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Yersinia enterocolitica cells, when cultured at 30 degrees C or below, are flagellated and motile. Cells cultured at 37 degrees C or above lack flagella and are non-motile. To identify flagellin genes that are a target of this temperature-dependent regulation, a library of Y. enterocolitica genomic inserts in a phage lambda vector was probed with the Salmonella typhimurium fliC (flagellin) gene. A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non-motile S. typhimurium fliC-fljB- (flagellin-minus) mutant. DNA sequence analysis shows that Y. enterocolitica contains three tandem flagellin genes, designated fleA, fleB and fleC. All three genes are co-ordinately transcribed at low, but not high, temperature from fliA-dependent (sigma F) promoters. Flagellin transcription arrests rapidly after upshift to 37 degrees C (host temperature). In contrast, flagellin transcription resumes only after several generations when cells cultured at 37 degrees C are downshifted to 28 degrees C.
Mol Microbiol 1995 Jul
PMID:Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes. 747 8

The genomes of three main biovars of Yersinia pestis were subjected to restriction fragment length polymorphism analysis using I-CeuI endonuclease. I-CeuI which is encoded by a mobile intron in Chlamydomonas engamenans recognizes a 25-bp site in the ribosomal RNA rrl gene and cuts DNA of most representatives of Enterobacteriaceae into seven fragments corresponding to the presence of seven rrn-operons. Glycerol-positive Y. pestis strains (biovars antiqua and mediaevalis) contain seven ribosomal operons which can be recognized by I-CeuI endonuclease. However, glycerol-negative strains of Y. pestis biovar orientalis expose only six restriction sites for I-CeuI. The restriction fragment length polymorphism patterns obtained with I-CeuI make it possible to distinguish between three biovars of Y. pestis. Use of another rare cutting restriction enzyme, Bln/I, permits differentiation between pigment-adsorbing and avirulent non-pigment-adsorbing Y. pestis. Still, due to homologous recombination between the two copies of IS 100 insertion sequence bracketing the pgm-locus, the mechanism of deletions in the pgm-locus seems to be confined only to strains of biovars antiqua and mediaevalis, and can be different in Y. pestis strains of biovar orientalis. The I-CeuI restriction patterns of two Yersinia strains isolated within a ten-year period in the port of St. Petersburg and originally identified as Y. pseudotuberculosis 01 turned out to be related to typical representatives of Y. pestis biovar antiqua. These strains could be exported from the same source or circulate among Rattus norvegicus population of the port as non-pigment-adsorbing avirulent immunogenic clone.
Mol Gen Mikrobiol Virusol
PMID:The established Yersinia pestis biovars are characterized by typical patterns of I-CeuI restriction fragment length polymorphism. 747 35

Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high-performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with approximately 20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5' transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N-acylhomoserine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two-dimensional SDS-PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.
Mol Microbiol 1995 Jul
PMID:Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules. 749 83

The inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37 degrees C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37 degrees C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild-type inv gene and the inv::phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23 degrees C. Y. enterocolitica grown at 37 degrees C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 degrees C. An increase in Na+ ions caused a slight increase in expression at 37 degrees C. However, expression at 37 degrees C was unaffected by anaerobiosis, growth medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyer's patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in Y. enterocolitica may remain elevated early during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.
Mol Microbiol 1994 Jan
PMID:Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene. 751 72

Campylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem-loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3' cleavage site maps within the putative stem-loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.
Mol Microbiol 1994 Oct
PMID:Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni. 753 Mar 17

The Pseudomonas syringae pathovars are composed of host-specific plant pathogens that characteristically elicit the defense-associated hypersensitive response (HR) in nonhost plants. P. s. pv. syringae 61 secretes an HR elicitor, harpinPss (HrpZPss), in a hrp-dependent manner. An internal fragment of the P. s. pv. syringae 61 hrpZ gene was used to clone the hrpZ locus from P. s. pv. glycinea race 4 (bacterial blight of soybean) and P. s. pv. tomato DC3000 (bacterial speck of tomato). DNA sequence analysis revealed that hrpZ is the second ORF in a polycistronic operon. The amino acid sequence identities of HrpZPss/HrpZPsg and HrpZPss/HrpZPst were 79 and 63%, respectively. Although none of the HrpZ proteins showed significant overall sequence similarity with other known proteins, HrpZPst contained a 24-amino acid sequence that is homologous with a region of the PopA1 elicitor protein of the tomato pathogen, Pseudomonas solanacearum GMI1000. hrpA, the upstream ORF, was highly divergent: The amino acid sequence identities of HrpAPss/HrpAPsg and HrpAPss/HrpAPst were 91 and 28%, respectively, and no HrpA sequence showed similarity to known proteins. In contrast, the predicted products of the downstream ORFs in P. s. pv. syringae and P. s. pv. tomato, hrpB, hrpC, hrpD, and hrpE showed varying levels of similarity to those of yscI, yscJ, yscK, and yscL. These are colinearly arranged genes in the virC locus of Yersinia spp., which are involved in the secretion of the Yop virulence proteins via the type III pathway. The similarity of the Ysc proteins was generally stronger in comparisons with the P. s. pv. tomato Hrp proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Plant Microbe Interact
PMID:The HrpZ proteins of Pseudomonas syringae pvs. syringae, glycinea, and tomato are encoded by an operon containing Yersinia ysc homologs and elicit the hypersensitive response in tomato but not soybean. 757 16

