Screening And Identifying Regulatory Factors Of The Chaperone/Usher Pathway In Myxococcus Xanthus

Objective:Chaperone/usher pathway is the most widespread and representative pathway of assembling pilus so far. CU systems can be classified into three families:classical, alternate and archaic. Among the three families, the archaic CU system is distributed most widely, with representatives being present in many important pathogenic and environmental microbes. Myxococcus xanthus is a Gram-negative bacterium characterized by social behaviour and a complex developmental cycle, so it is a favorable model of studying prokaryotic development. Our previous work revealed that the archaic CU gene cluster mcuABCD (MXAN3885-3882, mcuABC constitutes an operon) of Myxococcus xanthus functions in spore coat protein secretion, suggesting that certain spore coats are evolutionarily related to most pili of Gram-negetive bacteria. c-di-GMP is an important second messenger among bacteria, playing role in many physiological activities. This thesis aims at showing the relationship between genes may related to c-di-GMP and Mcu, consummating the regulation of archaic CU system, and providing a new way to block the synthesis of adhesive pathogenic factor on cell surface.Method:1. Detect expression of genes may related to c-di-GMP metabolism during the development in Myxococcus xanthus by RT-PCR;2. Analyse the expression profile of expressive genes MXAN2424 and MXAN5199 by report gene lacZ during development;3. Construct of MXAN2424 and MXAN5199 in-frame deletion mutants by homologous recombination;4. Detect the expression of mcuABC in wild type and in-frame deletion mutants by reporter gene、Western blot and so on and anslyse whether MXAN2424 or MXAN5199 is necessary for the expression of mcuABC; observe the developmental phenotype of the in-frame deletion mutants;5. Quantify c-di-GMP in wild type and in-frame deletion mutants by HPLC, combining with the proof of site-directed mutagenesis、genetic complementation and enzymatic analysis, and ascertain preliminary whether MXAN2424 and MXAN5199 are c-di-GMP metabolic enzymes.Results:1.MXAN2424、MXAN2807、MXAN3705、MXAN4232、MXAN4675、MXAN5199 and MXAN5340 are expressive during development;2. MXAN2424 and MXAN5199 are expressive during vegetative stage and developmental stage;3. Construct of MXAN2424 and MXAN5199 in-frame deletion mutants (△MXAN2424 and △MXAN5199) succsssfully, and the expression of mcuA is delayed in △MXAN2424 and AMXAN5199; on the phenotype of development, cell aggregation in △MXAN2424 is no difference from wild type, but in △MXAN5199 is delayed about 18 h, and the time of cell aggregation is close to that of mcuA expression;4. c-di-GMP lever in △MXAN2424 is higher than wild type, but in △MXAN5199 is lower at 18 h and 24 h during development, suggesting MXAN2424 and MXAN5199 may posses PDE (phosphodiesterase) and DGC (diguanylate cyclase) activity, respectively;5. Purified His-tagged MXAN2424 protein can hydrolyze bis-pNPP, a synthetic substrate of PDE, E.coli yahA, a well known PDE encoding gene, can partially complement the mcuA expression deficiency, suggesting MXAN2424 may be PDE further and affect Mcu by c-di-GMP content; but mcuA expression does not change when conserved amino acid(E59) is replaced, so, we speculate that it has a small effect when change a amino acid or Glu is not in active center of enzyme;6. mcuA expression does not change when conserved amino acid(E221) is replaced, so, we speculate that it has a small effect when change a amino acid or Glu is not in active center of enzyme; or in spite of MXAN5199 is DGC, its regulation does not rely on c-di-GMP lever and it may have other function; when the conserved amino acid(R55) of FHA is replaced, mcuA expression is delayed, showing the importance of FHA.Conclusion:1. There are a few genes whose products predicted to be involved in c-di-GMP metabolism are expressed during M. xanthus development;2. MXAN2424 and MXAN5199 are necessary for the expression of M. xanthus CU system; in addition, MXAN5199 is necessary for M. xanthus development;3. MXAN2424 is a PDE, and partly regulates mcu expression by altering intracellular c-di-GMP level;4. MXAN5199 might have DGC activity, and it may affect mcu expression through its regulatory effect on M. xanthus development. It remains unknown whether the DGC activity of MXAN5199 is necessary for regulating M. xanthus development and consequently the expression of Mcu pathway in this bacteria.