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Five transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR-inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions. Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant-pathogenic bacteria include proteins encoded by hrp genes. For Hrp-related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solancearum, include proteins encoded by fli and flh genes of S. typhimurium, Bacillus subtilis and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.
Mol Microbiol 1995 Mar
PMID:The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex. 762 65

Expression of invasion genes encoded by the large 230-kb plasmid of Shigella flexneri is controlled by the virB gene, which is itself activated by another regulator, virF. Transcription of the invasion genes is temperature regulated, since they are activated in bacteria grown at 37 but not at 30 degrees C. Recently, we have shown that the thermoregulated expression of invasion genes is mediated by thermal activation of virB transcription (T. Tobe, S. Nagai, B. Adler, M. Yoshikawa, and C. Sasakawa, Mol. Microbiol. 5:887-893, 1991). It has also been shown that a mutation that inactivates H-NS, the product of virR (hns), derepresses transcription of virB. To elucidate the molecular mechanisms underlying virB activation, we determined the location of the transcription start site and found it to be 54 bp upstream of the 5' end of the virB coding sequence. Deletion analysis revealed that transcriptional activation by virF requires a DNA segment of 110 bp extending upstream of the transcription start site. By using a protein binding assay with crude extracts of S. flexneri harboring the malE'-'virF fusion gene, which was able to activate virB transcription, two protein species, one of 70 kDa (MalE'-'VirF fusion) and another of 16 kDa (H-NS), were shown to bind specifically to the virB promoter region. DNA footprinting analysis indicated that the VirF fusion and H-NS proteins bound to the upstream sequence spanning from -17 to -117 and to the sequence from -20 to +20, in which virB transcription starts, respectively. In an vitro transcription assay, the VirF fusion protein was shown to activate virB transcription while the H-NS protein blocked it. virB activation was seen only when negatively supercoiled DNA was used as a template. In in vivo studies, virB transcription was significantly decreased by adding novobiocin, a gyrase inhibitor, into the culture medium while virB transcription was increased by mutating hns. These in vitro and in vivo studies indicated that transcription of virB is activated through VirF binding to the upstream sequence of the virB promoter in a DNA-topology-dependent manner and is directly repressed by H-NS binding to the virB transcription start site.
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PMID:Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: activation by virF and repression by H-NS. 769 91

In search for a rational way to convert the information encoded in peptide structures into peptidomimetics, major progress could be made by coupling the power of selection methods, now enormously increased in number as a result of the development of combinatorial peptide libraries, with the rational design of structure-inducing templates for the selectable sequences. The availability of libraries of peptides with predetermined structure would enable selection-driven peptidomimetic design, whereby a conformational model for the peptide pharmacophore would be directly derived from the screening, allowing the design of a suitable non-peptidic scaffold to replace the peptide backbone. We describe here the first example of a conformationally homogeneous combinatorial peptide library, which yields ligands with the expected structure upon selection. The library was built by randomising five positions in the alpha-helical portion of a 26 amino acid Cys2His2 consensus "zinc-finger" motif. Since in zinc-fingers metal coordination and folding are coupled, in our library metal-dependent binding represents a built-in control against the selection of structurally undefined sequences. The alpha-helical library was produced as both fusion with the pVIII protein of filamentous phage and soluble peptides by chemical synthesis, the latter enabling the expansion of the selectable repertoire by the inclusion of non-coded amino acids. The two libraries were independently screened with the same receptor (a monoclonal IgA reactive against the lipopolysaccharide of the human pathogen Shigella flexneri), yielding a very similar consensus. In particular, the peptides defined by both methods showed very strong, zinc-dependent binding to the IgA. The geometrical arrangement of the side-chains of the selected peptide pharmacophore was shown by circular dichroism, Co(II)-complex absorption and high-resolution NMR to be structurally invariant with respect to the parent zinc-finger.
J Mol Biol 1995 Mar 24
PMID:A conformationally homogeneous combinatorial peptide library. 770 66

Growth of Escherichia coli K-12 in low-phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (P1). This response is controlled by a two-component regulatory system of which PhoB and PhoR are the response-regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for P1, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the E. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site-directed mutagenesis revealed that one of these deviations, i.e. Leu-172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.
Mol Microbiol 1995 Jan
PMID:The pho regulon of Shigella flexneri. 774 46

