Isolation And Identification Of Nitrogen-fixing Bacilli And Tentative Study Of GlnB Gene

In this study, 5 rhizosphere soil samples of wheat, maize, ryegrasses and willow tree were collected from campus of China Agricultural University and Beijing chaoyangqu, respectively. A total of 29 isolates were selectively obtained from these rhizosphere soli samples based on their growth on nitrogen-free medium and their resistance to 100℃ for 10-15 min. PCR amplification using 2 degenerate primers for nitrogenase Fe protein gene was performed with chromosomal DNA isolated from the 29 isolates. The result suggested that a nifH amplicon of 323 nucleotides was detected in 7 isolates and the 7 isolates are C4 C5 G1 G2, W5 T1 and T7 . These PCR amplified fragments were cloned, and sequenced. Translated PCR sequences were aligned with NifH sequences from other proteobacteria, revealing high conservation. The NifH sequence similarity between isolates C5, Tl, W5, G2 and Klebsiella pneumoniae were 97-98% , respectively. The NifH sequence similarity between isolates C4, G1 and Azospirillum lipoferum, Azospirillum brasilense, Burkholderia brasilensis were 91-100% , respectively. The NifH sequence similarity between isolates T7 and Paenibacillus odorifer was 97%. The 7 isolates with nifli gene were tested for their nitrogenase activity using acetylene reduction assay (ARA). Isolates C4, G2, T7 and W5 showed nitrogenase activity, whereas nitrogenase activity was not detected in the isolates C5, Gl and T1 and suitable conditions for demonstrating nitrogenase activity for them remain to be found.The 7 isolates were identified based on physiological and biochemical characterization, 16S rDNA sequence, G+C mol% and DNA-DNA hybridization. Isolate Tl was identified as Bacillus cereus. Isolate Gl, C4 and C5 were identified as Bacillus megaterium. Isolate W5 and G2 had high similarity with Bacillus marisflavi and Paenibacillus polymyxa, respectively, and their taxonomic positions need to be determined in the future. Although the 16S rDNA sequence similarity of isolate T7 with Paenibacillus illinoisensis, Paenibacillus pabuli and Paenibacillus amylolyticus was 94%, 95% and 94%, respectively, DNA relatedness between isolate T7 and P. illinoisensis, P. pabuli and P. amylolyticus was 22.32%, 9.14% and 50.62%, respectively. Isolate T7 also had some unique physiological and biochemical characters. Thus isolate T7 may represent a novel species of Paenibacillus and the name Paenibacillus salicis sp. nov. was proposed.glnB gene encoding PII protein were obtained from G2 and Tl by PCR method using 2 degenerate primers. The deduced amino acid similarity of PII among G2, Tl, E. coli, and B. subtilis et al. were 69.47%. Southern blotting showed that there may be 2 copies of glnB genes in the DNA genome of G2.