Transposon mutagenesis of Mycobacterium marinum identifies a locus linking pigmentation and intracellular survival

Abstract

Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood. Mycobacterium marinum has recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship to Mycobacterium tuberculosis and because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of the M. marinum model, we have developed efficient transposon mutagenesis of the organism by using a Drosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous to Rv2603c and Rv2604c of M. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation with M. tuberculosis genomic DNA encompassing Rv2603c to Rv2606c corrected the pigmentation and growth defects of the mutant. These data demonstrate the utility of mariner-based transposon mutagenesis of M. marinum and that M. marinum can be used to study the function of M. tuberculosis genes involved in intracellular survival and replication.

FIG. 1.

Map of the mariner transposon mutagenesis vector pM272B (A) and the M⁴ procedure (B). (B) Transposon insertion mutants were isolated by a two-step selection procedure, first for Kan^r and second for Kan^r and Suc^r. The numbers in parentheses indicate the frequencies of the occurrence of resistant colonies. Mm, M. marinum; Ec-ori, E. coli origin of replication; WT, wild type; RT, room temperature; sacB⁻, sacB deficient; mts-ori⁻, mts-ori deficient.

FIG. 2.

Pigmentation of M. marinum mutants in the presence or absence of light. M. marinum colonies were grown in the dark (left) and then exposed to light for 48 h (right). Wild-type (WT) M. marinum is pigmented only after light exposure. R01, MmR01; R02, MmR02; R03, MmR03; R04, MmR04; W01, MmW01; W03, MmW03; W04, MmW04.

FIG. 3.

M. marinum carotenoid biosynthesis and regulatory genes, their genomic organization, and location of transposon insertions. (A) The known carotenoid biosynthesis locus in M. marinum is shown as a gene cluster from crtI to ORF-4. Below these genes the biosynthetic pathway is depicted; GGPP represents the initial substrate, geranylgeranylpyrophosphate. The novel crtR and crtP genes encode a putative repressor and a positive regulator, respectively, for the carotenoid biosynthesis locus. crtP indicates the locus containing the Rv2606c to Rv2603c homologues. The star symbols represent putative light-inducible promoters. Triangles indicate sites of transposon insertion, and arrows represent the orientation of the Kan^r gene in the chromosome. R01, MmR01; R02, MmR02; R03, MmR03; R04, MmR04; W01, MmW01; W02, MmW02; W03, MmW03; W04, MmW04. (B) The location of transposon insertion in mutant MmR03 is shown relative to those in other pigmentation mutants as detected by a PCR that amplifies the genomic sequence between insertions. The distance of the transposon insertion in MmW04 from others in the carotenoid biosynthesis locus is insufficiently detected by PCR, and the results were not shown.

FIG. 4.

Examination of resistance to light induction of methylene blue (A) and intracellular replication within J774 macrophages (B) for M. marinum pigmentation mutants and the complemented strain. (A) Dilutions of M. marinum strains were plated onto 7H10 plates containing 25 μM methylene blue and incubated in the dark or in ambient light for 7 days. The data are percentages of CFU in the dark divided by CFU in ambient light for each strain. Data for the wild type (WT), three pigmentation mutants, and the complemented MmW04 strain are graphed. The data are means of results from two experiments under the same conditions. (B) CFU associated with J774 cells were enumerated at time zero and after 96 h of intracellular growth. Data for the same strains as those used for panel A are graphed. The means and standard deviations of results from quadruplicate experiments performed on two different days are depicted. pRv2603c-Rv2606c indicates complementation with M. tuberculosis genes encompassing Rv2603c to Rv2606c. Differences between MmW04 and the WT or the complemented strain are statistically significant (P < 0.05); the levels of growth of the WT and the complemented mutant are not different.

FIG. 5.

Complementation of carotenoid biosynthesis for M. marinum mutants. (A) A plasmid expressing the M. marinum crtB gene was introduced into both MmW01 (W01) and MmR02 (R02), but it restored pigment synthesis only in MmW01. (B) A plasmid expressing M. tuberculosis genes Rv2603c to Rv2606c was used to complement the MmW04 (W04) mutant to produce wild-type pigment. Both plates were exposed to light for 48 h prior to imaging.