Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules

We show that the 53-nucleotide RNA molecule encoded by gene dicF blocks cell division in Escherichia coli by inhibiting the translation of ftsZ mRNA. Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome-binding region of the ftsZ mRNA. An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ-lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated. Partial inhibition of FtsZ synthesis leads to increased cell size. However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly. Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.     

ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex

A gene conferring low-level isoniazid (INH) resistance on Mycobacterium smegmatis was isolated from a cosmid library of the genome of an INH-resistant Mycobacterium bovis strain. The gene had good homology with ahpC , the product of which is a subunit of alkyl hydroperoxide reductase, and also with a family of thiol-specific antioxidant enzymes. A mutation was found in the promoter upon comparison with the equivalent DNA sequence from the INH-sensitive parent strain. Promoter sequences from other INH-sensitive and INH-resistant M. bovis and Mycobacterium tuberculosis strains were sequenced and the mutation was found only in the INH-resistant strains. An INH-resistant M. tuberculosis strain also had an additional mutation in the promoter region. The wild-type promoter and promoters with one and two mutations were ligated into a reporter plasmid containing the lacZ gene. The presence of the first mutation resulted in a sixfold induction of β-galactosidase activity, and the presence of both mutations caused a 10-fold induction. Increased expression of AhpC may account for some of the INH resistance of strains of the M. tuberculosis complex.     

Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.     

The alternative sigma factor SigH regulates major components of oxidative and heat stress responses in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyte Mycobacterium smegmatis. In this work we investigated the role of sigH in the M. tuberculosis response to heat and oxidative stress. We determined that a M. tuberculosis sigH mutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genes trxB2/trxC and a gene of unknown function, Rv2466c, is regulated by sigH via expression from promoters directly recognized by SigH. We also determined that the sigH mutant is more susceptible to heat stress and that inducible expression of the heat shock genes dnaK and clpB is positively regulated by sigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role of sigH in the M. tuberculosis stress response, we investigated the regulation of the stress-responsive sigma factor genes sigE and sigB. We determined that inducible expression of sigE is regulated by sigH and that basal and inducible expression of sigB is dependent on sigE and sigH. These data indicate that sigH plays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important in M. tuberculosis pathogenesis.     

Overlapping functional units in a cell division gene cluster in Escherichia coli

The ftsZ ( sulB ) coding sequence is preceded by two promoters, at least one of which lies within the coding sequence of the neighboring gene, ftsA . This region of the ftsA gene is required for full biological activity of ftsZ .     

PE_PGRS proteins are differentially expressed by Mycobacterium tuberculosis in host tissues

Characterization of PE_PGRS gene expression will help define the role of this protein family in the biology of Mycobacterium tuberculosis. In this report, quantitative real-time RT-PCR (QRT-PCR) was implemented to assess expression of three PE_PGRS genes (rv0746, rv1651c and rv1818c) under different experimental conditions. The three PE_PGRS genes showed a similar expression profile in axenic cultures, with a significant up-regulation occurring at late log and early stationary phases. rv1651c gene expression increased following intracellular growth in bone marrow-derived macrophages but not in type-II human pneumocytes, while rv0746 was induced in both in vitro systems. Following the infection of mice with M. tuberculosis, expression levels of rv1651c and rv0746 normalized to ftsZ and 16S rRNA were highest in the spleen tissue during the chronic stages of murine tuberculosis, with a >20- and >30-fold up-regulation, respectively. Levels of expression remained lower in the lung over the same time period. Expression of the rv1818c gene did not change significantly under different experimental conditions tested. The results of this study indicate that M. tuberculosis can differentially regulate expression of PE_PGRS genes and that genes such as rv0746 and rv1651c are significantly induced while M. tuberculosis persists in host cells and tissues.     

The pathogen Mycobacterium marinum, a faster growing close relative of Mycobacterium tuberculosis, has a single rRNA operon per genome

Although Mycobacterium marinum and Mycobacterium tuberculosis are very closely related they differ significantly in their growth rates. The Type strain of M. marinum and one clinical isolate were investigated and, like M. tuberculosis, were found to have a single rRNA (rrn) operon per genome located downstream from murA gene and controlled by two promoters. No sequence differences were found that account for the difference in the growth rates of the two species. We infer that M. tuberculosis has the capacity to synthesize rRNA much faster than it actually does; and propose that the high number of insertion sequences in this species attenuate growth rate to lower values.     

Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo

The Mycobacterium tuberculosis FtsZ (FtsZ(TB)), unlike other eubacterial FtsZ proteins, shows slow GTP-dependent polymerization and weak GTP hydrolysis activities [E.L. White, L.J. Ross, R.C. Reynolds, L.E. Seitz, G.D. Moore, D.W. Borhani, Slow polymerization of Mycobacterium tuberculosis FtsZ, J. Bacteriol. 182 (2000) 4028-4034]. In an attempt to understand the biological significance of these findings, we created mutations in the GTP-binding (FtsZ(G103S)) and GTP hydrolysis (FtsZ(D210G)) domains of FtsZ and characterized the activities of the mutant proteins in vitro and in vivo. We show that FtsZ(G103S) is defective for binding to GTP and polymerization activities, and exhibited reduced GTPase activity whereas FtsZ(D210G) protein is proficient in binding to GTP, showing reduced polymerization activity but did not show any measurable GTPase activity. Visualization of FtsZ-GFP structures in ftsZ merodiploid strains by fluorescent microscopy revealed that FtsZ(D210G) is proficient in associating with Z-ring structures whereas FtsZ(G103S) is not. Finally, we show that Mycobacterium smegmatis ftsZ mutant strains producing corresponding mutant FtsZ proteins are non-viable indicating that mutant FtsZ proteins cannot function as the sole source for FtsZ, a result distinctly different from that reported for Escherichia coli. Together, our results indicate that optimal GTPase and polymerization activities of FtsZ are required to sustain cell division in mycobacteria and that the same conserved mutations in different bacterial species have distinct phenotypes.