Introducing the Escherichia coli topA20::Tn10 allele to Shigella flexneri results in osmotic sensitivity, a reduced growth rate, an increase in reporter plasmid supercoiling (all common to the E. coli mutants), an inability to grow on MacConkey agar and a loss of virulence gene expression. E. coli mutants harbouring this topA allele often compensate for the loss of DNA topoisomerase I by amplifying the genes coding for topoisomerase IV. Unlike the E. coli topA mutants, derivatives of S. flexneri harbouring this topA allele did not appear to acquire any compensatory mutations. We investigated the possibility that this was due in part to an inability of the S. flexneri topoisomerase IV genes to compensate for loss of DNA topoisomerase I when overexpressed. The S. flexneri genes encoding the alpha- and beta subunits of topoisomerase IV were detected and cloned in separate multicopy plasmids. These plasmids complemented well-characterized Salmonella typhimurium temperature-sensitive topoisomerase IV mutations, showing that the S. flexneri and S. typhimurium proteins are capable of combining to form active complexes. When the S. flexneri topoisomerase IV genes were cloned in the same multicopy plasmid and introduced into a S. flexneri topA mutant, the plasmid restored osmotic tolerance, improved the growth rate, allowed growth on MacConkey indicator plates and resulted in a relaxation of reporter plasmid supercoiling. The same plasmid also partially restored S. flexneri virulence gene transcription. These data show that overexpression of the S. flexneri topoisomerase IV genes can compensate for the loss of topoisomerase I in terms of general viability of the cell, DNA supercoiling, and (partially) virulence gene expression. The fact that S. flexneri does not exploit topoisomerase IV gene amplification as E. coli does points to a significant difference in the abilities of these species to adapt to the loss of topoisomerase I.
Mol Microbiol 1995 Feb
PMID:Overexpression of the Shigella flexneri genes coding for DNA topoisomerase IV compensates for loss of DNA topoisomerase I: effect on virulence gene expression. 778 21

We have isolated two transposon insertion mutations of the pst-phoU operon which result in the constitutive expression of the phoA gene product, alkaline phosphatase. The two mutations also render Escherichia coli invasive towards cultured HEp-2 cells and define a novel Pho-regulated invasion pathway. The presence of the large 'invasion' plasmid derived from an entero-invasive E. coli (EIEC) clinical isolate in these mutants leads to enhanced invasiveness toward cultured HEp-2 cells, a phenomenon referred to as the 'hyper-invasive' phenotype. Transduction of a pst-phoU insertion mutation into clinical isolates of EIEC and Shigella flexneri results in constitutive PhoA expression and coupled hyper-invasiveness in the former but not the latter. We speculate that the Pho-regulated invasion pathway described here, while silent in bacteria grown in standard laboratory rich media, may become functional in the host when invasive bacteria encounter nutrient starvation and/or other related stress conditions.
Mol Microbiol 1993 Dec
PMID:Hyper-invasive mutants define a novel Pho-regulated invasion pathway in Escherichia coli. 793 62

Endotoxin is abortifacient. Abortion may be due to maternal, fetal or combined endotoxemia. The present study was performed to evaluate if fetal rat endotoxemia was induced by maternal endotoxemia in late gestation. An intraperitoneal injection of smooth lipopolysaccharide (Escherichia coli LPS and Shigella flexneri LPS) or rough LPS (Rc mutant Escherichia coli LPS) induced Limulus activity in maternal plasma, but not fetal plasma. These results suggest that fetal rat endotoxemia is not induced during maternal endotoxemia. Thus, the abortion may not be due to fetal endotoxemia.
Res Commun Mol Pathol Pharmacol 1994 Jul
PMID:LPS injected into the pregnant rat late in gestation does not induce fetal endotoxemia. 795 89

