Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.     

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli . The recombinant protein bound to the promoter regions of the M. leprae lexA , M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis . Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis . Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti- M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.     

Bacterial chromosome segregation: structure and DNA binding of the Soj dimer--a conserved biological switch

Soj and Spo0J of the Gram-negative hyperthermophile Thermus thermophilus belong to the conserved ParAB family of bacterial proteins implicated in plasmid and chromosome partitioning. Spo0J binds to DNA near the replication origin and localises at the poles following initiation of replication. Soj oscillates in the nucleoid region in an ATP- and Spo0J-dependent fashion. Here, we show that Soj undergoes ATP-dependent dimerisation in solution and forms nucleoprotein filaments with DNA. Crystal structures of Soj in three nucleotide states demonstrate that the empty and ADP-bound states are monomeric, while a hydrolysis-deficient mutant, D44A, is capable of forming a nucleotide 'sandwich' dimer. Soj ATPase activity is stimulated by Spo0J or the N-terminal 20 amino-acid peptide of Spo0J. Our analysis shows that dimerisation and activation involving a peptide containing a Lys/Arg is conserved for Soj, ParA and MinD and their modulators Spo0J, ParB and MinE, respectively. By homology to the nitrogenase iron protein and the GTPases Ffh/FtsY, we suggest that Soj dimerisation and regulation represent a conserved biological switch.     

A protein kinase inhibitor as an antimycobacterial agent

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.     

ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria

The RecA-dependent DNA damage response pathway (SOS response) appears to be the major DNA repair mechanism in most bacteria, but it has been suggested that a RecA-independent mechanism is responsible for controlling expression of most damage-inducible DNA repair genes in Mycobacterium tuberculosis. The specific reparative responses and molecular mediators involved in the DNA repair mechanism remain largely unclear in this pathogen and its related species. In this study, a mycobacterial ClpR-like regulator, corresponding to Rv2745c in M. tuberculosis and to Ms2694 in M. smegmatis mc(2)155, was found to interact with the promoter regions of multiple damage-inducible DNA repair genes. Specific binding of the ClpR-like factor to the conserved RecA-independent promoter RecA-NDp motif was then confirmed using in vitro electrophoretic mobility shift assays as well as in vivo chromatin immunoprecipitation experiments. The ClpR knock-out experiments, in combination with quantitative real time PCR assays, demonstrated that the expression of these RecA-independent genes were significantly down-regulated in the mutant strain of M. smegmatis in response to a DNA-damaging agent compared with the wild type strain. Furthermore, the ClpR-like factor was shown to contribute to mycobacterial genomic stability. These results enhance our understanding of the function of the ClpR regulator and the regulatory mechanism of RecA-independent DNA repair in mycobacteria.     

Physical and functional interactions between 3-methyladenine DNA glycosylase and topoisomerase I in mycobacteria

DNA glycosylases play important roles in DNA repair in a variety of organisms, including humans. However, the function and regulation of these enzymes in the pathogenic bacterium Mycobacterium tuberculosis and related species are poorly understood. In the present study, the physical and functional interactions between 3-methyladenine DNA glycosylase (MAG) and topoisomerase I (TopA) in M. tuberculosis and M. smegmatis were characterized. MAG was found to inhibit the function of TopA in relaxing supercoiled DNA. In contrast, TopA stimulated the cleavage function of MAG on a damaged DNA substrate that contains hypoxanthine. The interaction between the two proteins was conserved between the two mycobacterial species. Several mutations in MAG that led to the loss of its interaction with and activity regulation of TopA were also characterized. The results of this study further elucidate glycosylase regulation in both M. smegmatis and M. tuberculosis.     

