Taxonomic Analysis Of Three Novel Bacteria And Cloning And Expression Of A Novel Heat-resisting Agarase

As the development of society and economy, terrestrial resources have been more and more exhausted. People turned their attention to the oceans which accounted for 70 percent of the total area of the earth for the further development of human beings. The exploration and utilization of marine resources become a theme of the national strategic development. The oceans are the biggest habitat for various organisms, especially the marine microorganisms which contains a great number of species.They play an important role in the ocean exploration and utilization for its unique metabolic mechanism formed under extreme environments. Therefore, it is valuable both theoretically and practically to analyse the ecological, physiological and genetic characteristics of marine microbes, as well as the bioactive substances produced by them. On the basis of the previous research about the diversity of bacteria, we studied the marine microbial resources from two aspects, the taxonomy of novel bacteria and the analysis of their bioactive products.A novel Gram-negative, strictly aerobic, agar-hydrolyzing bacterium designated YM01T isolated from the Yellow Sea of coastal region of Qingdao was studied using polyphasic approach. Cells were rod-shaped, peritrichously flagellated and formed long chains by end-to-end. This isolate absolutely required Na⁺ ions but not sea water for growth. YM01^T was able to hydrolyze cellulose, starch, aesculin and Tween 80, but not egg yolk, gelatin, urea and casein. It contained menaquinone 7 as the predominant isoprenoid quinone and C_{16 : 0} (38.3 %), C_{16 : 1}ω7c and/or iso-C_{15 : 0} 2-OH (29.0 %), C_{18 : 1}ω7c (9.3 %) and C_{10 : 0} 3-OH (8.2 %) as the major cellular fatty acids. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and aminophospholipid (PN) were the major constituents of the phospholipids. The DNA G+C content was 44.8 mol%. 16S rDNA sequence analysis demonstrated that this isolate was unique, showing only <91.0 % sequence similarities to all published species and formed a distinct monophyletic clade closely related to the species of the family Alteromonadaceae within the group of Alteromonas-like Gammaproteobacteria. So, strain YM01T was considered to represent a novel genus and species in the Gammaproteobacteria, for which the name Catenovulum agarivorans gen. nov. sp. nov. is proposed.Another psychrotolerant (growth occurs at 0–33℃) , moderately halophilic (optimal NaCl concentration of 7–8 %) and obligately heterotrophic marine bacterium, designated ZS2-25 28^T, was isolated from intertidal sediment samples collected from the coastal regions of the Chinese Antarctic Zhongshan Station on the Larsemann Hills. phylogenetic analysis based on 16S rDNA sequences indicated that strain ZS2-28^T formed a distinct evolutionary lineage within the clade of genera Roseibaca, Roseinatronobacter and Rhodobaca of the Alphaproteobacteria with only <91.0 % sequence similarities to those neighbors. This isolate contained bacteriochlorophyll a. Poly-β-hydroxy butyrate accumulation and slime production can be observed. It contained Q-10 as the predominant isoprenoid quinone and C_{18 : 1}ω7c as the major cellular fatty acids. The main polar lipids were phosphatidylglycerol, phospatidylethanolamine, phosphatidylcholine and an unidentified aminolipid. The G+C content of genomic DNA was 63.3 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain ZS2-28^T was considered to represent a novel genus and species, for which the name Roseicitreum antarcticum gen. nov. sp. nov. is proposed.A novel bacterial strain, designated MY15^T, was isolated from human saliva. Phylogenetic analyses based on 16S rDNA gene sequences showed that the novel strain was most closely related to Myroides marinus JS-08^T, Myroides odoratimimus LMG 4092^T and Myroides profundi D25^T with 96.5 %, 96.3 % and 96.1 % sequence similarities, respectively. Cells were Gram-straining-negative, rod-shaped, lack flagella and non-motile. Flexirubin-type pigment is absent. This isolate contained menaquinone 6 as the major respiratory quinone, and C_15:0 iso (51.2 %), C_17:0 iso 3-OH (12.9 %) and C_13:0 iso (10.5 %) as the dominant fatty acids. The G+C content of the DNA was 34.3 mol%. On the basis of this polyphasic study, strain MY15^T represents a novel species of the genus Myroides, for which the name Myroides phaeus sp. nov. is proposed.On the basis of taxonomic study of Catenovulum agarivorans YM01^T, further research about the agar-hydrolyzing enzymes was performed. The results showed that this isolate produced a high temperature- and proteinase K- resistant agarase. After treatment at 100 and 90℃for 120 and 240 min respectively, the agarase remained the agar-hydrolyzing capacity for 33.3 and 44.4 %. In addition, one of the genes for the expression of agarase, aga A, was cloned by using degenerate PCR and hiTail-PCR. Aga A contained 781 amino acids and the theoretical molecular weight was 89 kDa. According to the result of SignalP analysis, aga A contained a 51 aa signal peptide. The conserved domain analysis revealed that aga A is a novel agarase belonging to the family glycoside hydrolase-42.