Preliminary Study On Duplicated PilA Genes' Function And Function Mechanism Of HGT Hdsp Gene

Analysis of myxobacterial genomic sequence showed that there were often duplicated genes in myxobacterial genome. Duplicated genes were reported that could be helpful in adjusting myxobacteria to its complicated social behavior. PilA gene could encode pilin protein, which is the subunit of type four pili (TFP) and TFP was reported as motility apparatus of social gliding motility. In Myxococcus xanthus DK1622, there was only one pilA gene while we found that there were multicopy pilAs in S.aurantiaca genome and S.cellulosum genome. In this resarch, we studied the function of four pilA genes in S.aurantiaca 17044 genome. Bioinformatics analysis indicated that three-dimensional structure of the four pilin protein were similar, but their C terminal sequences were highly variabled in contrast to conserved N terminal. We transferred each pilA gene of S.aurantiaca 17044 into M.xanthus DK10410 (△pilA) respectively by using E.coli-M.xanthus shuttle vector pZJY41. Reverse-transcription PCR (RT-PCR) and Western blot results showed that the four pilA genes transcribed, translated and assembled into TFP in each anaplerosis strain. Characteristics analysis showed that exopolysaccharides (EPS) production and social gliding motility of the four anaplerosis strains do not change greatly compared to DK10410. Interestingly, when food is exhasusted, the strain YL1101, which contains pilA-1 gene of S.aurantiaca 17044, can form separate and mature fruiting bodies compared to DK10410, of which fruiting bodies are enlongated and immature. In addition, sporulation rate of YL1101 is also higher than the other three anaplerosis strains. When mixed with DK1622, YL1101 recognized it as non-self but YL1102, YL1103 and YL1104 could not. To surprise, when incubated with S.aurantiaca 17044, strain YL1102, YL1103 and YL1104 also could form fruiting bodies. We presumed that certain components (such as EPS, etc.) in S.aurantiaca 17044, may interact with TFP of YL1102, YL1103, YL1104 and stimulate these strains to form fruiting bodies when food is exhasusted. In summary, we found that there were duplicated pilAs in genome of S.aurantiaca 17044, showed that function of these pilAs were not same and manifested pilA-1 played an important role in myxobactirial development and reorganization.Hdsp, which was reported as a horizontally transferred gene required for social bebaviors in salt-tolerant Myxococcus fulvus HW-1, did not exist in terrestrial myxococcus such as M.xanthus DK1622, FB and so on. YLH0401, of which hdsp gene was inserted by pMiniHimarl-lacZ in previous research, reduced EPS production and social gliding motility obviously. Moreover, YLH0401 could not form fruiting bodies and sporulate in TPM plates. When inoculated in liquid CTT medium, YLH0401 grew dispersed while HW-1 aggregated.Considering no genetic manipulation could carried to HW-1, we integrated hdsp gene into genome of M.xanthus DK1622 by using vector pSWU30. RT-PCR result showed that hdsp gene could properly expressed in DK1622. Characteristics analysis results indicated that DK1622 containing hdsp gene increased EPS production compared to wild type. Considering characteristics'changes of YLH0401, we speculated that hdsp positively regulated EPS production of myxococcus.Bioinformatics analysis showed that Hdsp protein contained four dispersed tetratricopeptide repeat domain (TPR domain). To study function mechanism of Hdsp in M.fulvus HW-1, we amplified the gene fragment which only encoding the four TPR domains of Hdsp and obtained truncated Hdsp protein in E.coli BL21 (DE3). By using truncated Hdsp as a bait and via GST-Pulldown protocol followed by LC-MS, we obtained proteins that interacted with Hdsp in M.fulvus HW-1. During these molecules, certain proteins which function in EPS biosynthesis and export were found. This study initially manifested function mechanism of hdsp gene in M.fulvus HW-1.