitrogen fixation tree. Based on the 16S rRNA sequence analysis, strain ptl- T 2 was classified as a member of the family Comamonadaceae The acetylene reduction assay was performed and found that of the class Betaproteobacteria. Sequence similarity analysis strain ptl-2T is positive for this test. Strain ptl-2T can fix demonstrated that the isolate belonged to the genus 274.52 nmol/mg/h of nitrogen in soil. In the nifH gene-based Pseudacidovorax with Pseudacidovorax intermedius as its phylogenetic tree, strain ptl-2T is placed along with other closest phylogenetic relative with 99.3 % similarity (Fig. 2). bacteria that are capable of dinitrogen fixation (Fig. 3). Fur- It showed <97 % similarity to species of the genera ther, it was confirmed that strain ptl-2T has nitrogen-fixing Acidovorax, Alicycliphilus, Xylophilus, Giesbergeria and ability. Simplicispira. DNA–DNA hybridization nifH sequence DNA–DNA hybridization experiments were performed in The nifH gene sequence was 329 bases long and it was used triplicate between strain ptl-2T and P. intermedius CCUG for similarity search and phylogenetic tree construction. The 54492T. The DNA–DNA hybridization data showed 45 % nifH gene of the strain ptl-2T showed the greatest degree of DNA–DNA relatedness between strain ptl-2T and similarity (99 %) to Azospirillum brasilense GenBank acces- P. intermedius CCUG 54492T. Percentage relatedness was sion no. GQ 161239 and with Pseudacidovorax intermedius calculated on the basis of data obtained. All values are mean (99 %) GenBank accession no. EU008822. % ± SD calculated from experiments performed in triplicates.

Fig. 2 16S rRNA gene sequence- based phylogenetic tree generated using the neighbour-joining method, showing the relationships between Pseudacidovorax austerolens and Pseudacidovorax intermedius and other closely related bacteria. Bootstrap values are shown as percentages of 1,000 replicates. Only bootstrap values above 70 % are shown. GenBank accession numbers are shown in parentheses. Hydrogenophaga caeni EMB71T (DQ372983) was used as outgroup 222 Ann Microbiol (2015) 65:217–223

Fig. 3 nifH gene sequence-based phylogenetic tree generated by the neighbour-joining method, showing the position of strain ptl- 2T. Bootstrap values are shown as percentages of 1,000 replicates. Only bootstrap values above 70 % are shown. GenBank accession numbers are shown in parentheses. Azohydromonas australica IAM 12664T (AB188121) was used as outgroup

The hybridization results were below the threshold of Description of Pseudacidovorax austerolens ptl-2T sp. nov 70 % set by Wayne et al. (1987) and Stackebrandt and Goebel (1994) for the delineation of bacterial species. It Pseudacidovorax austerolens (au.ste.ro’lens. L. adj. austerus - clearly indicated that strain ptl-2T and P. intermedius are a -um, pungent; L. part. adj. olens, having an odor; N.L. part. representatives of two distinct novel species of the genus adj. austerolens) pungent smelling. Pseudacidovorax. Bacterial colonies are circular and white in colour. The colonies are smooth with a shiny surface and opaque, and elevation is convex. Colony size is 1–2mmindiameteronJH ERIC-PCR agar plate after 48 h of incubation and it has a circular form with an entire margin. Strain ptl-2T has Gram-negative rod Enterobacterial Repetitive Intergenic Consensus (ERIC) se- shaped cells. Cells size is 971±50 nm×450±50 nm in length quences PCR is a method of fingerprinting used for and 450±50 nm in width. Cells bear one polar flagella. distinguishing among bacterial strains. The specific band pat- Bacteria grow at 27–40 °C (optimum 37 °C) and pH 6–9 tern of amplified PCR products obtained using these se- (optimum 7). The optimum salt concentration was found to quences is a powerful molecular identification tool. ERIC- be 1 % (%), and can tolerate up to 3 % NaCl. Fatty acid profile PCR gel picture showed three prominent DNA bands below consists of straight chain saturated and unsaturated fatty acids 500 bp in P. intermedius while only one prominent band below with 16:00 and 18:1ω7c as the major fatty acid. This strain 500 bp was observed in ptl-2T. This distinct banding pattern of cannot utilize citrate, glucose, adonitol, or lactose, as the sole both the bacteria showed clear distinction in ptl-2T and carbon source. Tests for ornithine decarboxylation, lysine P. intermedius. decarboxylation, phenylalanine deamination, nitrate reduc-

tion, ONPG, Voges P, H2S production and urease activity were negative. Utilization of arabinose, rhamnose, xylose, Percent G + C content trehalose, melibion, malinose, saccharose, cellobion, adonitol, raffinose, indole, lactose and sorbitol were also found to be The DNA G + C content of the strain ptl-2T was 67 mol %. negative. Oxidase test and utilization of arabinose were inter- This data is in agreement with the values (70.1 mol %) report- mediate. Strain ptl-2T was sensitive to chloramphenicol ed previously by Kämpfer et al. (2008)forP. intermedius. (30 μg), cefuroxime (30 μg), roxithromycin (30 μg), netillin On the basis of genotypic (DNA-DNA hybridization, (30 μg), cefaclor (30 μg), cephadroxil (30 μg), ampicillin/ ERIC-PCR, DNA G + C content), phylogenetic, phenotypic cloxacillin (10 μg), and amoxycillin (10 μg). It was interme- and chemotaxonomic data the strain ptl-2T was found to be a diate to norfloxacin (10 μg), gentamicin (10 μg), ciprofloxa- novel species of the genus Pseudacidovorax for which the cin (5 μg), cefaperazone (75 μg), co-trimoxazole (25 μg), name Pseudacidovorax austerolens sp. nov. is proposed. cephotaxime (30 μg), penicillin (10 Units), sparfloxacin Ann Microbiol (2015) 65:217–223 223