The genetic determinants required for invasion of epithelial cells by Shigella flexneri and for the subsequent bacterial spreading are encoded by the large virulence plasmid. Expression of the virulence genes is under the control of various genes on the large plasmid as well as on the chromosome. We previously identified one of the virulence-associated loci near phoBR in the NotI-C fragment of the chromosome of S. flexneri 2a YSH6000 and designated the locus vacC. The vacC mutant showed decreased levels of IpaC, and IpaD proteins as well as transcription of ipa, an operon essential for bacterial invasion (N. Okada, C. Sasakawa, T. Tobe, M. Yamada, S. Nagai, K. A. Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa, Mol. Microbiol. 5:187-195, 1991). To elucidate the molecular nature of the vacC locus, we cloned the vacC region from YSH6000 on a 1.8-kb SalI-BamHI DNA fragment. The nucleotide sequence of the 1,822-bp vacC clone was highly (> 98%) homologous to the tgt region of Escherichia coli K-12, which is located at 9.3 min on the linkage map. Complementation tests indicated that the vacC function was encoded by an open reading frame expressing a 42.5-kDa protein, which corresponded to the tgt gene of E. coli K-12, coding for tRNA-guanine transglycosylase (Tgt) (K. Reuter, R. Slany, F. Ullrich, and H. Kersten, J. Bacteriol. 173:2256-2264, 1991). The cloned tgt gene from E. coli K-12 restored the virulence phenotype to the vacC mutant of YSH6000. Characterization of the vacC mutant indicated that levels of VirG, a protein essential for bacterial spreading, and VirF, the positive regulator for the expression of the virG and ipaBCD operons, decreased significantly compared with those of the wild type. Similar phenotypic changes occurred in vacC mutants constructed by insertion of a neomycin resistance gene in shigellae and enteroinvasive E. coli strains, consistent with the hypothesis that the vacC (tgt) gene contributes to the pathogenicity of Shigella flexneri.
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PMID:vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12. 804 93

Expression of the virB gene, the transcriptional regulator for the invasion genes encoded by the large plasmid of Shigella flexneri, is temperature-regulated. virB transcription is under the control of VirF and H-NS, which act as positive and negative regulators, respectively, and is highly responsive to changes in DNA superhelicity. To further investigate the molecular mechanisms underlying the thermoregulation of virB transcription, a mutant which expressed an invasion phenotype at both 30 degrees C and 37 degrees C was isolated using miniTn10-kan (miniKAN) random insertion mutagenesis. The insertion site was mapped to the rho gene, and resulted in the addition of 11 amino acids to the C-terminus of the Rho protein. Consequently, decreased transcription termination activity at a rho-dependent terminator, lambda tL1, was observed. In the rho mutant, both the transcription of virB and expression of invasion genes were activated at 30 degrees C and were less responsive to changes in temperature. The deregulation of virB expression by the mutation was dependent upon the virB promoter, since the effects of the mutation on virB transcription were abolished when its promoter region was replaced by the tac promoter. Temperature-responsive changes in DNA topology, as determined by linking numbers of a reporter plasmid, showed that changes in DNA superhelicity in the rho mutant were smaller than that in the wild type. Furthermore, when the mutant was grown in medium containing novobiocin, an inhibitor of DNA gyrase, virB transcription at 30 degrees C as well as at 37 degrees C was greatly diminished. These results indicated that Rho protein could have a profound effect on topological temperature-dependent changes in DNA structure, thus contributing to thermoregulation of virB transcription.
Mol Microbiol 1994 Apr
PMID:Deregulation of temperature-dependent transcription of the invasion regulatory gene, virB, in Shigella by rho mutation. 805 51

Flagellin genes (fliC) were detected in two species of the genus Shigella. The fliCSF gene cloned from Shigella flexneri produced normal-type flagella in an Escherichia coli delta fliC strain while the fliCSS genes from two Shigella sonnei strains produced curly-type flagella and their expression is repressible by Salmonella FljA repressor. The fliCSF gene (1650 bp) shared high similarity with the E. coli fliCE gene not only in the 5' and 3' constant sequences but also in the upstream and downstream sequences. The fliCSS genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCS gene in the operator and 3' constant sequences and also shared high similarity with the fliCE gene in the downstream sequence, suggesting that the fliCSS gene has undergone horizontal transfer and recombination. Differences in nucleotide sequences of the central variable regions among the four fliC genes, including fliCE and fliCS, suggest that they started differentiation in each lineage approximately 80 million years ago. Loss of motility in Shigella seems to be evolutionarily a recent event.
Mol Microbiol 1994 Apr
PMID:Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella. 805 52


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