Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcriptional regulation of genes involved in the biosynthesis of cell wall lipids of Mycobacterium tuberculosis is poorly understood. The gene encoding mycocerosic acid synthase (mas) and fadD28, an adjoining acyl coenzyme A synthase gene, involved in the production of a virulence factor, dimycocerosyl phthiocerol, were cloned from Mycobacterium bovis BCG, and their promoters were analyzed. The putative promoters were fused to the xylE reporter gene, and its expression was measured in Escherichia coli, Mycobacterium smegmatis, and M. bovis BCG. In E. coli, the fadD28 promoter was not functional but the mas promoter was functional. Both fadD28 and mas promoters were functional in M. smegmatis, at approximately two- and sixfold-higher levels, respectively, than the BCG hsp60 promoter. In M. bovis BCG, the fadD28 and mas promoters were functional at three- and fivefold-higher levels, respectively, than the hsp60 promoter. Primer extension analyses identified transcriptional start points 60 and 182 bp upstream of the translational start codons of fadD28 and mas, respectively. Both promoters contain sequences similar to the canonical -10 and -35 hexamers recognized by the sigma(70) subunit of RNA polymerase. Deletions of the upstream regions of both genes indicated that 324 bp of the fadD28 and 228 bp of the mas were essential for promoter activity. Further analysis of the mas promoter showed that a 213-bp region 581 bp upstream of the mas promoter acted as a putative transcriptional enhancer, promoting high-level expression of the mas gene when present in either direction. This represents the identification of a rare example of an enhancer-like element in mycobacteria.
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PMID:Regulation of expression of mas and fadD28, two genes involved in production of dimycocerosyl phthiocerol, a virulence factor of Mycobacterium tuberculosis. 1244 29

Multidrug resistance among new cases of tuberculosis (TB) is increasingly becoming a significant problem in countries with a high prevalence of TB and with inadequate therapies for TB. Rifampin resistance is widely used as a marker for multidrug-resistant (MDR) TB; therefore, a new approach to the retrospective measurement of rifampin resistance without the need of a viable culture has been introduced. In many developing countries culture is unavailable and diagnosis relies on clinical manifestations and the results of Ziehl-Neelsen staining of sputum smears. We determined rifampin resistance directly with DNA extracts from Ziehl-Neelsen-stained slides by identification of mutations in the rpoB gene using reverse line blot hybridization and DNA sequencing. Analysis of the rpoB gene revealed that samples containing rifampin-resistant Mycobacterium tuberculosis carried altered codons representing amino acid positions 516, 526, and 531 of the RNA polymerase. Although the sensitivities of both methods were equal (84%), sequencing of the rpoB gene was more accurate in identifying mutations in the core region of the rpoB gene. Sequence analysis of the rpoB gene in extracts from Ziehl-Neelsen-stained slides may be used to quantify more precisely the magnitude of MDR TB and, more importantly, provide information on trends in the development of resistance on a global scale. The nature of rifampin resistance and the genotype can be determined by analysis of Ziehl-Neelsen-stained slides in a laboratory equipped for sequencing and spoligotyping without the need to ship biohazardous materials.
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PMID:Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis. 1262 36

Sudden infant death syndrome (SIDS) has been associated with the volatilization of arsenic, antimony or phosphorus compounds from infants' bedding material by micro-organisms, the so-called 'toxic gas hypothesis'. The volatilization of arsenic by aerobic micro-organisms isolated from new sheepskin bedding material, as well as on material used by a healthy infant and by an infant who perished of SIDS, was examined. Three fungi were isolated from a piece of sheepskin bedding material on which an infant perished of SIDS, which methylated arsenic to form trimethylarsenic(V) species, precursors to volatile trimethylarsine. These three fungi were identified as Scopulariopsis koningii, Fomitopsis pinicola and Penicillium gladioli by their 26S-ribosomal RNA polymerase chain reaction products. These fungi were not previously known to methylate arsenic. The volatilization of arsenic by these three fungi was then examined. Only P. gladioli volatilized arsenic and only under conditions such that the production of sufficient trimethylarsine to be acutely toxic to an infant is unlikely. S. brevicaulis grew on the sheepskin bedding material and evolved a trace amount of trimethylarsine. Known human pathogens such as Mycobacterium neoaurum and Acinetobacter junii were isolated from used bedding.
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PMID:Arsenic methylation by micro-organisms isolated from sheepskin bedding materials. 1285 55

