Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis

Santangelo, María de la Paz; Goldstein, Jorge; Alito, A.; Gioffré, Andrea; Caimi, Karina; Zábal, Osvaldo; Zumárraga, Martı N; Romanó, Maria; Cataldi, Angel; Bigi, Fabiana

doi:10.1099/00221287-148-10-2997

Cited by 45 publications

(42 citation statements)

References 29 publications

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“…Further comparison of microarray transcriptome data reveals that the four M. tuberculosis mce operons are not coregulated (3,19,30,31). This is not surprising in view of the fact that a TetR family transcriptional regulator divergently transcribed from the mce3 operon (Mce3R, Rv1963) has been shown to negatively affect regulation of this operon (28) and that a third probable transcriptional regulator (Rv0586) is associated with the mce2 operon (5). The differential expression levels of these operons may reflect their different roles.…”

Section: Discussionmentioning

confidence: 99%

Regulation of the Mycobacterium tuberculosis mce1 Operon

2006

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In the murine model of infection, a Mycobacterium tuberculosis mce1 operon mutant elicits an aberrant granulomatous response, resulting in uncontrolled replication and failure to enter a persistent state. In this study, we demonstrate that the mce1 genes can be transcribed as a 13-gene polycistronic message encompassing Rv0166 to Rv0178. Quantitative reverse transcriptase PCR and immunoblot analyses revealed that the mce1 genes and proteins are expressed during in vitro growth but are significantly down-regulated in intracellular bacilli isolated from murine macrophages. A homologue of the FadR subfamily of GntR transcriptional regulators, Rv0165c (designated Mce1R), lies upstream and is divergently transcribed from the operon. To investigate whether this gene plays a role in regulation of mce1 expression, we created an M. tuberculosis mce1R deletion mutant. There was no difference in expression of mce1 operon genes in ⌬mce1R compared to expression in the wild type during logarithmic growth in vitro. However, in bacilli isolated from murine macrophages, expression of mce1 genes was significantly higher in ⌬mce1R. In addition, overexpression of mce1R resulted in repression of the mce1 genes. These data demonstrate that Mce1R is a negative regulator that acts intracellularly to repress expression of the mce1 operon. We propose that Mce1R facilitates balanced temporal expression of the mce1 products required for organized granuloma formation, which is both protective to the host and necessary for the persistence of M. tuberculosis.Tuberculosis causes approximately two million deaths annually, and it has been estimated that two billion people are currently infected with the causative agent, Mycobacterium tuberculosis (10). Infection is initiated by inhalation of the bacilli into the lung, where they are ultimately ingested by alveolar macrophages. In the typical immunocompetent host, the cellmediated immune response results in the formation of a granuloma, the pathological hallmark of tuberculosis. In the majority of cases, this response is sufficient to contain the infection and prevent disease; however, viable bacilli remain within granulomas and can cause active disease years later.In the past decade, dramatic advances facilitating the molecular biological manipulation of mycobacteria have increased our knowledge of the mechanisms that tubercle bacilli use to survive within macrophages (33); however, little remains known about the bacterial determinants of infection outcome. Previous studies suggest that the M. tuberculosis mce1 operon is important in establishing a persistent infection in mice (32). It was shown that M. tuberculosis mce1 mutants failed to induce a characteristic proinflammatory response in ex vivo-infected murine macrophages. This phenotype was manifested in vivo by aberrant inflammatory cell migration and poor granuloma formation, leading to uncontrolled bacterial growth and rapid death of mutant-infected mice.The six Mce1 proteins (Mce1A to Mce1F) contain putative signal sequences indicative o...

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Section: Discussionmentioning

confidence: 99%

Regulation of the Mycobacterium tuberculosis mce1 Operon

2006

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“…We have previously reported that Mce3R, a TetR family transcriptional regulator, downregulates the mce3 operon during in vitro growth of M. tuberculosis (Santangelo et al, 2002). We have found that this regulation is specific for the mce3 operon (among all the mce genes) and that the Mce3R repressor regulates its own expression (Santangelo et al, 2008).…”

Section: Introductionmentioning

confidence: 94%

Mce3R, a TetR-type transcriptional repressor, controls the expression of a regulon involved in lipid metabolism in Mycobacterium tuberculosis

Santangelo¹,

Klepp²,

Nuñéz-García

et al. 2009

Microbiology

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The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each of which has 8-13 ORFs. Although the function of the Mce protein family has not been clearly established, its members are believed to be membrane lipid transporters. Based on functional experiments, we found that the regulator of the mce3 locus, Mce3R, negatively regulates the expression of the Rv1933c-Rv1935c and Rv1936-Rv1941 transcriptional units. These operons are adjacent to one another and divergently transcribed. The predicted functions of most of these genes are related to either lipid metabolism or redox reactions. Bioinformatic analysis of the 59 UTR sequences of the differentially expressed genes allowed us to define a putative Mce3R motif. Importantly, the Mce3R motif was present six and three times in the mce3R-yrbE3A and Rv1935c-Rv1936 intergenic regions, respectively. Two occurrences of this motif mapped within the two regions of the mce3 operon that were protected by Mce3R in a footprinting analysis, thus indicating that this motif is likely to serve as an operator site for the Mce3R regulator in the promoter. In addition, alterations in the lipid content of M. tuberculosis were detected in the absence of Mce3R. Taken together, these results suggest that Mce3R controls the expression of both the putative transport system encoded in the mce3 operon and the enzymes implicated in the modification of the Mce3-transported substrates.

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“…Two mutants of Brucella with a disrupted gene involved in rhizopine metabolism are attenuated (18,36), supporting the hypothesis that Brucella and Rhizobium could share common strategies to interact with their host (61). Members of the Crp, LysR, and TetR families of transcriptional regulators are involved in virulence and in symbiosis (26,33,51,53,65). Some regulators of the DeoR family are involved in sugar metabolism, but many proteins of that family…”

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Systematic Targeted Mutagenesis ofBrucella melitensis16M Reveals a Major Role for GntR Regulators in the Control ofVirulence

et al. 2005

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In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.Bacteria have selected mechanisms to express only the appropriate subset of genes conferring a growth or survival advantage in a given situation (10). The expression of many bacterial genes is regulated at the initiation of transcription by regulators which, in response to specific environmental and/or cellular signals, bind at the promoter of target genes to activate or repress them (23).The availability of complete bacterial genome sequences has opened up doors to the development of new strategies to study the biology of microorganisms (59). For instance, the complete Brucella melitensis sequence (15) allowed us to conduct a systematic study of several families of regulators in this organism. Bacteria of the genus Brucella are facultative intracellular gram-negative coccobacilli that are pathogenic for domestic animals and occasionally for humans (57). Some transcriptional regulators of Brucella that have a role in the control of virulence have already been identified. Indeed, the two-component system BvrR/BvrS controls cell invasion, intracellular survival, and expression of two outer membrane proteins (25, 58). Some transcriptional regulators required for Brucella's virulence were also identified in several screens in cellular models and in BALB/c mice (19,31,35,36). Random screening for attenuated mutants may not be viewed as a comprehensive analysis that would allow the identification of all regulators involved in the virulence of the model tested. We...

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Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis

Cited by 45 publications

References 29 publications

Regulation of the Mycobacterium tuberculosis mce1 Operon

Regulation of the Mycobacterium tuberculosis mce1 Operon

Mce3R, a TetR-type transcriptional repressor, controls the expression of a regulon involved in lipid metabolism in Mycobacterium tuberculosis

Systematic Targeted Mutagenesis ofBrucella melitensis16M Reveals a Major Role for GntR Regulators in the Control ofVirulence

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