Competition between DivIVA and the nucleoid for ParA binding promotes segrosome separation and modulates mycobacterial cell elongation

Although mycobacteria are rod shaped and divide by simple binary fission, their cell cycle exhibits unusual features: unequal cell division producing daughter cells that elongate with different velocities, as well as asymmetric chromosome segregation and positioning throughout the cell cycle. As in other bacteria, mycobacterial chromosomes are segregated by pair of proteins, ParA and ParB. ParA is an ATPase that interacts with nucleoprotein ParB complexes – segrosomes and non‐specifically binds the nucleoid. Uniquely in mycobacteria, ParA interacts with a polar protein DivIVA (Wag31), responsible for asymmetric cell elongation, however the biological role of this interaction remained unknown. We hypothesised that this interaction plays a critical role in coordinating chromosome segregation with cell elongation. Using a set of ParA mutants, we determined that disruption of ParA‐DNA binding enhanced the interaction between ParA and DivIVA, indicating a competition between the nucleoid and DivIVA for ParA binding. Having identified the ParA mutation that disrupts its recruitment to DivIVA, we found that it led to inefficient segrosomes separation and increased the cell elongation rate. Our results suggest that ParA modulates DivIVA activity. Thus, we demonstrate that the ParA‐DivIVA interaction facilitates chromosome segregation and modulates cell elongation.     

ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS

Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB-parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein-DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein-DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition.     

Physical and functional interaction between D-ribokinase and topoisomerase I has opposite effects on their respective activity in Mycobacterium smegmatis and Mycobacterium tuberculosis

d-ribose is an essential component of multiple important biological molecules and must first be phosphorylated by ribokinase before entering metabolic pathways. However, the function and regulation of ribokinases in Mycobacterium tuberculosis, the causative agent of tuberculosis, and its related species are largely unknown. In this study, we have characterized the activities of two putative ribokinases, Rv2436 and Ms4585, from M. tuberculosis and Mycobacterium smegmatis, respectively. The mycobacterial topoisomerase I (TopA) was found to physically interact with its ribokinase both in vitro and in vivo. By creating two ribokinase mutants that showed defective interactions with TopA, we further showed that the interaction between ribokinase and TopA had opposite effects on their respective function. While the interaction between the two proteins inhibited the ability of TopA to relax supercoiled DNA, it stimulated ribokinase activity. A cross-regulation assay revealed that the interaction between the two proteins was conserved in the two mycobacterial species. Thus, we uncovered an interplay between ribokinase and topoisomerase I in mycobacteria, which implies the existence of a novel regulatory strategy for efficient utilization of d-ribose in M. tuberculosis that may be useful in stressful environments with restricted access to nutrients.     

Importance of uracil DNA glycosylase in Pseudomonas aeruginosa and Mycobacterium smegmatis,G+C-rich bacteria,in mutation prevention,tolerance to acidified nitrite,and endurance in mouse macrophages

Uracil DNA glycosylase (Ung (or UDG)) initiates the excision repair of an unusual base, uracil, in DNA. Ung is a highly conserved protein found in all organisms. Paradoxically, loss of this evolutionarily conserved enzyme has not been seen to result in severe growth phenotypes in the cellular life forms. In this study, we chose G+C-rich genome containing bacteria (Pseudomonas aeruginosa and Mycobacterium smegmatis) as model organisms to investigate the biological significance of ung. Ung deficiency was created either by expression of a highly specific inhibitor protein, Ugi, and/or by targeted disruption of the ung gene. We show that abrogation of Ung activity in P. aeruginosa and M. smegmatis confers upon them an increased mutator phenotype and sensitivity to reactive nitrogen intermediates generated by acidified nitrite. Also, in a mouse macrophage infection model, P. aeruginosa (Ung-) shows a significant decrease in its survival. Infections of the macrophages with M. smegmatis show an initial increase in the bacterial counts that remain for up to 48 h before a decline. Interestingly, abrogation of Ung activity in M. smegmatis results in nearly a total abolition of their multiplication and a much-decreased residency in macrophages stimulated with interferon gamma. These observations suggest Ung as a useful target to control growth of G+C-rich bacteria.     

