SummaryPaired two-component regulatory systems consisting of a sensor kinase and a response regulator are the major means by which bacteria sense and respond to different stimuli. The role of essential response regulator, MtrA, in Mycobacterium tuberculosis proliferation is unknown. We showed that elevating the intracellular levels of MtrA prevented M. tuberculosis from multiplying in macrophages, mice lungs and spleens, but did not affect its growth in broth. Intracellular trafficking analysis revealed that a vast majority of MtrA overproducing merodiploids were associated with lysosomal associated membrane protein (LAMP-1) positive vacuoles, indicating that intracellular growth attenuation is, in part, due to an impaired ability to block phagosome-lysosome fusion. A merodiploid strain producing elevated levels of phosphorylation-defective MtrA (MtrA D53N ) was partially replicative in macrophages, but was attenuated in mice. Quantitative real-time PCR analyses revealed that expression of dna A, an essential replication gene, was sharply upregulated during intramacrophage growth in the MtrA overproducer in a phosphorylation-dependent manner. Chromatin immunoprecipitation using anti-MtrA antibodies provided direct evidence that MtrA regulator binds to dna A promoter in vivo indicating that dna A promoter is a MtrA target. Simultaneous overexpression of mtr A regulator and its cognate mtr B kinase neither inhibited growth nor sharply increased the expression levels of dna A in macrophages. We propose that proliferation of M. tuberculosis in vivo depends, in part, on the optimal ratio of phosphorylated to nonphosphorylated MtrA response regulator.

SummaryThe genetic factors responsible for the regulation of cell division in Mycobacterium tuberculosis are largely unknown. We showed that exposure of M. tuberculosis to DNA damaging agents, or to cephalexin, or growth of M. tuberculosis in macrophages increased cell length and sharply elevated the expression of Rv2719c, a LexA-controlled gene. Overexpression of Rv2719c in the absence of DNA damage or of antibiotic treatment also led to filamentation and reduction in viability both in broth and in macrophages indicating a correlation between Rv2719c levels and cell division. Overproduction of Rv2719c compromised midcell localization of FtsZ rings, but had no effect on the intracellular levels of FtsZ. In vitro, the Rv2719c protein did not interfere with the GTPdependent polymerization activity of FtsZ indicating that the effects of Rv2719c on Z-ring assembly are indirect. Rv2719c protein exhibited mycobacterial murein hydrolase activity that was localized to the N-terminal 110 amino acids. Visualization of nascent peptidoglycan (PG) synthesis zones by probing with fluoresceinated vancomycin (Van-FL) and localization of green fluorescent protein-Rv2719c fusion suggested that the Rv2719c activity is targeted to potential PG synthesis zones. We propose that Rv2719c is a potential regulator of M. tuberculosis cell division and that its levels, and possibly activities, are modulated under a variety of growth conditions including growth in vivo and during DNA damage, so that the assembly of FtsZ-rings, and therefore the cell division, can proceed in a regulated manner.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.

The well-recognized phospholipids (PLs) of Mycobacterium tuberculosis (Mtb) include several acidic species such as phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG), is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF)-lysyl-tRNA synthetase (lysU) protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein–positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection.

(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). SOS mutagenesis is regulated by at least two sequential reactions. First, RecA protein is activated by DNA damage to mediate the proteolytic cleavage of the LexA repressor for SOS-controlled genes, including umuC and umuD (2, 13). In a second RecA-dependent proteolytic step, UmuD is processed to UmuD', the active agent in mutagenesis (9,15,16). Based on direct evidence for a physical interaction, the UmuC-UmuD' complex is presumed to be the functional unit for mutagenesis (17). Genetic experiments have indicated that RecA probably has a third, more direct role in SOS mutagenesis, in addition to the two regulatory functions (9)(10)(11)(12).Based on the available evidence, the pathway for SOS mutagenesis has been presumed to involve replication past the lesion by DNA polymerase in an altered low-fidelity mode mediated by UmuC, UmuD', and RecA (14,[17][18][19] Genetic studies indicate that DNA polymerase I is not required (22,23). DNA polymerase II exhibits the interesting property of SOS induction (24)(25)(26); however, deletion and insertion mutations in the gene for polymerase II do not alter SOS mutagenesis (C. Bonner, S. Creighton and M.F.G., unpublished work).Together, the studies noted above give clear indications that SOS mutagenesis is a consequence of replicative bypass of DNA lesions mediated by a damage-localized nucleoprotein complex involving RecA, UmuC-UmuD', and pol 17). However, direct evidence for such a pathway has been lacking in the absence of a defined biochemical system. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Replication was initiated by the addition of deoxynucleoside triphosphates (60 AuM each). Replication was carried out for 10 min at 370C, followed by quenching with 20 Al of 20 mM EDTA in 95% formamide. The DNA was then denatured by heating, and the replicated primer products were separated by electrophoresis in 10%6 polyacrylamide gels containing urea and were visualized by autoradiography of dried gels. RESULTS Assay for the RepUcative Bypass of an Abasic Lesion. To develop a biochemical system to study replicative bypass of DNA lesions, we used an assay system that is highly sensitive to limited bypass. In principle, an oligonucleotide with a lesion at a specific site fulfills this need (24,27,33). However, assembly of pol III holoenzyme on a primer-template DNA requires at least 36 nucleotides from the 3' and the 5' end of the primer (M.O., unpublished data). To circumvent this problem, we used a 5.4-kb ssDNA substrate with an abasic site located 30 nucleotides upstream from the 5' end (Fig. 1).The 5.4-kb linear ssDNA was replicated from two 20-mer oligonucleotide primers. Primer 1 (P1), which anneals to the substrate 65 nucleotides upstream from the abasic site, was labeled at the 5' end with ['t-32P]ATP. Replication from the unlabeled primer 2 (P2), located 347 nucl...