Dehalogenation of haloalkanes by Mycobacterium tuberculosis H37Rv and other mycobacteria

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.     

Rv0802c acetyltransferase from Mycobacterium tuberculosis H37Rv

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.     

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.Abbreviations DNA deoxyribonucleic acid - td delay in initiation - OD optical density - CAM chloramphenicol - RIF rifampicin     

Rv1915 and Rv1916 from Mycobacterium tuberculosis H37Rv form in vitro protein-protein complex

BackgroundMycobacterium tuberculosis (Mtb) isocitrate lyase (ICL) is an established drug target that facilitates Mtb persistence. Unlike other mycobacterial strains, where ICL2 is a single gene product, H37Rv has a split event, resulting in two tandemly coded icls - rv1915 and rv1916. Our recent report on functionality of individual Rv1915 and Rv1916, led to postulate the cooperative role of these proteins in pathogen's survival under nutrient-limiting conditions. This study investigates the possibility of Rv1915 and Rv1916 interacting and forming a complex.MethodsPull down assay, activity assay, mass spectrometry and site directed mutagenesis was employed to investigate and validate Rv1915-Rv1916 complex formation.ResultsRv1915 and Rv1916 form a stable complex in vitro, with enhanced ICL/MICL activities as opposed to individual proteins. Further, activities monitored in the presence of acetyl-CoA show significant increase for Rv1916 and the complex but not of Rv0467 and Rv1915Δ90CT. Both full length and truncated Rv1915Δ90CT can form complex, implying the absence of its C-terminal disordered region in complex formation. Further, in silico analysis and site-directed mutagenesis studies reveal Y64 and Y65 to be crucial residues for Rv1915-Rv1916 complex formation.ConclusionsThis study uncovers the association between Rv1915 and Rv1916 and supports the role of acetyl-CoA in escalating the ICL/MICL activities of Rv1916 and Rv1915Δ90CT-Rv1916 complex.General significancePartitioning of ICL2 into Rv1915 and Rv1916 that associates to form a complex in Mtb H37Rv, suggests its importance in signaling and regulation of metabolic pathway particularly in carbon assimilation.     

Fatty acid regulation of adenylyl cyclase Rv2212 from Mycobacterium tuberculosis H37Rv

Adenylyl cyclase Rv2212 from Mycobacterium tuberculosis has a domain composition identical to the pH-sensing isoform Rv1264, an N-terminal regulatory domain and a C-terminal catalytic domain. The maximal velocity of Rv2212 was the highest of all 10 mycobacterial cyclases investigated to date (3.9 micromol cAMP.mg(-1).min(-1)), whereas ATP substrate affinity was low (SC(50) = 2.1 mm ATP). Guanylyl cyclase side activity was absent. The activities and kinetics of the holoenzyme and of the catalytic domain alone were similar, i.e. in distinct contrast to the Rv1264 adenylyl cyclase, in which the N-terminal domain is autoinhibitory. Unsaturated fatty acids strongly stimulated Rv2212 activity by increasing substrate affinity. In addition, fatty acids greatly enhanced the pH sensitivity of the holoenzyme, thus converting Rv2212 to a pH sensor adenylyl cyclase. Fatty acid binding to Rv2212 was modelled by homology to a recent structure of the N-terminal domain of Rv1264, in which a fatty acid-binding pocket is defined. Rv2212 appears to integrate three cellular parameters: ATP concentration, presence of unsaturated fatty acids, and pH. These regulatory properties open the possibility that novel modes of cAMP-mediated signal transduction exist in the pathogen.     

Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K _m and V _max of enzyme was determined to be 21.29 U mg^?1 protein, 714.28 μM and 62.5 μmol ml^?1 min^?1, respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser⁵⁰, Asp¹⁸⁰ and His²⁰³ residues by site-directed mutagenesis.     

Biosynthesis of nucleic acid purines in Mycobacterium tuberculosis H37Rv

1. The biosynthesis of nucleic acid purine in Mycobacterium tuberculosis H(37)R(v) has been studied by using (14)C-labelled precursors. 2. The results indicate that C-2 and C-8 of the purine ring are derived most efficiently from serine and glycine and not from formate. 3. [(14)C]Methionine is not incorporated into the ureide carbon atoms of the purine ring.     

Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv

Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis. complement.     

Purification and properties of DNA polymerase from Mycobacterium tuberculosis H37Rv

DNA polymerase has been purified approximately 2000-fold from Mycobacterium tuberculosis H37Rv. The purified preparation was homogeneous by electrophoretic criteria and has a molecular weight of 135 000. The purified enzyme resembles Escherichia coli polymerase I in its properties, being insensitive to sulfhydryl drugs and possessing 5',3'-exonuclease activity in addition to polymerase and 3',5'-exonuclease activities. However, it differs from the latter in its sensitivity to higher salt concentration and DNA intercalating agents such as 8-aminoquinoline. The polymerase exhibited maximal activity between 37--42 degrees C and pH 8.8--9.5. The polymerase was stable for several months below 0 degree C. However, the 5',3'-exonuclease activity was more labile. The effects of different metal ions, polyamines and drugs on the polymerase activity are presented.     