Pseudomonas syringae pv. syringae 61 contains a 25-kb hrp cluster that is sufficient to elicit the hypersensitive response (HR) in nonhost plants. Previous studies have shown that mutations in complementation groups VIII, IX, and XI in the hrp cluster abolished the ability of the bacterium to cause the HR. The sequence of a 3.7-kb SmaI-SstI fragment covering groups VIII and IX now reveals five open reading frames (ORFs) in the same transcript, designated as hrpU, hrpW, hrpO, hrpX, and hrpY, and predicted to encode proteins of 14,795, 23,211, 9,381, 28,489, and 39,957 Da, respectively. The hrpU, hrpW, hrpO, hrpX, and hrpY genes are homologous and arranged colinearly with the yscQ/spa33/spaO, yscR/spa24/spaP, yscS/spa9/spaQ, yscT/spa29/spaR, and yscU/spa40/spaS genes of Yersinia spp., Shigella flexneri, and Salmonella typhimurium, respectively. These proteins also show similarity to Fli/Flh proteins of Bacillus and enteric bacteria. The Ysc and Spa proteins are involved in the secretion of virulence factors, like the Yop and Ipa proteins. Fli/Flh proteins are involved in flagellar biogenesis. The sequence of a 2.9-kb EcoRV-EcoRI DNA fragment containing mainly group XI revealed five ORFs, designated hrpC, hrpD, hrpE, hrpF, and hrpG, predicted to encode proteins of 29,096, 15,184, 21,525, 7,959, and 13,919 Da, respectively. The first three genes belong to an operon containing hrpZ, which encodes an extracellular protein that elicits the HR. hrpF and hrpG are two potential ORFs upstream of hrpH in the hrpH operon. HrpC is homologous to Yersinia YscJ, Pseudomonas solanacearum HrpI, Xanthomonas compestris pv. vesicatoria HrpB3, and Rhizobium fredii NolT. HrpE is similar to YscL of Yersinia spp. P. s. pv. syringae 61 Hrp proteins are most similar to Ysc proteins among those homologs. TnphoA insertions in hrpC, hrpE, hrpW, hrpX, and hrpY abolished the ability of P. s.pv. syringae 61 to secrete HrpZ (harpinPss), as determined by immunoblot analysis of cell-bound and culture supernatant fractions. Thus, many of the proteins required for flagellar biogenesis and virulence protein secretion in plant and animal pathogens may have a common ancestry.
Mol Plant Microbe Interact
PMID:The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for harpinPss secretion that are arranged colinearly with Yersinia ysc homologs. 757 17

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

We characterized a bacterium-host cell interaction that is mediated by the Yersinia adhesin YadA. Derivatives of the virulence plasmid pIB1 harboring mutations in yadA, yopE, or yopH or in a low-calcium-response regulatory locus were introduced into a Yersinia pseudotuberculosis YPIII strain defective for Inv. The mutant strains were tested for the capacity to attach to and enter HEp-2 cells and express the cytotoxic activities of YopE and YopH. As previously shown, expression of YadA was necessary for bacterial attachment and Yop activity in the absence of Inv (R. Rosqvist, A. Forsberg, M. Rimpilainen, T. Bergman, and H. Wolf-Watz, Mol. Microbiol. 4:657-667, 1990). In addition, bacterial entry into HEp-2 cells occurred efficiently when YadA was expressed in the absence of YopE and YopH. These results demonstrated that YadA mediates intimate attachment of Y. pseudotuberculosis to HEp-2 cells and that phagocytic uptake of bacteria by this pathway is inhibited by the synergistic activities of YopH and YopE. A role for beta 1 integrins as host cell receptors for this bacterial attachment and entry mechanism was supported by HEp-2 cell adhesion and monoclonal antibody neutralization studies.
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PMID:The Yersinia pseudotuberculosis adhesin YadA mediates intimate bacterial attachment to and entry into HEp-2 cells. 768 42

The Yersinia enterocolitica O:3 lipopolysaccharide O-antigen is a homopolymer of 6-deoxy-L-altrose. The cloned rfb region was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementation experiments showed that eight of the genes, organized into two operons, rfbABC and rfbDEFGH, are essential for O-antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar to Salmonella proteins involved in the dTDP-L-rhamnose biosynthesis. Rhamnose and 6-deoxy-L-altrose are C3-epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT of Escherichia coli. This and transposon mutagenesis showed that RfbD and RfbE function as O-antigen exporters.
Mol Microbiol 1993 Jul
PMID:Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. 769 17


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