A monoclonal antibody that inhibits mycobacterial DNA gyrase by a novel mechanism

DNA gyrase is a DNA topoisomerase indispensable for cellular functions in bacteria. We describe a novel, hitherto unknown, mechanism of specific inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis DNA gyrase by a monoclonal antibody (mAb). Binding of the mAb did not affect either GyrA-GyrB or gyrase-DNA interactions. More importantly, the ternary complex of gyrase-DNA-mAb retained the ATPase activity of the enzyme and was competent to catalyse DNA cleavage-religation reactions, implying a new mode of action different from other classes of gyrase inhibitors. DNA gyrase purified from fluoroquinolone-resistant strains of M.tuberculosis and M.smegmatis were inhibited by the mAb. The absence of cross-resistance of the drug-resistant enzymes from two different sources to the antibody-mediated inhibition corroborates the new mechanism of inhibition. We suggest that binding of the mAb in the proximity of the primary dimer interface region of GyrA in the heterotetrameric enzyme appears to block the release of the transported segment after strand passage, leading to enzyme inhibition. The specific inhibition of mycobacterial DNA gyrase with the mAb opens up new avenues for designing novel lead molecules for drug discovery and for probing gyrase mechanism.     

ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities

ParA and ParB of Caulobacter crescentus belong to a conserved family of bacterial proteins implicated in chromosome segregation. ParB binds to DNA sequences adjacent to the origin of replication and localizes to opposite cell poles shortly following the initiation of DNA replication. ParA has homology to a conserved and widespread family of ATPases. Here, we show that ParB regulates the ParA ATPase activity by promoting nucleotide exchange in a fashion reminiscent of the exchange factors of eukaryotic G proteins. Furthermore, we demonstrate that ADP-bound ParA binds single-stranded DNA, whereas the ATP-bound form dissociates ParB from its DNA binding sites. Increasing the fraction of ParA-ADP in the cell inhibits cell division, suggesting that this simple nucleotide switch may regulate cytokinesis.     

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.     

Mycobacterium smegmatis RecA protein is structurally similar to but functionally distinct from Mycobacterium tuberculosis RecA

In eubacteria, RecA proteins belong to a large superfamily of evolutionarily conserved, filament-forming, functional homologs of DNA strand exchange proteins. Here, we report the functional characterization of Mycobacterium smegmatis (Ms) and Mycobacterium tuberculosis (Mt) RecA proteins. Although in some respects Ms and Mt RecA proteins are structural and functional homologs of Escherichia coli (Ec) RecA, there are significant differences as well. The single-stranded DNA-binding property of RecA proteins was analyzed by electrophoretic mobility shift assays. We observed that Ms or Mt RecA proteins bound single-stranded DNA in a manner distinct from that of Ec RecA: The former two were able to form protein-DNA complexes in the presence of high salt. Further experiments indicated that Ms or Mt RecA proteins catalyzed adenosine triphosphate hydrolysis at approximately comparable rates across a wide range of pHs. Significantly, DNA strand invasion promoted by Ms or Mt RecA proteins displayed similar kinetics but distinctly different pH profiles. In contrast to MtRecA, MsRecA by itself was unable to form joint molecules across a wide range of pHs. However, regardless of the order in which SSB was added, it was able to stimulate MsRecA to form joint molecules within a narrow pH range, indicating that SSB is a required accessory factor. Together, these results provide a source of sharp contrast between EcRecA and mycobacterial RecAs on the one hand and Mt and Ms RecA proteins on the other.     

