Decreased Methylenetetrahydrofolate Reductase Activity Leads to Increased Sensitivity to para-Aminosalicylic Acid in Mycobacterium tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most fatal diseases in the world. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the production of 5-methyltetrahydrofolate (5-CH3-THF), which is required for the de novo biosynthesis of methionine in bacteria. Here, we identified Rv2172c as an MTHFR in M. tuberculosis through in vitro and in vivo analyses and determined that the protein is essential for the in vitro growth of the bacterium. Subsequently, we constructed rv2172c R159N and L214A mutants in M. tuberculosis and found that these mutants were more sensitive to the antifolates para-aminosalicylic acid (PAS) and sulfamethoxazole (SMX). Combining biochemical and genetic methods, we found that rv2172c R159N or L214A mutation impaired methionine production, leading to increased susceptibility of M. tuberculosis to PAS, which was largely restored by adding exogenous methionine. Moreover, overexpression of rv2172c in M. tuberculosis could increase methionine production and lead to PAS resistance. This research is the first to identify an MTHFR in M. tuberculosis and reveals that the activity of this enzyme is associated with susceptibility to antifolates. These findings have particular value for antitubercular drug design for the treatment of drug-resistant TB.

Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity.

Mycobacterium tuberculosis (Mtb) has evolved multiple strategies to counter the human immune system. The effectors of Mtb play important roles in the interactions with the host. However, because of the lack of highly efficient strategies, there are only a handful of known Mtb effectors, thus hampering our understanding of Mtb pathogenesis. In this study, we probed Mtb proteome microarray with biotinylated whole-cell lysates of human macrophages, identifying 26 Mtb membrane proteins and secreted proteins that bind to macrophage proteins. Combining GST pull-down with mass spectroscopy then enabled the specific identification of all binders. We refer to this proteome microarray-based strategy as SOPHIE (Systematic unlOcking of Pathogen and Host Interacting Effectors). Detailed investigation of a novel effector identified here, the iron storage protein BfrB (Rv3841), revealed that BfrB inhibits NF-κB-dependent transcription through binding and reducing the nuclear abundance of the ribosomal protein S3 (RPS3), which is a functional subunit of NF- κB. The importance of this interaction was evidenced by the promotion of survival in macrophages of the mycobacteria, Mycobacterium smegmatis, by overexpression of BfrB. Thus, beyond demonstrating the power of SOPHIE in the discovery of novel effectors of human pathogens, we expect that the set of Mtb effectors identified in this work will greatly facilitate the understanding of the pathogenesis of Mtb, possibly leading to additional potential molecular targets in the battle against tuberculosis.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.

Global Profiling of PknG Interactions Using a Human Proteome Microarray Reveals Novel Connections with CypA.

Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb-host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF-κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb-host interactions.

Deletion of nudB Causes Increased Susceptibility to Antifolates in Escherichia coli and Salmonella enterica.

Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in increased susceptibility to both SMX and TMP in Salmonella enterica Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.

Deletion of sigB Causes Increased Sensitivity to para-Aminosalicylic Acid and Sulfamethoxazole in Mycobacterium tuberculosis.

Although the de novo folate biosynthesis pathway has been well studied in bacteria, little is known about its regulation. In the present study, the sigB gene in Mycobacterium tuberculosis was deleted. Subsequent drug susceptibility tests revealed that the M. tuberculosis ΔsigB strain was more sensitive to para-aminosalicylic acid (PAS) and sulfamethoxazole. Comparative transcriptional analysis was performed, and downregulation of pabB was observed in the ΔsigB strain, which was further verified by a quantitative reverse transcription-PCR and Western blot assay. Then, the production levels of para-aminobenzoic acid (pABA) were compared between the sigB deletion mutant and wild-type strain, and the results showed that sigB deletion resulted in decreased production of pABA. In addition, SigB was able to recognize the promoter of pabB in vitro Furthermore, we found that deleting pabC also caused increased susceptibility to PAS. Taken together, our data revealed that, in M. tuberculosis, sigB affects susceptibility to antifolates through multiple ways, primarily by regulating the expression of pabB To our knowledge, this is the first report showing that SigB modulates pABA biosynthesis and thus affecting susceptibility to antifolates, which broadens our understanding of the regulation of bacterial folate metabolism and mechanisms of susceptibility to antifolates.

Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis.

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.

The S28H mutation on mNeptune generates a brighter near-infrared monomeric fluorescent protein with improved quantum yield and pH-stability.

For living deep-tissue imaging, the optical window favorable for light penetration is in near-infrared wavelengths, which requires fluorescent proteins with emission spectra in the near-infrared region. Here, we report that a single mutant Ser28His of mNeptune with a near-infrared (≥650 nm) emission maxima of 652 nm is found to improve the brightness, photostability, and pH stability when compared with its parental protein mNeptune, while it remains as a monomer, demonstrating that there is still plenty of room to improve the performance of the existing near infrared fluorescence proteins by directed evolution.

