Bacterial Diversity In Different Aged Luzhou Laojiao Pit Muds And Determing The Taxonomy Of Four Bacterial Strains

In this paper,59purified strains were isolated form eight pid muds sample(Lj2, Lj4,Lj,8,Lj10, Lj18,Lj19, Lj22)collected from pit wall and the pit bottom in Luzhou Laojiao. BOX-AIR,16S rDNA PCR, PCR-RFLP, representative strains sequence analysis method were used to study the genetic diversity and phylogeny of the tested bacteria, and four putative new species were choosed. Through morphological, physiological and biochemical characteristics,16S rDNA, whole-cell fatty acid component, in combination with DNA homology analysis and methods of (G+C) mol%,2strains of bacillus and2strains of Paenibacillus’taxonomic status were established. The conclusions were as follows:(1) BOXAIR-PCR results showed that, at the level of66%similarity, all the strains got together in a group; there were8isolates grouped singly, and the others divided into9genetic groups at the similarity of80%. The results also showed that the genetic diversity was existed.(2) According to the results of BOXAIR-PCR, fifty four strains were chosen for16S rRNA PCR-RFLP analysis. The results showed that these strains were divided into eight genetic groups at95%of the similarity level, except fourteen strains grouped singly. All bacteria, isolated from Ij8together into two groups, the separation from Lj4, Lj10gathered into four groups, separation from Lj12, Lj19, Lj3gathered into three groups, separated from Lj22gathered into five groups, separated from Lj10gathered into seven groups.(3) Based on the results above,31representative strains were selected to do16S rRNA gene sequences analysis, and phylogenetic tree was coned by MEGA4.0software, the results suggested that the strains were belong to genus of bacillus and Paenibacillus, and Bacillus distributed in nine levels of branches, showed the abundant genetic diversity.(4) The polyphasic taxonomy technology is used to determine the classification of the four bacteria in pit mud. The details are as follows:Two novel spore-forming bacteria, strains LZ003and LZ033were isolated. Cells of both isolates were rod-shaped bacilli and each contained subterminal, ellipsoidal spores and in the intermediate ellipsoidal spores. Strains were facultatively anaerobic, catalase-positive, oxidase-positive and only LZ003motile with flagella. Major fatty acid found in the strain was anteiso-C15:0. The G+C contents of the DNA were43.70and43.50mol%.16S rRNA gene sequences placed them in the Paenibacillus, according to phylogenetic analysis. The type strains most closely related to LZ003were Paenibacillus favisporus LMG20987T, with16SrRNA gene sequence similarities of99.7%. Levels of DNA-DNA relatedness between strain LZ003and Paenibacillus favisporus LMG20987T and Paenibacillus azoreducens LMG21668T were49.43%and32.87%, respectively. The type strains most closely related to LZ033were Paenibacillus cookii LMG18419T, Paenibacillus chibensis9905T, with16S rRNA gene sequence similarities of99.3%and98.3%, respectively. Levels of DNA-DNA relatedness between strain LZ033and Paenibacillus cookii LMG18419T, Paenibacillus chibensis9905T were36.60%and23.13%, respectively.Two novel spore-forming bacteria, strains LZ013and LZ035were isolated. The G+C contents of the DNA were49.70and56.80mol%.16S rRNA gene sequences placed them in the bacillus, according to phylogenetic analysis. The type strains most closely related to LZ013were Bacillus sonorensis12232T, with16SrRNA gene sequence similarities of97.7%. Levels of DNA-DNA relatedness between strain LZ013and Bacillus sonorensis12232T and Bacillus licheniformis2505T were37.08%and36.04%, respectively. The type strains most closely related to LZ035were Bacillus coagulans2257T, with16SrRNA gene sequence similarities of99.1%. Levels of DNA-DNA relatedness between strain LZ035and Bacillus coagulans IAM2257T and Bacillus acidiproducens IAM14638T were48.39%and43.47%, respectively.