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In a previous work, we demonstrated that the Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc. In this study, the orthologous genes from Mycobacterium smegmatis mc 2 155 were inactivated and mutants analyzed. Rv2358 protein was purified and found to bind upstream of the Rv2358 gene. Binding was inhibited by Zn 2؉ ions.The long-recognized phenomenon of nutritional immunity, in which the sequestration of iron and possibly other metals occurs as a nonspecific host response to infection (1), hints in general terms at the possibility of a keen competition between host and parasite for essential metal ions. Two major mechanisms play the prominent roles in governing metal ion homeostasis and resistance. The first one involves the uptake or efflux of specific heavy metal ions across biomembranes (15), while the second involves the specific chelation of metals by intracellular chaperons, e.g., metallothioneins (12). In prokaryotes, the expression of these genes is tightly controlled by specific "metal-sensing" transcriptional regulators (3, 5) clustered in four distinct families: Fur (4), DtxR (10), MerR (2), and SmtB/ArsR (3). In each family, representative members are dimers, with each subunit containing an amino-terminal helix-turn-helix DNA-binding domain and an adjacent metalbinding domain.Mycobacterium tuberculosis genome encodes two regulators of the Fur family (FurA and FurB), two regulators of the DtxR family (IdeR and SirR), three regulators of the MerR family (Rv1674c, Rv1994c, and Rv3334), and seven regulators of the SmtB/ArsR family (Rv0324, Rv0576, Rv2034, Rv2358, Rv2640c, Rv2642, and Rv3744) (http://genolist.pasteur.fr/TubercuList/). IdeR is the only one whose role has been well characterized. Like Fur and DtxR, IdeR binds iron and then interacts with a specific sequence in the operator region of iron-regulated genes to control their transcription (11, 13).The genetic linkage of furA and katG in all species of mycobacteria has suggested that FurA may control katG expression. Indeed, recent studies have demonstrated that it is a negative regulator of katG (18). Further, we showed that transcription of both genes is induced upon oxidative stress and that FurA negatively controls transcription of its own gene (7,14).In mycobacteria, the furB gene is located immediately downstream of the Rv2358 gene, encoding a putative regulator of the SmtB/ArsR family. Using Mycobacterium smegmatis as a model system, we found that the M. tuberculosis Rv2358 and furB genes are cotranscribed by a common promoter which is induced by zinc (8), thus suggesting that this operon is involved in zinc homeostasis. In this study, we report results on inactivation of Ms2358 (the Rv2358 ortholog) and furB genes in M. smegmatis and the in vitro interaction between the Rv2358 protein and a palindromic sequence located immediately upstream of the transcriptional start site of the Rv2358-furB operon.Ms2358 and furB knockout strains and zinc tolerance. To investigate the physiological role of these genes, we produced two M. sm...

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Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.

The nucleotide sequence and mechanism of action of a tetracycline resistance gene from Mycobacterium smegmatis were determined. Analysis of a 2.2-kb sequence fragment showed the presence of one open reading frame, designated tet(V), encoding a 419-amino-acid protein (molecular weight, 44,610) with at least 10 transmembrane domains. A database search showed that the gene is homologous to membrane-associated antibiotic efflux pump proteins but not to any known tetracycline efflux pumps. The steady-state accumulation level of tetracycline by M. smegmatisharboring a plasmid carrying the tet(V) gene was about fourfold lower than that of the parental strain. Furthermore, the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone blocked tetracycline efflux in deenergized cells. These results suggest that the tet(V) gene codes for a drug antiporter which uses the proton motive force for the active efflux of tetracycline. By primer-specific amplification the gene appears to be restricted toM. smegmatis and M. fortuitum.