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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 hybridi- zation 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.
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