Regulatory Mechanisms Underlying The Expression Of The Chaperone/Usher-like Pathway In Myxococcus Xanthus

Objective:The chaperone-usher (CU) pathway is the most representative pili assembly pathway which is used to secret some proteins to the cell surface. The CU pathway can be classified into three families:classical, alternate and archaic which is the most widely distributed CU family. In our previous work, we have confirmed that the mcuABCD (i.e. MXAN3885-3882) gene cluster of Myxococcus xanthus encodes a member of the archaic CU pathway and the mcuABC genes are located in the same operon. In addition, we have already confirmed the structure and functional mechanism of mcuABC operon and identified an upstream regulatory gene--MXAN2872. In this study we intend to screen more regulatory genes of mcuABC and do some further research upon MXAN2872. This study will help us to find new components of regulatory network of the CU pathway expression in M.xanthus and lay the foundation for revealing the regulatory mechanism of archaic CU pathway. It can also provide us some new ideas to block the biogenesis of adhesive pathogenic factors on cell surfaces by the archaic CU pathway.Method:1. Insert the transposon mariner(KmR) into the transcriptional fusion strain DK1622/pMP-MXAN3883 by electroporation and screen the upstream regulatory genes of mcuABC by blue-white spot on the nutritional deficiency media TPM containing X-gal,then locate the transposon insertion site by sequencing.2. Confirm the expression map of the candidate regulatory gene in DK1622 strain and the effect of the candidate regulatory gene deletion on mcuABC expression by reporter gene and Western blot.3. Explore the effects of the putative regulatory gene on mcuABC expression and multicellular development by gene in-frame deletion and site-directed mutation.4. Explore the possible relationship between the candidate regulatory gene and sulfur metabolism by sulfate deficiency.5. Do some further research about MXAN2872 biochemical characteristics by the way of site-directed mutation and UV-visible spectroscopy with the his-tagged MXAN2872 protein purified by Ni2+-NTA.Results:1. We screened a transposon mutant in which the mcuABC expression is abnormal and western blot result indicated the McuA protein expression in this mutant is later than wild type cell. And the transposon insertion site is located at MXAN3487.2. According to the β-galactosidase activity determination we confirmed the MXAN3487 expression in wild type cell DK1622 begins after 12h induced on development plate and it reaches the peak at 48h,and then its expression level begins to decrease; The mcuABC expression level in AMXAN3487 strain was obviously lower than in wild type and it does not express until induced for 24h which is much later than it in wild type.3. The phenotype observation showed us delayed multicellular development process and abnormal fruiting bodies.Western bolt suggested that the McuA protein in AMXAN3487 strain was detectable until the developmental time point at 72h.4. We found the regulatory role of MXAN3487 needs the existence of conserved P-loop (phosphate binding loop) through site-directed mutation on the basis of bioinformatics prediction.5. The McuA expression in ΔMXAN3487 and DK1622 strains were not affected by sulfate deficiency; the expression map of MXAN3487 was not affected when the cells induced on development plate without SO42-.6.Site-directed mutation upon the conserved histidine of the Baeyer-Villiger monooxygenases (BVMOs)-identifying sequence in MXAN2872 resulted the delayed fruiting body development process as well as the McuA expression.7. The UV-visible absorbance spectra suggested MXAN2872 could bind FAD and NADPH.Conclusion:1. We screened an upstream positive regulatory gene MXAN3487 which could regulate the CU pathway expression and multicellular development.2. The conserved P-loop is an essential element for MXAN3487 to play its regulatory role upon CU pathway and this suggests MXAN3487 is likely to encode a nucleotide-binding enzyme.3. Although the expressions of CU pathway and MXAN3487 were not affected by the lack of sulfate, it can not be ruled out that MXAN3487 may be involved in the sulfur metabolism during the multicellular development period.4. MXAN2872 probably belongs to the BVMOs and its conserved BVMO-identifying sequence motif is indispensable for its regulatory role while its Rossmann fold structure is needed for binding FAD.