Genome Analysis Of A Nitrogen Fixing Bacterium And The Study On Its WrbA Gene Function

In our previous study,we collected eucalyptus forest soil from Dongmen(Fusui,Chongzuo,Guangxi),and found a strong nitrogen fixing bacteria by using the nitrogen free medium.The bacteria were identificated by Biolog microbial identification system as Klebsiella oxytoca,so this strain was named KO108.Then its mutant library was sdtablished through the random insertion of transposon[mTn5gusA-pgfp21].In the process to build a symbiotic system of eucalyptus and nitrogen fixing bacteria,one of the mutant named MA can be stably attached to the root surface of eucalyptus.In previous studies,the sequence around the MA insertion site was identified by reverse PCR.In this study,the whole genome sequence of KO108 was sequenced and then annotated.The specific results have been uploaded to NCBI(accession:NZ_LQMR00000000).Consider the advice of the sequence company,the results of Biolog detection and the reference,this paper compared rpoB,gyrA,mdh,phoE,infB,nifH gene of several bacteria under the Klebsiella,Raoultella and Escherichia.Confirmed that the strain variable habitat of Klebsiella,and the strain was named as KV321.Sequence alignment confirmed the insertion site of MA,the KV321 insertional mutant strain,which encodes a tryptophan repressor binding protein(WrbA).This paper finishes the homologous comparison between the KV321 WrbA protein sequence and the similar protein sequence of other species,predicts the molecular weight,hydrophobicity,secondary and tertiary structure of this protein,lays a foundation for the future analysis of this protein.The results of Blast showed that the similarity of KV321 WrbA sequence and Escherichia coli WrbA sequence was 88%,due to Klebsiella variicola WrbA protein is rarely reported,this paper mainly uses the WrbA protein of Escherichia coli as a reference.In order to verify the function of WrbA,a complementary strain MA of mutant MB was successfully constructed by homologous recombination.Crude protein of KV321,MA and MB was extracted by osmotic shock method.The relative enzyme activity of KV321 crude protein in the quinone-NADH system reached 24.99,while the relative enzyme activity of MB crude protein in the same system xwas 17.16,and the same index of MA crude protein was only 7.19.For ferricyanide-NADH system,the relative enzyme activity of KV321 crude protein was 1.87,MA crude protein was 1.36,and MB crude protein was 2.41.For the quinone-NADPH system,the relative enzyme activity of KV321 crude protein was 10.75,MA crude protein was 5.16,and MB crude protein was 10.43.For ferricyanide-NADPH system,the relative enzyme activity of KV321 crude protein was 0.59,MA crude protein was 0.10,and MB crude protein was 0.41.The results were consistent with the results of Escherichia coli pure WrbA protein:the catalytic effect of WrbA protein on quinone was better than that of potassium ferricyanide,and the catalytic effect on NADH was higher than that of NADPH.This result confirms that the WrbA protein of Klebsiella is also a quinone oxidase and may play a role in responding to oxidative stress.In this study,the expression vector pEtW of KV321 WrbA protein was successfully constructed,and the expression of WrbA protein was induced.The zeta potential of KV321,MA and MB strains was measured,KV321 was-8.15mV,MA was-11.00mV,and MB was-7.53mV.The potential of mutant strain MA is lower than that of KV321 and MB.Because the surface potential of bacteria is closely related to the adhesion of bacteria,it is the first time that WrbA is related to the surface potential of bacteria and the adhesion of bacteria.But more evidence is needed to support this conclusion.