The T120P or M172V mutation on rv2172c confers high level para-aminosalicylic acid resistance in Mycobacterium tuberculosis.

Although para-aminosalicylic acid (PAS) has been used to treat tuberculosis for decades, mechanisms of resistance to this drug in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates have not been thoroughly investigated. Previously, we found that decreased methylenetetrahydrofolate reductase (MTHFR) activity of Rv2172c led to increased sensitivity to antifolates in M. tuberculosis. In this study, we collected the genome-sequencing data of 173 PAS-resistant and 803 PAS-sensitive clinical isolates and analyzed rv2172c mutations in those 976 isolates. The results showed that two mutations (T120P and M172V) on rv2172c could be identified in a certain proportion (6.36%) of PAS-resistant isolates. The results of AlphaFold2 prediction indicated that the T120P or M172V mutation might affect the enzymatic activity of Rv2172c by influencing nicotinamide adenine dinucleotide (NADH) binding, and this was verified by subsequent biochemical analysis, demonstrating the role of residues Thr120 and Met172 on NADH binding and enzymatic activity of Rv2172c. In addition, the effect of rv2172c T120P or M172V mutation on methionine production and PAS resistance was determined in M. tuberculosis. The results showed that both T120P and M172V mutations caused increased intracellular methionine concentrations and high level PAS resistance. In summary, we discovered new molecular markers and also a novel mechanism of PAS resistance in M. tuberculosis clinical isolates and broadened the understanding of the NADH-dependent MTHFR catalytic mechanism of Rv2172c in M. tuberculosis, which will facilitate the molecular diagnosis of PAS resistance and also the development of new drugs targeting Rv2172c.

Competition between H4PteGlu and H2PtePAS Confers para-Aminosalicylic Acid Resistance in Mycobacterium tuberculosis.

Tuberculosis remains a serious challenge to human health worldwide. para-Aminosalicylic acid (PAS) is an important anti-tuberculosis drug, which requires sequential activation by Mycobacterium tuberculosis (M. tuberculosis) dihydropteroate synthase and dihydrofolate synthase (DHFS, FolC). Previous studies showed that loss of function mutations of a thymidylate synthase coding gene thyA caused PAS resistance in M. tuberculosis, but the mechanism is unclear. Here we showed that deleting thyA in M. tuberculosis resulted in increased content of tetrahydrofolate (H4PteGlu) in bacterial cells as they rely on the other thymidylate synthase ThyX to synthesize thymidylate, which produces H4PteGlu during the process. Subsequently, data of in vitro enzymatic activity experiments showed that H4PteGlu hinders PAS activation by competing with hydroxy dihydropteroate (H2PtePAS) for FolC catalysis. Meanwhile, over-expressing folC in ΔthyA strain and a PAS resistant clinical isolate with known thyA mutation partially restored PAS sensitivity, which relieved the competition between H4PteGlu and H2PtePAS. Thus, loss of function mutations in thyA led to increased H4PteGlu content in bacterial cells, which competed with H2PtePAS for catalysis by FolC and hence hindered the activation of PAS, leading to decreased production of hydroxyl dihydrofolate (H2PtePAS-Glu) and finally caused PAS resistance. On the other hand, functional deficiency of thyA in M. tuberculosis pushes the bacterium switch to an unidentified dihydrofolate reductase for H4PteGlu biosynthesis, which might also contribute to the PAS resistance phenotype. Our study revealed how thyA mutations confer PAS resistance in M. tuberculosis and provided new insights into studies on the folate metabolism of the bacterium.

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation.

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.

CycA-Dependent Glycine Assimilation Is Connected to Novobiocin Susceptibility in Escherichia coli.

