Expression of the plague plasminogen activator in Yersinia pseudotuberculosis and Escherichia coli

Enteropathogenic yersiniae (Yersinia pseudotuberculosis and Yersinia enterocolitica) typically cause chronic disease as opposed to the closely related Yersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage by Y. pestis of a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared approximately 70-kb low-calcium-response, or Lcr, plasmids (pCD in Y. pestis and pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (alpha-Pla) and slightly smaller (beta-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only alpha-Pla accumulates in Escherichia coli LE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble alpha and beta forms possessing biological activity. This process also converted cell-bound alpha-Pla to beta-Pla and smaller forms in Y. pestis KIM/pPCP1 and Y. pseudotuberculosis PB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops in Y. pseudotuberculosis PB1/+/pPCP1 occurred more slowly than that in Y. pestis but was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP by Y. pseudotuberculosis did not significantly influence virulence in mice.     

Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli.

Previously, the gene for heat-stable enterotoxin II (STII) has been mapped by transposon Tn5 insertion mutagenesis in the chimeric R-Ent plasmid pCG86 (Mazaitis, A. J., R. Maas, and W. K. Maas, J. Bacteriol. 145:97-105, 1981). DNA segments containing this gene were cloned from the wild-type and STII-insertion-mutant plasmid. The position of the Tn5 insertion was determined, and a 530-base-pair-long segment of the wild-type plasmid corresponding to the Tn5 insertion site was sequenced. An open reading frame for the STII gene was identified and is characterized by typical promoter and ribosome binding site sequences. The deduced STII structural gene codes for a protein 71 amino acids long, including a typical signal peptide of 23 amino acids and a mature protein of 48 amino acids. The size and overall structure of STII are similar to those of STI, but the amino acid compositions of the two heat-stable enterotoxins are completely different.     

Ribosomal Vaccines I. Immunogenicity of Ribosomal Fractions Isolated from Salmonella typhimurium and Yersinia pestis

The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein.     

Comparison of the structure and regulation of the udp gene of Vibrio cholerae, Yersinia pseudotuberculosis, Salmonella typhimurium, and Escherichia coli

The nucleotide sequences of the udp gene encoding uridine phosphorylase of Yersinia pseudotuberculosis and Vibrio cholerae are presented and compared with the udp sequences of Salmonella typhimurium and Escherichia coli. Both genes contain 759 bases and encode a 253 amino acid polypeptide, which is the same as for E. coli and S. typhimurium. The amino acid sequence derived from S. typhimurium gene was more similar to the derived E. coli sequence, with only a 7 amino acid difference. The Y. pseudotuberculosis and V. cholerae uridine phosphorylases presented a higher degree of divergence in their amino acid sequence as compared to the corresponding E. coli amino acid sequence, with 20 and 64 changes, respectively. The promoter regions of the udp gene for S. typhimurium (udpPSt), Y. pseudotuberculosis (udpPYp) and V. cholerae (udpPVc) were identified by primer extension analysis. Comparative analysis of the udpP promoter region from Y. pseudotuberculosis, V. cholerae, S. typhimurium and E. coli revealed that location, spacing and orientation of putative binding sites for CRP protein are highly conserved, whereas CytR protein recognition sequences of udpPYp and udpPVc deviate markedly from the E. coli and S. typhimurium CytR binding site. In vitro studies demonstrated that the CytR protein from E. coli shows different affinity for each promoter region analyzed. According to this, the degree of CytR derepression after introduction of heterologous promoters into E. coli cells is different.     

Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells.

Salmonella typhimurium, enteroinvasive Escherichia coli, Yersinia pseudotuberculosis, and Yersinia enterocolitica possess the ability to enter intestinal epithelial cells. We used a quantitative tissue culture model employing HEp-2 cells to compare the abilities of these bacteria to enter epithelial cells. S. typhimurium and Yersinia species were highly infective for HEp-2 cells but were unable to replicate extensively intracellularly. Enteroinvasive E. coli exhibited low infectivity but replicated extensively intracellularly. The growth of enteroinvasive E. coli led to destruction of the HEp-2 monolayer, whereas Yersinia spp. and S. typhimurium were maintained intracellularly for prolonged periods without damage to the monolayer. The ability of enteroinvasive E. coli to enter HEp-2 cells required prior growth at 37 degrees C; neither S. typhimurium nor Yersinia spp. exhibited this temperature dependence for cell entry. An E. coli K-12 derivative containing a 230-kilobase plasmid from enteroinvasive E. coli was constructed. This derivative shared all the invasive characteristics of the parental enteroinvasive strain, suggesting that determinants required for cell entry and intracellular multiplication were at least partially plasmid encoded. An HB101 derivative containing a cloned invasion determinant from Y. pseudotuberculosis was constructed in our laboratory. HEp-2 monolayers were coinfected with these two K-12 derivatives to compare invasion determinants from enteroinvasive E. coli with those of Y. pseudotuberculosis in a common genetic background. Results from these experiments suggest that these organisms reside within separate intracellular compartments.     

Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli.

The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues.     

Nucleotide sequence of the Salmonella typhi groEL heat shock gene

Heat shock proteins (HSP) have been shown to elicit a strong immune response during infection by a variety of pathogens. The HSP60 gene of Salmonella typhi was amplified using oligonucleotide primers based on the Escherichia coli groEL sequence. The nucleotide sequence of the amplified fragment was determined and used to predict the amino acid sequence of S. typhi GroEL. The E. coli and S. typhi proteins were found to be highly similar; however, several non-conservative substitutions near the carboxy-termini of the two polypeptides were found. Knowing the amino acid sequence of the S. typhi HSP60 homologue will enhance our knowledge of host immune recognition of HSP produced by bacterial pathogens.     

Ribosomal-RNA patterns of Escherichia coli, Salmonella typhimurium and related Enterobacteriaceae

rRNA sequences are usually highly conserved among species. In Enterobacteriaceae we have shown that Salmonella typhimurium does not have an equivalent to the 23S rRNA of Escherichia coli but its 23S rRNA is cleaved in vivo into two smaller species. This cleavage appears to be a result of a difference between the S. typhimurium and E. coli rRNA sequences rather than to differences in ribonuclease activity. We have surveyed a wide range of Enterobacteriaceae for the presence or absence of 23S rRNA and found this rRNA species to be present in all strains of E. coli, Shigella and Citrobacter and all salmonellae examined except S. typhimurium. All S. typhimurium cultures, isolated at different times and from several different countries, lack an intact 23S rRNA. Thus, the presence or absence of this rRNA species is an excellent diagnostic characteristic for S. typhimurium.     

Genotoxicity of quinoxaline 1,4-dioxide derivatives in Escherichia coli and Salmonella typhimurium

The mutagenicities of 5 quinoxaline 1,4-dioxide (QdO) derivatives were tested by 2 bacterial assays using forward mutation with Escherichia coli WP2uvrA/pKM101 and reverse mutation with Salmonella typhimurium TA100 and TA98. Potent mutagenic activities of all QdOs tested were observed in both mutation assays. Mutagenicities of these compounds were varied by addition of S9 mix. Their SOS-inducing activities were examined with a ‘Rec-lac test’ that has been newly developed by us for detecting genotoxins. A high level of SOS-inducing activity was observed in all samples tested. These results suggest that the mutagenicity of QdOs results from the error-prone repair involved in SOS responses.     

Direct expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli and Salmonella typhimurium aroA.

Nonfused (i.e., nonhybrid) filamentous hemagglutinin (FHA) of Bordetella pertussis was efficiently expressed in Escherichia coli K-12 and Salmonella typhimurium aroA at levels higher than those found in wild-type B. pertussis when the upstream signals of the gene were replaced and the translation initiation region was engineered to optimize translational efficiency. Inclusion of part of the C-terminal FHA open reading frame, whose translation product does not appear to be part of the major secreted species of FHA, was shown to be important in achieving protein expression in both E. coli and S. typhimurium aroA; removal of the downstream gene sequence abolished recombinant FHA production. The levels of expression observed varied widely according to the construct and host bacterium used.     

Integration of the Vwa plasmid into the chromosome of Yersinia pestis strains harboring F'' plasmids of Escherichia coli.

The stimulus for the migration of polymorphonuclear leukocytes (PMNs) in acute chlamydial infection was studied in vitro by examining the chemotaxigenic effect of L2 and DE Chlamydia trachomatis elementary bodies (EB) upon the plasma of three healthy donors. In each individual experiment, chemotactic response was assessed with PMNs and plasma from the same respective donor, and no specific antibodies against C. trachomatis were detected in the plasma of any donor. Chemotaxis was observed in an agarose plate assay and was quantitated as the chemotactic differential, or CD (directed migration of PMNs minus random movement of PMNs). For each donor, the mean CD was significantly greater (P less than 0.005) when plasma preincubated for 2 h with L2 EB was used as the chemoattractant than when (i) plasma alone, (ii) plasma preheated to 56 degrees C for 30 min before incubation with L2 EB, or (iii) L2 EB in phosphate-buffered saline (PBS) was used as the potential chemoattractant. Similarly, in the one donor in whom DE EB were studied, the mean CD was also significantly greater (P less than 0.005) for plasma preincubated with DE EB as compared with (i) plasma alone or (ii) DE EB in PBS. Complement activation by C. trachomatis EB was assessed by radioimmunoassay for C5a des-arginine in all chemoattractant preparations used in the chemotaxis assay. Mean C5a des-arginine levels were high in plasma samples preincubated with L2 EB (171.00 +/- 10.64, 107.00 +/- 4.76, and 89.70 +/- 1.74 ng per ml) or DE EB (37.40 +/- 15.76 ng per ml) but were undetectable (less than 4.0 ng per ml) in (i) plasma alone, (ii) preheated plasma incubated with L2 EB, and (iii) PBS containing L2 EB. Thus, L2 EB and DE EB of C. trachomatis exert a chemotaxigenic effect upon normal antibody-negative plasma, and this effect is at least in part a result of complement activation and generation of the potent chemotaxin C5a.     

