SummaryDeletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrowderived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA , lprQ , whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA :: lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro . In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.
Drug resistance in Mycobacterium tuberculosis complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR) M. tuberculosis complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of M. bovis. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS6110. Here, we mapped and sequenced the regions flanking the two copies of IS6110 in the B strain. Ligation-mediated PCR showed that one of these IS6110 copies is located within the promoter region of phoP, a transcriptional regulator that is essential for M. tuberculosis virulence. We used PCR to screen 219 MDR M. tuberculosis complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS6110 in the phoP promoter. To determine whether IS6110 affects phoP promoter activity in the B strain, we individually cloned the phoP gene and its promoter region (including IS6110 from the B strain and the equivalent region from M. tuberculosis without IS6110 as a control) into a mycobacterial replicative plasmid and transformed M. smegmatis with the resulting plasmid. Primer extension analysis showed that phoP transcription was strongly upregulated when the promoter region contained IS6110, as in the case of the B strain.Tuberculosis (TB) is currently one of the leading causes of mortality throughout the world (8,25,27). The human immunodeficiency virus-AIDS pandemic, the deterioration of public health systems in developing countries, and the emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains have further contributed to the spread of TB. Knowledge of the molecular mechanisms involved in the bacillus-host cell interaction is essential for developing adequate strategies for TB control. Recent advances in the genetic manipulation of mycobacteria (2, 29) combined with the publication of the complete M. tuberculosis genome sequence (6) have made it possible to study the contribution of individual genes to M. tuberculosis virulence (5, 7). However, little is known about the regulatory and expression mechanisms that determine the virulence of clinical isolates of M. tuberculosis.Insertion sequence (IS) 6110 has been extensively used for molecular typing of M. tuberculosis strains. Restriction fragment length polymorphism (RFLP) analysis using IS6110 as a probe is currently the most common molecular method used to type M. tuberculosis complex strains. However, the physiological role and impact of specific IS6110 insertions on the biology of bacilli are not well known. IS6110 fingerprinting studies have demonstrated heterogeneity between virulence and transm...
Forty-five mycobacterial strains isolated from 23 Colombian HIV-positive patients were identified as members of the Mycobacterium avium complex (MAC) and were characterized using different molecular approaches. Seven of the isolates showed characteristic features that allowed them to be differentiated from other members of the complex. The isolates had a novel 16S-23S rRNA internal transcribed spacer (ITS 1) gene sequence which is described as a new sequevar, MAC-X. All of the seven novel isolates gave a positive result with the MAC-specific AccuProbe (Gen-Probe), but tested negative for Mycobacterium avium and Mycobacterium intracellulare species-specific probes (64 and 100 % of the isolates, respectively). The novel isolates could be differentiated phenotypically from other members of the MAC on the basis of the production of urease and by a consistent mycolic acid pattern. The novel isolates shared some characteristics with M. avium, such as the avium variant I (av-I) pattern of the hsp65 gene as determined by PCR restriction analysis and a positive PCR result for the mig (macrophage-induced) gene. However, the novel isolates showed a unique 16S rRNA gene sequence. DNA-DNA relatedness values, from 24 to 44 %, confirmed the distinction of the novel isolates from other members of the MAC at the genetic level and their status as members of a separate species. The novel isolates are proposed as representatives of a novel species, Mycobacterium colombiense sp. nov., that is closely related to M. avium within the MAC. The type strain is 10B T (=CIP 108962Members of the Mycobacterium avium complex (MAC) are currently identified on the basis of positive results with the commercial MAC-specific probe, AccuProbe (Gen-Probe). Originally, two species were identified within the MAC, namely Mycobacterium avium and Mycobacterium intracellulare. However, recent studies have drawn attention to the wide diversity of isolates that can be detected in the complex (Mijs et al., 2002;Smole et al., 2002;Lebrun et al., 2005). Analysis of the 16S-23S rRNA internal transcribed spacer (ITS 1) sequence, a molecular approach frequently used for the identification of these bacteria, has revealed the existence of more than 30 different sequevars (Tortoli et al., 2004). Only a few of these sequevars belong to members of M. avium (seven sequevars) or M. intracellulare (four sequevars). Recently, members of the MAC represented by sequevar MAC-A were described as the novel species Mycobacterium chimaera. This novel species has similar characteristics to M. intracellulare (Tortoli et al., 2004).During a study to characterize MAC isolates from HIVpositive Colombian patients, a group of distinct strains was identified. These isolates showed several features that allowed them to be differentiated from other members of the MAC and recognized as a separate novel species.Abbreviations: ITS 1, 16S-23S rRNA internal transcribed spacer; MAC, Mycobacterium avium complex; PRA, PCR restriction analysis; RAPD, randomly amplified polymorphic DNA.The GenBank/E...
Since 2004, a series of localized skin and soft tissue infections caused by rapidly growing mycobacteria have occurred in Brazil in patients who have undergone invasive procedures, such as laparoscopic, arthroscopic, plastic surgery, or cosmetic interventions (12,16,23,37). In 4 years, more than 2,000 cases were officially reported to Brazilian federal authorities, who consider this problem an epidemiological emergency (5). Almost all isolates studied so far have belonged to the Mycobacterium chelonae-M. abscessus group (39). The majority of them were identified as members of two recently described emerging pathogens, Mycobacterium massiliense (3) and Mycobacterium bolletii (1), both of which belong to the Mycobacterium chelonae-M. abscessus group.All five members of the Mycobacterium chelonae-M. abscessus group, M. chelonae, M. abscessus (21), Mycobacterium immunogenum (40), M. massiliense (3), and M. bolletii (1), are nearly indistinguishable phenotypically. Common features include growth in less than 7 days, the absence of pigmentation, better growth at 30°C than at 35°C, a positive 3-day arylsulfatase test result, a negative nitrate reductase test result, and a negative iron uptake test result (41). Two biochemical tests, sodium chloride tolerance and the utilization of citrate, are useful in distinguishing the five members (1,3,40,41).Antimicrobial susceptibility can also be used to differentiate the members of the M. chelonae-M. abscessus group. M. abscessus is generally susceptible to cefoxitin (MIC Ͻ 16 g/ml)
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