In a systematic approach to understand the transcriptional machinery of mycobacteria, we had previously isolated and characterized mycobacterial promoter regions. In this study, we have investigated molecular interactions between mycobacterial RNA polymerase holoenzyme, reconstituted with different sigma subunits and the promoter element of the Mycobacterium tuberculosis gene pknH (Rv1266c), a representative of promoters belonging to the 'extended -10' class. In vitro transcription assays using the pknH promoter and reconstituted RNA polymerase holoenzyme demonstrated that transcription from the pknH promoter is specifically initiated by sigmaA, the principal sigma factor of mycobacteria. DNase I protection assay and deletion studies with the pknH promoter revealed that the minimal region required for optimal transcription carries the sequence from position -37 to position +6. Moreover, mutation in the TGN motif of the pknH promoter resulted in the loss of >75% of its activity. Binding of RNA polymerase with wild-type promoter as well as its TG- mutant revealed that the TGN motif is required for the transition from a close complex into an open complex. Further, it was observed that the presence of the TGN motif reduces the thermal energy required for the conversion of a close complex into an open complex, necessary for initiation of transcription.
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PMID:Role of 5'-TGN-3' motif in the interaction of mycobacterial RNA polymerase with a promoter of 'extended -10' class. 1290 24

In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.
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PMID:Towards a phosphoproteome map of Corynebacterium glutamicum. 1292 88

The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein.
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PMID:PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response. 1457 53

In this study, the expression of the Mycobacterium tuberculosis PE, PE_polimorphic GC-rich sequences (PGRS) gene family encoding approximately 99 glycine-rich proteins was assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) in M. tuberculosis H37Rv, Mycobacterium canettii and two clinical isolates of M. tuberculosis. Restriction analyses and sequencing of the RT-PCR products showed that all the strains expressed the PE Rv1172c gene while the PE_PGRS Rv3652 gene was only expressed by one of the M. tuberculosis clinical isolates, and the PE_PGRS Rv0578c was not expressed by M. canettii. It was also determined that the PE_PGRS Rv0278c and Rv0279c were not expressed by any of the studied strains. The data presented in this report show that the PE, PE_PGRS genes are differentially expressed in M. tuberculosis strains during in vitro growth. These findings suggest that PE, PE_PGRS genes may play a role in protein variation between M. tuberculosis strains.
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PMID:Differential expression of PE and PE_PGRS genes in Mycobacterium tuberculosis strains. 1458

We report the crystal structure of N-utilizing substance A protein (NusA) from Thermotoga maritima (TmNusA), a protein involved in transcriptional pausing, termination, and antitermination. TmNusA has an elongated rod-shaped structure consisting of an N-terminal domain (NTD, residues 1-132) and three RNA binding domains (RBD). The NTD consists of two subdomains, the globular head and the helical body domains, that comprise a unique three-dimensional structure that may be important for interacting with RNA polymerase. The globular head domain possesses a high content of negatively charged residues that may interact with the positively charged flaplike domain of RNA polymerase. The helical body domain is composed of a three-helix bundle that forms a hydrophobic core with the aid of two neighboring beta-strands. This domain shows structural similarity with one of the helical domains of sigma(70) factor from Escherichia coli. One side of the molecular surface shows positive electrostatic potential suitable for nonspecific RNA interaction. The RBD is composed of one S1 domain and two K-homology (KH) domains forming an elongated RNA binding surface. Structural comparison between TmNusA and Mycobacterium tuberculosis NusA reveals a possible hinge motion between NTD and RBD. In addition, a functional implication of the NTD in its interaction with RNA polymerase is discussed.
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PMID:Crystal structure of NusA from Thermotoga maritima and functional implication of the N-terminal domain. 1462 88

Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial beta subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited <2% partial rpoB gene sequence divergence from 1 of 20 species under study and were regarded as correctly identified at the species level. Mycobacterium abscessus and Mycobacterium mucogenicum isolates were clearly distinguished from Mycobacterium chelonae; Mycobacterium mageritense isolates were clearly distinguished from "Mycobacterium houstonense." Four isolates were not identified at the species level because they exhibited >3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM.
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PMID:rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria. 1466 64

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences ( approximately 500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.
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PMID:Novel mass spectrometry-based tool for genotypic identification of mycobacteria. 1471 74


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