Many environmental microbes produce melanins, and melanin pigments are also an inherent phenotype of a broad range of eukaryotic microorganisms. The melanin in these strains may confer resistance to unfavorable environmental factors, host immunity, and even play a role in virulence expression. Therefore, melanin may confer a survival advantage on these natural pigment-producing V. cholerae strains in the estuary niche, and pathogenicity in the host.
Previously, V. cholerae strains with a pigmented phenotype were induced under stress or by chemical mutagenesis. In this study, we describe certain O139 and O1 isolates that can produce pigment under normal experimental growth conditions. Though the mutations in these O1 and O139 pigment-producing strains are different, both of them involve the dysfunction of HGO, the product of the VC1345 gene of V. cholerae. In our study, gene complementation of the mutant VC1345 confirmed the role of its dysfunction in pigment production. As a consequence, the disruption of the balance between the enzymes encoded by VC1344 and VC1345 causes homogentisate accumulation and spontaneous oxidation. The pigment production mechanism in these wild-type strains is same as in the chemically induced pigmented mutants [15, 18].
With the exception of the 10-bp deletion in the VC1345 gene of the El Tor strain 3182, which appears to be a random mutation, it is interesting that the same 15-bp deletion in the VC1345 gene occurs in all the studied O139 pigment-producing strains, despite the fact that these strains were isolated over a period spanning the years 1995 to 2006. The deletion boundaries contain short direct repeats; therefore, it is possible that these commonly occurring recombinations gave rise to the mutant strains. It is not clear, however, how the 15-bp fragment affects the activity of the HGO enzyme. In the crystal structure of human HGO , the homologous amino acid residues encoded by this 15 bp form a small turn in the protein surface. Although it is not included in the predicted active sites or the 20 missense mutations that have been identified in the HGO from AKU patients , structural change in this mutant protein could be assumed.
The genes VC1344, VC1345, VC1346, and VC1347 comprise an operon, and the products of all four genes are predicted to be involved in tyrosine catabolism. The nucleotide and amino acid sequence variations in these genes are, however, inconsistent; VC1344 is highly conserved, although its nucleotide sequence varies among the different strains, only a single amino acid residue difference is present at the protein level, which suggests that it plays an important role in the tyrosine pathway, and is conserved despite undergoing different stress selections. In contrast, VC1345 is considerably more variable, and different deletion mutations result in dysfunction of its product. This suggests that the accumulation of homogentisate, and the subsequent melanin production instead of complete decomposition of the amino acid in the routine pathway, may have survival benefits for the mutants in certain specific environments, thus the mutations will be retained. Variation and even dysfunction of the VC1345 product may shift the metabolic production of tyrosine and produce strains that are adapted to surviving in rigorous environments.
It is also interesting that the molecular types of the O139 pigment strains are indistinguishable or quite similar, suggesting the high clonality of these strains, even though they were obtained over a span of at least 12 years and from different regions. They have the same mutation in the tyrosine metabolism pathway. Additionally, compared to the high variance of the VC1344 to VC1347 genes, the sequences in all the six O139 pigment-producing strains were highly consistent. These data suggest that the O139 pigment-producing strains originate from one distinctive clone. The wide distribution of such strains in the environment may suggest their survival advantage. The signature of the 15-bp deletion within the homogentisate 1,2-dioxygenase gene (VC1345) in the O139 pigmented strains, or the mutation of VC1345 in the melanin-producing strains of V. cholerae when the El Tor biotype is included, is one of the biomarkers for the pigment-producing strains, and may be of significance in biological research and epidemiological tracing.
We observed that the nontoxigenic O139 pigment-producing strains exhibited a rb4 ribotype. In our previous study, the rb4 isolates were cholera toxin gene-negative O139 strains, and this ribotype is clearly different from the other patterns of the toxigenic O139 strains that are cholera toxin gene positive . All of the rb4 strains were isolated from patients, and an unknown pathogenic mechanism is presumed . Though the O139 pigment-producing strains examined in this study were isolated from environmental water samples, their possible pathogenicity should not be excluded, particularly since such strains are isolated successively in some years. The study showed that the pigment-producing strain expressed more toxin-coregulated pilus and cholera toxin, by possibly mechanism which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide . Strain 3182 is the toxigenic strain associated with the seventh pandemic, and it is speculated that this strain is more virulent than other strains on account of its pigment production, based on its role in V. cholerae virulence factor expression .