V cholerae has been proposed to be a useful prokaryotic model of

V. cholerae has been proposed to be a useful prokaryotic model of alterations in L-tyrosine catabolism and has been used to study the molecular basis of diseases related to L-tyrosine catabolism [15]. However, to date, all the research on melanogenesis in V. cholerae has been based on chemically

induced mutants or mutants generated using transposons. During our cholera surveillance, some O139 and O1 strains that produced soluble brown pigments were isolated from environmental water samples and patients. Unusually, these strains can produce pigment under the normally used experimental growth conditions [Luria-Bertani (LB) nutrient agar or broth without temperature limitation].

Using transposon Vorinostat order mutagenesis, we determined that the p-hydroxyphenylpyruvate dioxygenase (HPD; VC1344 in the N16961 genome) in the tyrosine catabolic pathway was responsible for the pigment production in these strains [24]. Further, the three genes in a cluster mTOR inhibitor downstream of VC1344 were found to correspond to the other three enzymes involved in tyrosine catabolism [23, 24]. In this study, we analyzed the sequence variance of the four genes involved SB-3CT in tyrosine catabolism and the functions of the mutant genes to determine the possible mechanism of pigment production in these

isolates. We also found a close relationship of clonality among these strains, even though they were isolated in different years and from different areas. The potentiality of clone selection and pathogenicity of such strains should be considered. 2. Methods 2.1 Strains In this study, 22 V. cholerae O1 and O139 toxigenic and nontoxigenic strains were used (Table 1). Among these isolates, 95-4, 98-200, JX2006135, JX2006136, JX2006175, GD200101012, and 3182 are pigment-producing strains. These strains were isolated in different years and from different provinces of China. The El Tor strain 3182 was isolated from patients and the other six O139 strains were isolated from environmental water. In addition to the reference strains, including N16961, 569B, and MO45, the controls included other non-pigment-producing strains that were isolated in the same province or at the same time as the pigmented strains. Strains were cultured in LB liquid medium shaking at 37°C or on LB agar plates (1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 1.5% agar).

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