A distinct role of formamidopyrimidine DNA glycosylase (MutM) in down-regulation of accumulation of G, C mutations and protection against oxidative stress in mycobacteria

Reactive oxygen species produced as a part of cellular metabolism or environmental agent cause a multitude of damages in cell. Oxidative damages to DNA or the free nucleotide pool result in occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA, and failure to replace it with the correct base results in a variety of mutations in the genome. Formamidopyrimidine DNA glycosylase (Fpg/MutM), a functionally conserved repair enzyme initiates the 8-oxoG repair pathway in all eubacteria. DNA in mycobacteria with G+C rich genomes is particularly vulnerable to the oxidative damage. In this study, we disrupted fpg gene in Mycobacterium smegmatis to generate an Fpg deficient strain. The strain showed an enhanced mutator phenotype and susceptibility to hydrogen peroxide. Analyses of rifampicin resistance determining region (RRDR) revealed that, in contrast to Fpg deficient Escherichia coli where C to A mutations predominate, Fpg deficient M. smegmatis shows a remarkable increase in accumulation of A to G (or T to C) mutations. Interestingly, exposure of the mutant to sub-lethal level of hydrogen peroxide results in a major shift towards C to G (or G to C) mutations. Biochemical analysis showed that mycobacterial Fpg; and MutY (which excises misincorporated A against 8-oxoG) possess substrate specificities similar to their counterparts in E. coli. However, the DNA polymerase assays with cell-free extracts showed preferential incorporation of G in M. smegmatis as opposed to an A in E. coli. Our studies highlight the importance and the distinctive features of Fpg mediated DNA repair in mycobacteria.     

Biochemical activities of the ParA partition protein of the P1 plasmid

The unit-copy P1 plasmid depends for stability on a plasmid-encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition-critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP-dependent, site-specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The role of the ATP-dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.     

The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli

The bacteria for which there is evidence that proteins of the ParAB family act in chromosome segregation also undergo developmental transitions that involve the ParAB homologues, raising the question of whether the partition activity is equivalent to that of plasmid partition systems. We have investigated the role in partition of the parAB locus of a free-living bacterium, Pseudomonas putida, not known to pass through developmental phases. A parAB deletion mutant, compared with wild type, showed slightly higher frequencies of anucleate cells in exponentially growing cultures but much higher frequencies in deceleration phase. This increase was growth medium dependent. Oversupply of ParA and ParB proteins also raised anucleate cell levels, specifically in the deceleration phase, in wild-type and mutant strains and regardless of medium, as well as generating abnormal cell morphologies. Absence or oversupply of ParAB function had either slight or considerable effects on growth rate, depending on temperature and medium. The need for the Par proteins in chromosome partition thus appears to be subject to the cell's physiological state. Three sequences similar to cis-acting stabilization sites of Bacillus subtilis are present in the P. putida oriC-parAB region. One was inserted into an unstable mini-F and shown to stabilize it in E. coli in a ParAB-dependent manner.     

Uncoupling of Nucleotide Hydrolysis and Polymerization in the ParA Protein Superfamily Disrupts DNA Segregation Dynamics

DNA segregation in bacteria is mediated most frequently by proteins of the ParA superfamily that transport DNA molecules attached via the segrosome nucleoprotein complex. Segregation is governed by a cycle of ATP-induced polymerization and subsequent depolymerization of the ParA factor. Here, we establish that hyperactive ATPase variants of the ParA homolog ParF display altered segrosome dynamics that block accurate DNA segregation. An arginine finger-like motif in the ParG centromere-binding factor augments ParF ATPase activity but is ineffective in stimulating nucleotide hydrolysis by the hyperactive proteins. Moreover, whereas polymerization of wild-type ParF is accelerated by ATP and inhibited by ADP, filamentation of the mutated proteins is blocked indiscriminately by nucleotides. The mutations affect a triplet of conserved residues that are situated neither in canonical nucleotide binding and hydrolysis motifs in the ParF tertiary structure nor at interfaces implicated in ParF polymerization. Instead the residues are involved in shaping the contours of the binding pocket so that nucleotide binding locks the mutant proteins into a configuration that is refractory to polymerization. Thus, the architecture of the pocket not only is crucial for optimal ATPase kinetics but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes.