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)

Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.
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PMID:The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants. 140 Feb 38

One of the earliest steps in the pathogenic cycle of the facultative intracellular pathogen Salmonella spp. is the invasion of the cells of the intestinal epithelium. We have previously identified a genetic locus, inv, that allows Salmonella spp. to enter cultured epithelial cells. invA is a member of this locus, and it is the first gene of an operon consisting of at least two additional invasion genes. We have constructed strains carrying nonpolar mutations in invA and examined the individual contribution of this gene to the invasion phenotype of Salmonella typhimurium. Nonpolar S. typhimurium invA mutants were deficient in invasion of cultured epithelial cells although they were fully capable of attaching to the same cells. In addition, unlike wild-type S. typhimurium, invA mutants did not alter the normal architecture of the microvilli of polarized epithelial cells nor did they cause any alterations in the distribution of actin microfilaments of infected cells. The invasion phenotype of invA mutants was readily rescued by wild-type S. typhimurium when cultured epithelial cells were simultaneously infected with both strains. On the contrary, in a similar experiment, the adherent Escherichia coli strain RDEC-1 was not internalized into cultured cells when coinfected with wild-type S. typhimurium. The invA locus was found to be located at about 59 min on the Salmonella chromosome, 7% linked to mutS. The nucleotide sequence of invA showed an open reading frame capable of encoding a polypeptide of 686 amino acids with eight possible membrane-spanning regions and a predicted molecular weight of 75,974. A protein of this size was visualized when invA was expressed in a bacteriophage T7 RNA polymerase-based expression system. The predicted sequence of InvA was found to be homologous to Caulobacter crescentus FlbF, Yersinia LcrD, Shigella flexneri VirH, and E. coli FlhA proteins. These proteins may form part of a family of proteins with a common function, quite possibly the translocation of specific proteins across the bacterial cell membrane.
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PMID:Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. 162 29

We have investigated the organization and expression of the Caulobacter crescentus flbF gene because it occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the 3' end of the flaO operon and the adjacent flbF promoter and structural gene was determined, and the organization of transcription units within this sequence was investigated. We located the 3' ends of the flaO operon transcript by using a nuclease S1 protection assay, and the 5' end of the flbF transcript was precisely mapped by primer extension analysis. The nucleotide sequence upstream from the 5' end of the flbF transcript contains -10 and -35 elements similar to those found in promoters transcribed by sigma 28 RNA polymerase in other organisms. Mutations that changed nucleotides in the -10 or -35 elements or altered their relative spacing resulted in undetectable levels of flbF transcript, demonstrating that these sequences contain nucleotides essential for promoter function. We identified a 700-codon open reading frame, downstream from the flbF promoter region, that was predicted to be the flbF structural gene. The amino-terminal half of the FlbF amino acid sequence contains eight hydrophobic regions predicted to be membrane-spanning segments, suggesting that the FlbF protein may be an integral membrane protein. The FlbF amino acid sequence is very similar to that of a transcriptional regulatory protein called LcrD that is encoded in the highly conserved low-calcium-response region of virulence plasmid pYVO3 in Yersinia enterocolitica (A.-M. Viitanen, P. Toivanen, and M. Skurnik, J. Bacteriol. 172:3152-3162, 1990).
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PMID:Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid. 173 19

A simple multiplex riboprobe system for detection of virulent Yersinia enterocolitica was developed using a pool of RNA probes specific for various chromosomal and plasmid-borne virulence gene sequences (yadA, virC, ail and yst). Riboprobes were synthesized by a rapid, simple and efficient technique involving in vitro transcription of polymerase chain reaction-generated templates incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the priming oligonucleotides. After dot blotting target DNA samples on nitrocellulose and hybridization with the riboprobes, the RNA: DNA hybrids formed were detected by a simple immunoenzymic assay involving sequential reactions with an anti-RNA : DNA hybrid monoclonal antibody, anti-mouse Ig-peroxidase conjugate and chromogenic or chemiluminescent enzyme substrate solution. This multiplex riboprobe system targeting both chromosomal and plasma-borne sequences permitted detection of virulent Y. enterocolitica, regardless of plasmid loss during handling of cultures, and was unreactive with a virulent Y. enterocolitica, other Yersinia and other bacteria. This system resulted in a significant improvement in the limit of detection in comparison to that obtained with individual probes.
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PMID:Multiplex riboprobes for the detection of virulent Yersinia enterocolitica and simple methods for their preparation. 759 16

The chromosome of Yersinia enterocolitica encodes a heat-stable enterotoxin called Yst and a surface antigen called Myf, which closely resembles enterotoxin-associated fimbriae. Both factors could act in conjunction to produce diarrhea. Production of the enterotoxin is regulated by temperature, osmolarity, and pH and occurs only when bacteria reach the stationary phase. Myf production is regulated by temperature and pH and, as we show in this work, also occurs after the exponential growth phase. In an attempt to understand the late-phase expression of yst and myf, we cloned, sequenced, and mutagenized the gene encoding RpoS, an alternative sigma factor of the RNA polymerase involved in expression of stationary-phase genes in other enterobacteria. An intact rpoS gene was necessary for full expression of yst in the stationary phase but not for the expression of myf and of pYV-encoded virulence determinants.
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PMID:The rpoS gene from Yersinia enterocolitica and its influence on expression of virulence factors. 772 93

The Yersinia enterocolitica surface antigen Myf is a fibrillar structure that resembles CS3 fimbriae. Gene myfA encodes the 21-kDa major subunit of the antigen, while genes myfB and myfC are required for the transport and assembly of pilin subunits at the bacterial cell surface. Here we show that the expression of Myf is regulated at the transcriptional level by temperature and pH. Gene myfA is transcribed at 37 degrees C and in acidic medium. The transcription start is preceded by a putative -10 box for the vegetative RNA polymerase as well as by sequences resembling the consensus sequence recognized by sigma 28. Thus, myfA could be transcribed either from a classical sigma 70 promoter or from a sigma 28 promoter. Transcription of myfA requires at least two genes, myfF and myfE, situated immediately upstream from myfA. The myfF product does not show similarity to any known regulatory protein. It is an 18.5-kDa protein with no typical helix-turn-helix motif and a unique hydrophobic domain in the NH2-terminal part. T7 expression, osmotic shock, fractionation experiments, and TnphoA fusion analyses carried out in Escherichia coli suggest that MyfF is associated with the inner membrane by means of its hydrophobic domain whereas the hydrophilic part protrudes in the periplasm. These features strikingly evoke ToxS, a protein involved in regulation of Tcp pilus production in Vibrio cholerae. MyfE resembles PsaE, a protein involved in regulation of pH6 antigen in Yersinia pestis. Genes myfF and myfE are presumably part of a whole regulatory network. MyfF could be an element of the signal transducing system.
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PMID:MyfF, an element of the network regulating the synthesis of fibrillae in Yersinia enterocolitica. 783 9

Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human colon and ileocecum. Two separate loci (tia and tib) that direct noninvasive E. coli HB101 to adhere to and invade intestinal epithelial cells have previously been cosmid cloned from ETEC H10407. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cellular fractions from tib-positive HB101 shows that the tib locus directs the synthesis of a 104-kDa outer membrane protein (the TibA protein). The tib locus was subcloned to a maximum of 6.7 kb and mutagenized with transposon Tn5. Production of TibA was directly correlated with the capacity of the subclones and Tn5 mutants to invade and adhere to epithelial cells, suggesting that TibA was required for these phenotypes. The position and direction of transcription of the tibA gene were identified by complementation and in vivo T7 RNA polymerase-promoter induction experiments. The role of the tib locus in epithelial cell invasion was confirmed by the construction of chromosomal deletion derivatives in H10407. These deletion mutants invaded epithelial cells at about 15% of the parental level and were fully complemented by plasmids bearing the tib locus. The size and function of the TibA protein are similar to those of invasin from Yersinia pseudotuberculosis (103 kDa). However, a tib probe did not hybridize with the gene encoding invasin. Hybridization analyses of genomic DNA from a wide variety of pathogenic and nonpathogenic bacteria, including Salmonella, Shigella, Yersinia, and Escherichia species, indicate that the tib locus is unique to specific ETEC strains.
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PMID:Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein. 803 17

Biogenesis of the toxin-coregulated pilus (TCP) of Vibrio cholerae 01 is essential for successful bacterial colonization of the small intestine. Pilus assembly requires the products of at least seven genes located on the chromosome adjacent to the pilin-encoding gene, tcpA. Previously reported TnphoA insertions in the TCP-assembly-deficient V. cholerae strains, KP2.21 and KP4.2, were isolated from the chromosome for further analysis. Nucleotide sequencing of the tcpE::phoA and tcpF::phoA fusions and corresponding clones of the region containing the intact genes revealed the presence of two open reading frames (ORFs) of 340 and 338 amino acids, designated TcpE and TcpF, respectively. The partial sequence of an ORF downstream from the TcpF coding sequence was determined to correspond to the global virulence regulator, ToxT. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Computer-generated alignment algorithms predict that a homology exists between TcpE and the Klebsiella pneumoniae pullulanase secretion proteins PulD and PulF, the Xanthomonas campestris extracellular enzyme secretion factor XpsF, the Bacillus subtilis DNA competence protein ComG-ORF2, and the Yersinia enterocolitica Yop secretion determinant YscC. These observations provide a model to investigate further the relationship between the secretion mechanisms utilized by these seemingly diverse virulence determinants. Additionally, an extreme C-terminal segment of TcpE shows striking homology to the transmembrane segment of the eukaryotic integrin beta-1 chain, which could imply a role for TcpE in not only TCP secretion, but also host cell interaction.
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PMID:Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems. 809 77

Our laboratories have independently identified a gene in Salmonella choleraesuis and Salmonella typhimurium that is necessary for efficient adherence and entry of these organisms into cultured epithelial cells. Introduction of a mutated gene into several Salmonella strains belonging to different serotypes rendered these organisms deficient for adherence and invasion of cultured cells. This effect was most pronounced in the host-adapted serotypes Salmonella gallinarum, S. choleraesuis, and Salmonella typhi. The nucleotide sequence of this gene, which we have termed invH, encodes a predicted 147-amino-acid polypeptide containing a signal sequence. The InvH predicted polypeptide is highly conserved in S. typhimurium and S. choleraesuis, differing at only three residues. The invH gene was expressed in Escherichia coli using a T7 RNA polymerase expression system and a polypeptide of approximately 16,000 molecular weight was observed, in agreement with the predicted size of its gene product. Upon fractionation, the expressed polypeptide was localized in the bacterial membrane fraction. Southern and colony hybridization analyses indicated that the invH gene is present in all Salmonella strains tested (91 strains belonging to 37 serotypes) with the exception of strains of Salmonella arizonae. No homologous sequences were detected in Yersinia, Shigella, Proteus, and several strains of enteroinvasive and enteropathogenic E. coli. Downstream from the S. choleraesuis (but not S. typhimurium) invH gene, a region with extensive homology to the insertion sequence IS3 was detected.
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PMID:Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells. 838 33

The RfaH protein controls the transcription of a specialized group of Escherichia coli and Salmonella operons that direct the synthesis, assembly and export of the lipopolysaccharide core, exopolysaccharide, F conjugation pilus and haemolysin toxin. RfaH is a specific regulator of transcript elongation; its loss increases transcription polarity in these operons without affecting initiation from the operon promoters. The operons of the RfaH-dependent regulon contain a short conserved 5' sequence, the ops element, deletion of which increases operon polarity to an extent similar to that caused by loss of RfaH. The ops element is also present upstream of polysaccharide gene clusters of Shigella flexneri, Yersinia enterocolitica, Vibrio cholerae and Klebsiella pneumoniae and the RP4 fertility operon of Pseudomonas aeruginosa, suggesting that this is a widely spread control system. The mechanistic coupling of RfaH and the ops element has been demonstrated in vitro and in vivo, and we suggest that the ops element recruits RfaH and potentially other factors to the RNA polymerase complex, modifying the complex to increase its processivity and allowing transcription to proceed over long distances.
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PMID:RfaH and the ops element, components of a novel system controlling bacterial transcription elongation. 942 23


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