(5 μg), nalidixic acid (10 μg), tobramycin (10 μg) and eryth- Chun J, Lee J-H, Jung Y,Kim S, Kim BK, Lim YW (2007) EzTaxon: a romycin (15 μg), and resistant to ceftazidime (30 μg), web based tool for the identification of prokaryotes based on 16 S μ μ ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57: clarithromycin (15 g), azithromycin (15 g), amikacin 2259–2261 (30 μg), ampicillin/sulbactam (10/10 μg), nitrofurantoin Gonzalez JM, Saiz-Jimenez C (2002) A fluorimetric method for the (300 μg) and vancomycin (30 μg) (Table 3). Strain ptl-2T estimation of G + C mol% content in microorganisms by thermal – gives one distinct band with ERIC-PCR primers. denaturation temperature. Environ Microbiol 4:770 773 T Kämpfer P, Thummes K, Chu H-I, Tan C-C, Arun AB, Chen W-M, Lai Strain ptl-2 is capable of dinitrogen fixation, and can fix W-A, Shen F-T, Rekha PD, Young C-C (2008) Pseudacidovorax 274.52 nmol/mg/h of nitrogen. intermedius gen. nov., sp. nov., a novel nitrogen-fixing Thetypestrainisptl-2T (= CCUG 58759T,=DSM betaproteobacterium isolated from soil sequences. Int J Syst Evol – 24877T). G + C content of the bacteria is 67 mol %. 16S Microbiol 58:491 495 Kimura M (1980) A simple method for estimating evolutionary rates of rRNA and nifH sequence of strain Pseudacidovorax base substitutions through comparative studies of nucleotide se- T T austerolens ptl-2 sp. nov. (= CCUG 58759 =DSM quences. J Mol Evol 16:111–120 24877T) are FJ581042 and GQ249664, respectively. The type Öhlinger R (1996) Methods in soil physics; methods in soil chemistry. In: – strain ptl-2T was isolated from soil at Chamba, Himachal Schinner F (ed) Methods in soil biology. Springer, NY, pp 383 396 Paisley R (1996) MIS whole cell fatty acid analysis by gas chromatogra- Pradesh, India. phy training manual. MIDI, Newark Pal R, Bala S, Dadhwal M (2005) Hexachlorocyclohexane-degrading Acknowledgements Financial assistance from the Department of Bio- bacterial strains Sphingomonas paucimobilis B90A, UT26 and technology (BT/PR-12522/BCE/08/05/2009) is highly acknowledged. Sp+, having similar lin genes, represent three distinct species, Shivani is thankful to the Council of Scientific and Industrial Research, Sphingobium indicum sp. nov., Sphingobium japonicum sp. nov. New Delhi, for providing fellowship as JRF/SRF to her during this work. and Sphingobium francense sp. nov., and reclassification of We would like to thank SIAF-DST (Sophisticated Analytical Instrumen- [Sphingomonas] chungbukensis as Sphingobium chungbukense – tation Facility - Department of Science and Technology, Department of comb. nov. Int J Syst Evol Microbiol 55:1965 1972 Anatomy, AIIMS) for providing the electron microscopy facility. We are Pearson WR, Lipman DJ (1988) Improved tools for biological sequence – highly thankful Mr. Jean P. Euzeby (Ecole Nationale Veterinaire, comparison. Proc Natl Acad Sci 85:2444 2448 Toulelouse, France) for etymological advice. We are also thankful to Poly F, Monrozier LJ, Bally R (2001) Improvement in the RFLP proce- Dr. Sangeeta Pal, Division of Microbiology, IARI, New Delhi for her dure for studying the diversity of nifH genes in communities of kind help during experimentation. nitrogen fixers in soil. Res Microbiol 152:95–103 Prakash O, Verma M, Sharma P, Kumar M, Kumari K, Singh A, Kumari H, Jit S, Gupta K, Khanna M, Lal R (2007) Polyphasic approach of bacterial classification—an overview of recent advances. Indian J References Microbiol 47:98–108 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Alam S, Brailsford SR, Whiley RA, Beighton D (1999) PCR-based Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for methods for genotyping viridans group streptococci. J Clin DNA-DNA reassociation and 16S rRNA sequence analysis in Microbiol 37:2772–2776 the present species definition in bacteriology. Int J Syst Evol Alexander SK, Strete D, Niles MJ (2003) Laboratory exercises in organ- Microbiol 44:846–849 ismal molecular microbiology. The McGraw-Hill, pp 77–83 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG Arden-Jones MP, McCarthy AJ, Cross T (1979) Taxonomic and (1997) The CLUSTAL_X windows interface: flexible strategies Serological studies on Micropolyspora faeni and Micropolyspora for multiple sequence alignment aided by quality analysis tools. strains from soil bearing the specific epithet rectivirgula. J Gen Nucleic Acids Res 25:4876–4882 Microbiol 115:343–354 Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Burris RH (1972) Nitrogen fixation assay – methods and techniques. Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Methods Enzymol 24:415–431 Stackebrandt E, Starr MP, Truper HG (1987) International commit- Cashion P, Hodler-Franklin MA, McCully J, Franklin M (1977) A rapid tee on systematic bacteriology. Report of the ad hoc committee on method for base ratio determination of bacterial DNA. Anal reconciliation of approaches to bacterial systematics. Int J Syst Biochem 81:461–466 Bacteriol 37:463–464