A crtB homolog essential for photochromogenicity in Mycobacterium marinum: isolation, characterization, and gene disruption via homologous recombination

Abstract

A gene essential for light-induced pigment production was isolated from the photochromogen Mycobacterium marinum by heterologous complementation of an M. marinum cosmid library in the nonchromogen Mycobacterium smegmatis. This gene is part of an operon and homologous to the Streptomyces griseus and Myxococcus xanthus crtB genes encoding phytoene synthase. Gene replacement at this locus was achieved via homologous recombination, demonstrating that its expression is essential for photochromogenicity. The ease of targeted gene disruption in this pathogenic Mycobacterium allows for the dissection of the molecular basis of mycobacterial pathogenesis.Mycobacterium marinum is a relatively rapidly growing animal-and human-pathogenic organism that is photochromogenic (4, 6, 10, 22, 30, 42a, 44, 46). When grown in the dark, M. marinum colonies are white, but upon exposure to visible light, they turn bright yellow (42a). The principal pigment responsible for this coloration is a beta-carotene, and the photoreceptor for the photoinduction process is likely a porphyrin (6, 36). Carotenoids are thought to protect cells against lethal photooxidative damage by quenching singlet oxygen and harmful radicals formed upon illumination (14, 54). Indeed, this has been shown to be the case for M. marinum, which is protected from lethal photooxidation by the presence of its pigment (36).There is evidence that the color is the result of a photoinduced synthesis of pigments in living cells rather than the photooxidation of a preformed compound (4). Unpigmented M. marinum killed by heat or UV light is incapable of developing pigment upon light exposure. Similarly, alcohol extracts from M. marinum grown in the dark do not turn yellow upon exposure to light. Also, the addition of chloramphenicol (an inhibitor of protein synthesis) to live cells inhibits light-induced pigment synthesis, suggesting that these pigments are not preformed (46,47). Taken together, these data suggest that interaction of the photoreceptor with light causes either the activation or the derepression of the gene(s) encoding the enzymatic machinery involved in carotenoid synthesis.The studies described above, coupled with those demonstrating that the light-induced carotenoid pigments of Myxococcus xanthus are controlled by a light-induced promoter (20,39,49), led us to examine the genetic basis of pigment formation in M. marinum. The isolation of the locus responsible for photochromogenicity in M. marinum was facilitated by the ability to transfer this phenotype to Mycobacterium smegmatis, a nonchromogenic mycobacterial species. A gene encoding a phytoene synthase homolog was found to be necessary for pigment formation. The easily discernible phenotype resulting from the expression of this gene made it an ideal locus to develop and test the feasibility of gene replacement via homologous recombination in this pathogenic Mycobacterium species. MATERIALS AND METHODSBacterial strains and growth conditions. M. marinum M (43) and M. smegmatis mc 2 155 (52) were used. M. marin...

Evolution of Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria from a Common Mycobacterium marinum Progenitor

“…For example, crtI encodes phytoene dehydrogenase, an enzyme essential for the production of carotenoid pigments in M. marinum (25). M. ulcerans Agy99 has the same crt locus as M. marinum, but it is nonpigmented, and this has been explained by a point mutation in crtI that introduces a premature stop codon and truncates the gene (see Table S4 in the supplemental material) (34).…”

Mycobacterium smegmatis whmD and its homologue Mycobacterium tuberculosis whiB2 are functionally equivalent

“…The gfp gene was then PCR-amplified from the vector pFPV27 (Ramakrishnan et al, 2000) using the primers gfpDatg-F (59-CGG-GGTACCTCTAAAGGTGAAGAAT-39) and gfp27-R (59-ACATGCA-TGCTTATTTGTACAATTCATC-39). The whmD fragment digested with EcoRV/KpnI was then ligated to KpnI/SphI-digested gfp amplified as above and EcoRV/SphI-digested pFPV27 in a three-way ligation.…”

“…However, the septation defect and filamentation observed in M. smegmatis cells lacking WhmD led us to hypothesize that WhmD perhaps plays a structural role in cell division by localizing to developing septa. To determine its subcellular localization, whmD was fused to the gene encoding green fluorescent protein (Gfp), in the vector pFPV27 (Ramakrishnan et al, 2000), with the fusion protein being expressed from the whmD promoter. A construct carrying the full-length whmD gene, and the empty vector, were used as negative controls.…”

“…WhmD belongs to the growing WhiB-like family of proteins shown to be involved in the regulation of significant cellular processes such as cell division , pathogenesis (Ramakrishnan et al, 2000;Steyn et al, 2002), antibiotic resistance (Morris et al, 2005) and response to oxidative stress (Kim et al, 2005). Each WhiB-like protein contains four invariant cysteine residues and is relatively short (76-139 residues).…”

Pigments and Pathogenesis

“…Pigment production may aid in the resistance to acidity and help these mycobacteria survive. The promoter driving expression of carotenoid synthesis genes has regions that respond transcriptionally to exposure to light in Mycobacterium marinum [22,23]. In M. smegmatis, exposure to acidity transcriptionally upregulates the homologue of this promoter region [17].…”