| At forepart, most of myxobacteria were isolated from soil, so the myxobacteria were considered to be typical soil microorganisms and no myxobactrium could survive in seawater. But with the development of molecular biology, the existence of the bacterial phylotypes closely related to myxobacteria has been testified by phylogenetic analyses of 16S rDNA fragments amplified from marine samples, and more and more myxobacteria have been isolated from marine environments since 1998. The marine myxobacteria, Myxococcus fulvus HW-1 (ATCC BAA-855) isolated from a seaside sample by our lab has a variety of different characteristics compared with terrestrial myxobacteria, such as cellular density-independent growth and spherical cells characteristics and etc.Two-component signal transduction pathways (two-component system) widely exist in bacteria, and play a major role in the response of bacteria to osmotic stress by regulating gene expression. Also, two-component system has been found controlling gene expression of myxobacteria such as the regulation of growth, development, social motility and so on. Although there is no report about the two-component system involved in osmotic stress response, but we can assume that the two-component signal transduction pathways also play a key part in the adaptation of the marine environment by regulating the amount of some outer membrane proteins serving as channel proteins or transporters and try to prove the assumption through gene expression profile analysis.First, we analyzed the incompletely released genome sequences of Myxococcus xanthus DK1622 and predicted 9314 ORFs by using Glimmer. After deleting ORFs with non-biological characteristics, we finally obtained 7885 ORFs.Genes of two-component system, outer membrane proteins, periplasmic proteins and extracellualr proteins were predicted by using SignalP, TMHMM , PSORTb and SMART, and we selected 386 of them for microarray design, in which there are 233 two-component system ORFs, 82 outer membrane proteins, 20 extracellualr proteins and 51 periplasmic proteins. Using Array Designer v3.01, we finally designed the primers of 360 ORFs for further research, and among these ORFs: 74 ORFs for outer membrane proteins, 42 ORFs for periplasmic proteins, 18 ORFs for extracellualr proteins, of which the functions are all unknown and the rest 226 ORFs for two-component systems. 60 genes related to myxobacteria motility were also added to the microarray.DK1622 genome DNA served as template, and 411 target genes were successfully amplified as the probes of microarray. Subsequently, we made a microarray containing the 411 genes mentioned above, and testified its validity in gene expression profile analysis of M. Julvus HW-1 by hybridizing the genome DNA of M. xanthus DK1622 and M. Julvus HW-1.After confirming the best isolation conditions of RNA, we compared the gene expression profile of HW-1 growing under the marine environment with the one under freshwater environment, and found 94 differentially expressed genes. Under the marine environment, 10 genes were highly expressed, in which there were 6 two-component systems genes, 1 outer membrane protein, 2 extracellualr proteins and 1 periplasmic protein; under the freshwater environment, 84 genes were highly expressed, in which there were 29 two-component systems genes, 19 outer membrane proteins, 5 extracellualr proteins and 14 periplasmic proteins.Analyzing the predicted functions and the levels of differential expression of those genes, we picked up two genes (T105, OM031) closely related to the adaptation of marine environment, and confirmed the validation of microarray data by quantitative real-time RT-PCR . The authentic functions of the two genes will be testified in the mutants. At present, we have successfully constructed the knock-out vector, and the following work are in process. |
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