Escherichia coli serine hydroxymethyltransferase (GlyA) converts serine to glycine, and glyA mutants are auxotrophic for glycine. CycA is a transporter that mediates glycine uptake. Deleting glyA in E. coli strain W3110 led to activation of CysB, which was related to novobiocin (NOV) susceptibility. Moreover, deleting glyA resulted in increased sensitivity to NOV, and this could be reversed by high concentrations of glycine. Reverse mutants of ΔglyA were selected and one of them had a mutation in yrdC, the gene encoding threonylcarbamoyl-AMP synthase. Subsequent proteome analysis showed that deleting glyA led to increased expression of TcyP and TdcB, making this bacterium dependent on CycA for glycine assimilation. Furthermore, deleting cycA in a ΔglyA background caused a severe growth defect on Luria-Bertani medium, which could be complemented by high concentrations of exogenous glycine. Mutation of yrdC led to decreased expression of TdcB but increased expression of ThrA/B/C and LtaE, which favored the conversion of threonine to glycine and thus avoided the dependence on CycA. Correspondingly, deleting of tcyP, tdcB, or gshA could reverse the NOV-sensitive phenotype of ΔglyA mutants. Overexpression of cycA resulted in increased sensitivity to NOV, whereas deleting this gene caused NOV resistance. Moreover, overexpression of cycA led to increased accumulation of NOV upon drug treatment. Therefore, inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which enhanced the accumulation of NOV and then made the bacterium more sensitive to this drug. These findings broaden our understanding of glycine metabolism and mechanisms of NOV susceptibility. IMPORTANCE Novobiocin (NOV) has been used in clinical practice as an ATPase inhibitor for decades. However, because it has been withdrawn from the market, pharmaceutical companies are searching for other ATPase inhibitors. Thus, probing the mechanisms of susceptibility to NOV will be beneficial to those efforts. In this study, we showed that inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which accompanied the accumulation of NOV and thereby increased the sensitivity to this drug. To date, this is the first report demonstrating the linkage between glycine assimilation and NOV susceptibility, and it is also the first report showing that YrdC is able to modulate the metabolic flux of threonine.

CobB regulates Escherichia coli chemotaxis by deacetylating the response regulator CheY.

The silent information regulator (Sir2) family proteins are NAD+-dependent deacetylases. Although a few substrates have been identified, functions of the bacteria Sir2-like protein (CobB) still remain unclear. Here the role of CobB on Escherichia coli chemotaxis was investigated. We used Western blotting and mass spectrometry to show that the response regulator CheY is a substrate of CobB. Surface plasmon resonance (SPR) indicated that acetylation affects the interaction between CheY and the flagellar switch protein FliM. The presence of intact flagella in knockout strains DeltacobB, Deltaacs, Delta(cobB) Delta(acs), Delta(cheA) Delta(cheZ), Delta(cheA) Delta(cheZ) Delta(cobB) and Delta(cheA) Delta(cheZ) Delta(acs) was confirmed by electron microscopy. Genetic analysis of these knockout strains showed that: (i) the DeltacobB mutant exhibited reduced responses to chemotactic stimuli in chemotactic assays, whereas the Deltaacs mutant was indistinguishable from the parental strain, (ii) CheY from the DeltacobB mutant showed a higher level of acetylation, indicating that CobB can mediate the deacetylation of CheY in vivo, and (iii) deletion of cobB reversed the phenotype of Delta(cheA) Delta(cheZ). Our findings suggest that CobB regulates E. coli chemotaxis by deacetylating CheY. Thus a new function of bacterial cobB was identified and also new insights of regulation of bacterial chemotaxis were provided.

MgrB Inactivation Confers Trimethoprim Resistance in Escherichia coli.

After several decades of use, trimethoprim (TMP) remains one of the key access antimicrobial drugs listed by the World Health Organization. To circumvent the problem of trimethoprim resistance worldwide, a better understanding of drug-resistance mechanisms is required. In this study, we screened the single-gene knockout library of Escherichia coli, and identified mgrB and other several genes involved in trimethoprim resistance. Subsequent comparative transcriptional analysis between ΔmgrB and the wild-type strain showed that expression levels of phoP, phoQ, and folA were significantly upregulated in ΔmgrB. Further deleting phoP or phoQ could partially restore trimethoprim sensitivity to ΔmgrB, and co-overexpression of phoP/Q caused TMP resistance, suggesting the involvement of PhoP/Q in trimethoprim resistance. Correspondingly, MgrB and PhoP were shown to be able to modulated folA expression in vivo. After that, efforts were made to test if PhoP could directly modulate the expression of folA. Though phosphorylated PhoP could bind to the promotor region of folA in vitro, the former only provided a weak protection on the latter as shown by the DNA footprinting assay. In addition, deleting the deduced PhoP box in ΔmgrB could only slightly reverse the TMP resistance phenotype, suggesting that it is less likely for PhoP to directly modulate the transcription of folA. Taken together, our data suggested that, in E. coli, MgrB affects susceptibility to trimethoprim by modulating the expression of folA with the involvement of PhoP/Q. This work broadens our understanding of the regulation of folate metabolism and the mechanisms of TMP resistance in bacteria.

Viral coat proteins as flexible nano-building-blocks for nanoparticle encapsulation.