Transfer of the virulence plasmid of Yersinia pestis to Yersinia pseudotuberculosis.

Transposon Tn5 insertion derivatives of the virulence plasmid pYV019 of Yersinia pestis were transferred by P1 transduction into a plasmid-free strain of Y. pseudotuberculosis. One of these plasmid derivatives conferred virulence upon the Y. pseudotuberculosis strain. This strain had the ability to express temperature-inducible plasmid-coded outer membrane proteins and was also found to be Ca2+ dependent.     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7–8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col^? segregants.     

The sequence of the malG gene from Salmonella typhimurium and its functional implications

The nucleotide sequence of the malG gene which is essential for maltose transport was determined in Salmonella typhimurium and compared to homologous genes from Escherichia coli and Enterobacter aerogenes. malG genes from S. typhimurium and E. aerogenes were expressed and their products were active in E. coli. The primary structure of the three MalG proteins was highly conserved. Changes were mainly clustered in a relatively large hydrophilic region of the protein (residues 40 to 75). In contrast, other hydrophilic segments were more conserved, and most remaining changes occurred in the hydrophobic putative transmembrane segments. This suggests that hydrophilic loops in this inner membrane protein may be functionally constrained. These results prove new insights into the functional sites in MalG.     

Selection of agar for use in Salmonella typhimurium and Escherichia coli mutation assays

The spontaneous and induced revertant frequency of four Salmonella typhimurium strains (TA1535, TA1537, TA98, and TA100) and Escherichia coli [WP2 uvrA (pKM101)] was evaluated using Vogel Bonner minimal plates prepared with ten different agars. In addition to the Difco Bacto agar originally recommended by Ames, Difco Noble, granulated and Bitek agars; BD grade A, BBL granulated and purified agars; Oxoid purified and No. 1 agars; and GIBCO select agar were tested. Several of these agars have been reported as acceptable alternatives for these Salmonella strains, but comparable studies with E. coli have not been done. The bacteria were treated with DMSO or an appropriate positive control in the presence or absence of an Aroclor 1254-induced rat liver activation system. With the exception of Noble agar in the presence of S9, there was little difference among the responses of the Salmonella strains on any of the agars. However, with E. coli the responses include either a reduction or an increase in spontaneous revertants numbers as well as a reduction in absolute and relative induced revertant frequency. Difco Bacto agar appears to be the most consistent agar for use with these strains. As an alternative, only BBL purified agar resulted in consistent results for all of these strains under all testing conditions. These results emphasize the need to evaluate the components of the standard mutation assay when incorporating additional bacterial strains. Suboptimal responses related to the agar or other components could compromise the detection of weak mutagens. Environ. Mol. Mutagen. 32:192–196, 1998 © 1998 Wiley-Liss, Inc.     

Nucleotide sequence and expression in Escherichia coli of the gene encoding the nonstructural protein NCVP2 of bovine rotavirus

Cloned DNA copy of rotavirus genome segment 5 from bovine rotavirus RF strain has been used to determine the nucleotide sequence of the gene that encodes for the nonstructural viral protein NCVP2. The sequence data indicated that segment 5 consists of 1581 base pairs and is A + T rich (66%). The positive strand of segment 5 contains a single open reading frame that extends 491 codons and possesses 5'- and 3'-terminal untranslated regions of 32 and 73 base pairs, respectively. The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 58,654, slightly higher than the apparent molecular weight of NCVP2 (MW 54,000). Although it is not evident whether the gene product is glycosylated, four potential glycosylation sites were found at positions 50, 168, 403, and 438. NCVP2 has been expressed in Escherichia coli using the inducible expression vector pKK233-2. Following IPTG induction high levels of full-length nonfused proteins were synthesized and accumulated in induced cells.