Physiological Characterization And Strain Identification Of A Microbe Isolated From An Extreme Environment

Microorganisms isolated from extreme conditions (including alkaline environments) have special characteristics due to their adaptation to special environmental conditions, and have become important bio-materials for scientific research in many aspects, such as isolation of new strains, cloning and application of extremoenzymes, characterization of adaptation mechanisms to the extreme environmental conditions and identification of novel biologically active substances, etc. Alkalophilic actinomycetes play very important roles among alkalophilic microbes because most of them possess complicated morphological differentiation and could accumulate a wide variety of secondary metabolites. HSL10, a microbe isolated from an alkaline lake in Xinjiang, was studied in the present work from several aspects as the following:1. Physiological characterization of HSL10:Firstly, we optimized solid media for HSL10 growth and selected TSA and GYM as the best growth media for HSL10. During the cultivation, this microbe was observed to accumulate yellow-pigments, probably related to the production of the secondary metabolism. Studies on the cultivating pH, temperature, NaCl-tolerance and sensitiveness to antibiotics showed that HSL10 was alkali-tolerant, mild halophilic, psychrotrophic and sensitive to two commonly used antibiotics—thiostrepton and chloramphenicol (the lowest selective concentrations were 10μg/mL for the former and 50μg/mL for the latter). The growth curve of HSL10 indicated that it grew very rapidly, reaching the stationary phase in 20 h during liquid cultivation.2. Bacterium identification of HSL10:Gram staining and electron microscope observation showed that HSL10 was short-rod-like Gram-positive bacterium and has no flagella. Cloning and sequencing analysis of the 16S rDNA revealed that HSL10 was Microbacterium oxydans belonging to actinomycetes and having 100% homology with the strain DSM 20578 isolated in normal terrestrial environment and stored in German Collection of Microorganisms and Cell Cultures (DSMZ). Like HSL10, we found that DSM 20578 was also alkali-tolerant and psychrotrophic, but these features have never been reported previously. In order to know whether these two strains isolated from different environments are identical species, we amplified and sequenced the Intergenic Spacer Region(ISR) of 16S rDNA-23S rDNA of HSL10 and DSM20578 by PCR respectively. Comparative analysis of the 16S-23S rDNA ISR from HSL10 and DSM 20578 revealed that some different and unique regions still exist in their respective sequences, indicating that these two strains are not identical.3. Genomic library construction of HSL10:In order to further clone and functionally characterize interesting genes located on the genome of HSL10,we constructed a genomic library of HSL10 through the extraction of genomic DNA, DNA end-repairing, purification of DNA fragments with proper size, ligation with the cosmid vector, packaging, transfection and screening recombinants.1920 recombinants were obtained, ensuring over 99% covering-probability of a given genome bacterium of HSL10. The quality of the resulted genomic library was tested by digestion of randomly-selected recombinant cosmids. Our data showed that the average size of insert DNAs on these cosmids is ca.40Kb, indicating that this genomic library was constructed successfully.4. Preliminary analysis of some interesting genes on the genome of HSL10:In order to study whether HSL10 has potential to accumulate natural products (whether there are some natural product synthesis genes on HSLIO genome, such as polyketide synthases, halidase, glycosyltransferase, glycosyl-synthase and so on), we used degenerate primers designed according to the conserved sequences of the interesting genes to amplify certain regions with HSL10 genome as template by PCR. Under the optimized PCR conditions and having positive controls in the reactions, no expected DNA fragments could be detected from HSL10 genome by PCR. In the experiment to test whether HSLIO has potential to accumulate extremoenzymes, we could not detect the cellulose gene by functional screening the genomic library of HSL10 or by PCR using degenerate primers and HSL10 genome as template. However, we found that HSLIO has obviousβ-galactosidase activity, but the inducer IPTG commonly used in E.coli could inhibit the activity of this enzyme to some extent, implying that the regulation system ofβ-galactosidase expression in HSL10 may be different from in E. coli.This work can set a foundation for further study of extremophiles HSL10.