The Role Of Melanin Production In The Survival Of Vibrio Cholerae In The Marine Environment

Abstract The marine bacterium Vibrio cholerae produces pyomelanin through the catabolism of L-tyrosine to homogentisic acid. Various types of melanins are used by microbes as defense mechanisms against a variety of environmental stresses. This thesis investigated the defensive role of V. cholerae pyomelanin against exogenous stresses, particularly hydrogen-peroxide (HzOz). A V. cholerae 569B melanin-deficient mutant was created by insertional inactivation of ppdA, which encodes for p-hydroxyphenyl pyruvate dioxygenase, with the aid of suicide vector pGP704 that contained an internal ppdA fragment. The melanin mutant was more sensitive than the wild type to the presence of 5 mM HzOz. Pyomelanin also protected melanised Escherichia coli expressing ppdA from the low copy number plasmid pAC Mel against HzOz. Although both microorganisms showed greater resistance to H20 2, the melanised bacteria always resulted in lower bacterial numbers (in the absence of HzOz) in comparison to non-melanised V. choLerae and E. coli cultures. Complementation of the ppdA mutation with ppdA expressed from the high copy number plasmid pCM302-16 did not restore resistance to H20 Z, but instead resulted in enhanced sensitivity to H20 2. Increased sensitivity also occurred in V. cholerae wild type and E. coli cells when melanin was produced in these strains from plasmid pCM302-16. The decline in the cell number of melanised cultures, together with the enhanced bactericidal effect of H20 2 on bacterial strains overproducing melanin, suggested that a by-product of melanin production was detrimental to cellular fitness and enhance·d killing by exogenous H20. It is known from published literature that HZ0 2 is formed during the autopolymerization of homogentisic acid, and that it causes oxidative DNA damage. However, we were unable to demonstrate DNA damage in melanised cultures using alkaline agarose gel electrophoresis. The V. choLerae ppdA mutant was more sensitive than the wild type strain to copper chloride, but not ferrisulphate. In contrast, V. choLerae 569B was more sensitive to silver nitrate and sodium hypochlorite than the ppdA mutant. The role of melanin as an antioxidant metabolite was further investigated in a V. choLerae strain that lacked the catalase-peroxidase PerA. PerA was found to be a heat-labile protein, 724 amino acids in length, and the sole catalase produced by V. cholerae 569B during the stationary phase of growth. The perA gene was isolated from a V. cholerae 569B genebank and used to insertionally inactivate perA. The perA V. cholerae mutant was more sensitive to 1 mM H20 2 compared to the wild type strain when the cells were either in log phase or were twenty-four hours old. In contrast, melanised V. cholerae perA mutant cultures, i.e. two day old cultures grown under melanininducing conditions, exhibited increased sensitivity to I mM H20 Z in comparison to the nona A we were not mutant was {'r,,"->t,'rI mutant was more of mutation to to V. It is known mutant was more mutant. was more 'UU'JH"" was was to by V. cholerae a V. cholerae mutant was more to 1 a V. to to against was of non-melanised perA mutant cultures. In addition, we observed a severe decline in melanised perA mutant cell numbers, even in the absence of exogenous H20 2, suggesting that H20 2 is indeed a byproduct of melanization in V. cholerae. We conclude that melanin protects V. cholerae against H20 2 only when homogentisic acid IS produced at physiological levels, that H20 2 is formed during melanogenesis and that PerA IS required for the removal of the H20 2 formed during melanogenesis. Finally, melanin protects V. cholerae against some, but not all, exogenous stresses.