Viral capsid-nanoparticle hybrid structures offer new opportunities for nanobiotechnology. We previously generated virus-based nanoparticles (VNPs) of simian virus 40 (SV40) containing quantum dots (QDs) for cellular imaging. However, as an interesting issue of nano-bio interfaces, the mechanism of nanoparticle (NP) encapsulation by viral coat proteins remains unclear. Here, four kinds of QDs with the same core/shell but different surface coatings are tested for encapsulation. All the QDs can be encapsulated efficiently and there is no correlation between the encapsulation efficiency and the surface charge of the QDs. All the SV40 VNPs encapsulating differently modified QDs show similar structures, fluorescence properties, and activity in entering living cells. These results demonstrate the flexibility of SV40 major capsid protein VP1 in NP encapsulation and provide new clues to the mechanism of NP packaging by viral shells.

Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120.

Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (P(i)) during P(i) starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during P(i) starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase AP(A), coded by all2843, is characterized as a metalloenzyme containing Mg2+ and Zn2+ with molar ratio of 1 : 2. Site-directed mutagenesis analysis indicated that, though the active center of AP(A) is highly conserved in comparison with EAP, differences do exist between AP(A) and EAP in metal ion coordination. Besides, biochemical analysis revealed that AP(A) is a monomeric protein and inactivated rapidly at 50 degrees C. These results suggest that AP(A) is the first monomeric heat-labile classical PhoA found in cyanobacteria.

Cloning and characterization of NAD-dependent protein deacetylase (Rv1151c) from Mycobacterium tuberculosis.

Sir2 family proteins are highly conserved and catalyze a well-characterized NAD-dependent protein deacetylation reaction that regulates multiple cellular processes including aging, gene silencing, cellular differentiation, and metabolic pathways. Little is known about Sir2 family proteins in bacteria. The Sir2 homolog Rv1151c of Mycobacterium tuberculosis was cloned and over-expressed in Escherichia coli, and the protein then purified by Ni(2+)-affinity chromatography to homogeneity. The purified recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide and other known Sir2 inhibitors. The optimal temperature and pH for activity of Rv1151c are 25 degrees C and pH 9 +/- 1, respectively. Rv1151c is capable of deacetylating the acetyl-CoA synthetase from M. tuberculosis. However, unlike Sir2 family proteins identified from other bacteria, Rv1151c shows a substrate-independent NAD glycohydrolase activity in accordance with its auto-ADP ribosylation activity.

Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase.

The nicotinamidase/pyrazinamidase (PncA) of Mycobacterium tuberculosis is involved in the activation of the important front-line antituberculosis drug pyrazinamide by converting it into the active form, pyrazinoic acid. Mutations in the pncA gene cause pyrazinamide resistance in M. tuberculosis. The properties of M. tuberculosis PncA were characterized in this study. The enzyme was found to be a 20.89 kDa monomeric protein. The optimal pH and temperature of enzymatic activity were pH 7.0 and 40 degrees C, respectively. Inductively coupled plasma-optical emission spectrometry revealed that the enzyme was an Mn(2+)/Fe(2+)-containing protein with a molar ratio of [Mn(2+)] to [Fe(2+)] of 1 : 1; furthermore, the external addition of either type of metal ion had no apparent effect on the wild-type enzymatic activity. The activity of the purified enzyme was determined by HPLC, and it was shown that it possessed similar pyrazinamidase and nicotinamidase activity, by contrast with previous reports. Nine PncA mutants were generated by site-directed mutagenesis. Determination of the enzymatic activity and metal ion content suggested that Asp8, Lys96 and Cys138 were key residues for catalysis, and Asp49, His51, His57 and His71 were essential for metal ion binding. Our data show that M. tuberculosis PncA may bind metal ions in a manner different from that observed in the case of Pyrococcus horikoshii PncA.

Escherichia coli mismatch repair protein MutL interacts with the clamp loader subunits of DNA polymerase III.

It has been hypothesized that DNA mismatch repair (MMR) is coupled with DNA replication; however, the involvement of DNA polymerase III subunits in bacterial DNA MMR has not been clearly elucidated. In an effort to better understand the relationship between these 2 systems, the potential interactions between the Escherichia coli MMR protein and the clamp loader subunits of E. coli DNA polymerase III were analyzed by far western blotting and then confirmed and characterized by surface plasmon resonance (SPR) imaging. The results showed that the MMR key protein MutL could directly interact with both the individual subunits delta, delta', and gamma and the complex of these subunits (clamp loader). Kinetic parameters revealed that the interactions are strong and stable, suggesting that MutL might be involved in the recruitment of the clamp loader during the resynthesis step in MMR. The interactions between MutL, the delta and gamma subunits, and the clamp loader were observed to be modulated by ATP. Deletion analysis demonstrated that both the N-terminal residues (1-293) and C-terminal residues (556-613) of MutL are required for interacting with the subunits delta and delta'. Based on these findings and the available information, the network of interactions between the MMR components and the DNA polymerase III subunits was established; this network provides strong evidence to support the notion that DNA replication and MMR are highly associated with each other.

The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA).

As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.