Cloning and characterization of aspartate-beta-semialdehyde dehydrogenase from Mycobacterium tuberculosis H37 Rv

AIMS: To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv. METHODS AND RESULTS: The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K(m) and V(max) values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined. CONCLUSIONS: Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K(cat) and specific activity of the enzyme were 8.49 s(-1) and 13.4 micromol min(-1) microg(-1) respectively. Significance and Impact of the Study: The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB.     

Expression and production of soluble Mycobacterium tuberculosis H37Rv mycosin-3

Mycobacteria encode five type VII secretion system (T7SS) or ESX for nutrient acquisition and virulence. Mycosins are membrane-anchored components of ESX with serine protease activity but an unidentified substrate range. Establishing the substrate specificity of individual mycosins will help to elucidate individual ESX functions. Mycosin-1 and -3 orthologues from two environmental mycobacterial species, Mycobacterium smegmatis and Mycobacterium thermoresistibile, have been heterologously produced, but mycosins from Mycobacterium tuberculosis (Mtb) remain to be studied. Here we describe the successful production of Mtb mycosin-3 as a first step in investigating its structure and function.     

Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.     

Molecular characterization of secretory proteins Rv3619c and Rv3620c from Mycobacterium tuberculosis H37Rv

Rv3619c and Rv3620c are the secretory, antigenic proteins of the ESAT-6/CFP-10 family of Mycobacterium tuberculosis H37Rv. In this article, we show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric complex with a dissociation constant (K(d)) of 4.8 × 10(-7) M. The thermal unfolding of the heterodimer was completely reversible, with a T(m) of 48 °C. The comparative thermodynamics and thermal unfolding analysis of the Rv3619c-Rv3620c dimer, the ESAT-6-CFP-10 dimer and another ESAT family heterodimer, Rv0287-Rv0288, revealed that the binding strength and stability of Rv3619c-Rv3620c are relatively lower than those of the other two pairs. Molecular modeling and docking studies predict the structure of Rv3619c-Rv3620c to be similar to that of ESAT-6-CFP-10. Spectroscopic studies revealed that, in an acidic environment, Rv3619c and Rv3620c lose their secondary structure and interact weakly to form a complex with a lower helical content, indicating that Rv3619c-Rv3620c is destabilized at low pH. These results, combined with those of previous studies, suggest that unfolding of the proteins is required for dissociation of the complex and membrane binding. In the presence of membrane mimetics, the α-helical contents of Rv3619c and Rv3620 increased by 42% and 35%, respectively. In mice, the immune response against Rv3619c protein is characterized by increased levels of interferon-γ, interleukin-12 and IgG(2a) , indicating a dominant Th1 response, which is mandatory for protection against mycobacterial infection. This study therefore emphasizes the potential of Rv3619c as a subunit vaccine candidate.     

差显技术分析结核杆菌H37Rv与H37Ra差异表达的基因

采用差异显示技术比较了结核分支杆菌强毒株H37Rv和弱毒株H37Ra在体外培养条件下的基因表达差异。通过20种引物组合进行mRNA差异显示,克隆到了两菌株间的20余个差异表达基因,经序列分析及杂交鉴定发现其中2个基因仅在H37Rv中表达。其中Rv1894c基因编码的可能是H37Rv中的一个新蛋白。而在H37Ra的基因组中含有这2个基因的编码序列,但均未检测到基因的表达。     

结核分枝杆菌H37Rv新基因Rv2742克隆表达及纯化

Rv2742是本课题组前期基于蛋白质基因组学策略从结核分枝杆菌Mycobacteriumtuberculosis H37Rv中发现、鉴定的遗漏注释基因。文中旨在建立结核分枝杆菌H37Rv漏注释蛋白Rv2742的可溶性诱导表达、纯化体系,为进一步探索Rv2742基因参与的生物学功能奠定基础。前期实验发现构建的pGEX-4T-2-Rv2742、pET-28a-Rv2742、pET-32a-Rv2742及pMAL-c2X-Rv2742原核表达载体均无法实现目的蛋白的诱导表达。但经密码子优化后,仅有pMAL-c2X-Rv2742载体能够实现目的蛋白的可溶性诱导表达。此外,通过比较不同宿主菌、温度及IPTG浓度对目的蛋白表达量的影响,发现目的蛋白在Rosetta (DE3)中,16℃及0.5mmol/LIPTG诱导条件下表达量最高。直链淀粉树脂(Amyloseresin)亲和层析柱纯化获得较纯的产物,经LC-MS/MS验证确认是Rv2742融合蛋白肽段序列。成功获得结核分枝杆菌H37Rv新基因Rv2742的重组蛋白,可进一步开展其潜在相互作用及免疫原性研究工作。