Search PubMed⌕ Search

Biomedical subjects

M Rhen

Publications and source records attributed to M Rhen.

At least 37 records · Page 2Linked to original sources
PubMed1995

Evidence for functional polymorphism of the spvR gene regulating virulence gene expression in Salmonella.

The expression of Salmonella enterica spv virulence genes was studied in serovariants Dublin and Typhimurium using Western blotting (immunoblotting), spv-lacZ operon fusions and Northern blotting. The SpvA protein was detected in immunoblots from stationary phase cultures of Dublin but not from the corresponding cultures of Typhimurium. Transcriptional measurements, using a spvA-lacZ operon fusion, indicated 8-10 times higher spvA transcription in Dublin. In an isogenic Escherichia coli chromosomal background, virulence plasmids from various Dublin strains systematically had a significantly higher induction level of the spvA-lacZ operon fusion than virulence plasmids from Typhimurium strains. The cloned spvR transcriptional activator gene of Dublin strain 2229 was found to activate both spvR-lacZ and spvA-lacZ operon fusions, as well as to raise spv mRNA levels in E. coli TG1. In contrast, the corresponding cloned gene of Typhimurium strain SL2965 possessed a lower induction potential and required higher spvR gene dosage for activation. A comparison of the nucleotide sequences of spvR genes from two Dublin and four Typhimurium strains revealed conserved, serovariant-associated basepair substitutions. Our results indicate that the spv virulence gene cluster possesses different functional alleles of the regulator gene spvR. This finding has important consequences for comparative studies of regulation and virulence in different serovariants of Salmonella.

Base Sequence↗
PubMed1995

The Escherichia coli G-fimbrial lectin protein participates both in fimbrial biogenesis and in recognition of the receptor N-acetyl-D-glucosamine.

The gafD gene encoding the N-acetyl-D-glucosamine-specific fimbrial lectin (adhesin) protein GafD of uropathogenic Escherichia coli was cloned and subjected to genetic analysis. The corresponding gene product was isolated as a MalE fusion protein. The lectin gene was identified with the aid of deletion mutagenesis; mutations in gafD impaired either receptor binding or both receptor binding and fimbria production, depending on the mutation created. All mutants converted to wild-type expressors when complemented in trans with the cloned intact gafD gene. The predicted 354-amino-acid sequence of GafD, deduced from the nucleotide sequence, is closely related to those of the fimbria-associated F17-G and F17b-G proteins coded for by enterotoxigenic and invasive E. coli strains. Isolated GafD was shown to recognize N-acetyl-D-glucosamine by virtue of specific binding to an immobilized receptor, thus proving directly that GafD is a sugar-binding protein. Our results indicate that GafD as such is sufficient for receptor recognition and that the protein also participates in fimbrial biogenesis.

Acetylglucosamine↗
PubMed1994

Role of rpoS in the regulation of Salmonella plasmid virulence (spv) genes.

Salmonella plasmid virulence (spv) genes are organized into two transcriptional units: one formed by the spvR gene and the other by the spvA, spvB, spvC and spvD genes. Transcription of both units is activated by SpvR, a regulatory protein of the LysR family. The effect of RpoS, a stationary phase-associated sigma factor, on the expression of spv genes was studied using lacZ transcriptional fusions to spvR and spvA in wild-type and rpoS Escherichia coli backgrounds. Mutant and wild-type SpvR proteins were expressed in trans from a multicopy plasmid. The results show that the combined action of rpoS and spvR is necessary for transcription of spvA and that this combination also enhances transcription of spvR. Interestingly, spvR can also be transcribed in an alternative manner, i.e. in the absence of rpoS or spvR or both. The possible role for SpvR as a repressor of its own transcription is discussed.

Cloning, Molecular↗
PubMed1994

Intermediate filament-like network formed in vitro by a bacterial coiled coil protein.

The TlpA protein encoded by the virulence plasmid of Salmonella enterica is an alpha-helical 371-amino acid protein possessing characteristics similar to eukaryotic coiled coil proteins (Koski, P., Saarilahti, H., Sukupolvi, S., Taira, S., Rikkonen, P., Osterlund, K., Hurme, R., and Rhen, M. (1992) J. Biol. Chem. 267, 12258-12265). In this paper we have investigated inter- and intramolecular associations and the morphology of structures formed by TlpA. Dynamics and temperature stability of TlpA dimers were studied by examining the feasibility and conditions in which TlpA would form an artificial heterodimer with its truncated derivative. Formation of heterodimers, bridged by Cu(2+)-catalyzed air oxidation of adjacent Cys residues, showed that TlpA dimers are dynamic chain exchanging structures at 37 degrees C, whereas they were nonexchanging at room temperature or on ice. Chemical cross-linking suggested higher order interaction between TlpA dimers. Electron microscopy studies revealed two levels of TlpA organization in vitro: thin filaments and rods, 2-5 nm in diameter, and a higher ordered filament network consisting of tonofilament-like formations with a diameter of 8-15 nm. Electron microscopy of thin-sectioned Escherichia coli over-producing TlpA showed an extraordinary intracellular assembly of proteinacious lamellae with a striated appearance and a 38-nm periodicity. This study describes for the first time a bacterial protein capable of organizing itself into an ordered and suspectedly dynamic intermediate filament-like architecture.

Air↗
PubMed1993

Transcriptional regulation of Salmonella enterica virulence plasmid genes in cultured macrophages.

The plasmid-carried spv genes promote virulence of salmonellae in mice by allowing bacterial growth in the reticuloendothelial tissue. When the bacteria are cultivated under normal laboratory conditions the spv genes appear dormant. This communication explores the transcriptional regulation of spv genes within murine macrophage-like J774-A.1 cells utilizing a new reporter system. Transcriptional fusions were constructed between promoter elements of the Salmonella enterica var. Typhimurium spv genes and the KS71A fimbrial gene cluster. The expression of KS71A fimbriae in fusion-carrying Escherichia coli strains was found to be under the control of the transcriptional activator gene spvR. In strains overproducing SpvR, KS71A fimbriae were assembled on the bacterial cell surface and could be detected by bacterial agglutination or immunofluorescence of intact bacteria; the reporter activity was quantified by estimating the percentage of fluorescent bacteria and by immunoblotting of cell lysates. The activity of the reporters, when transformed into the parent Typhimurium TML R66, was low and revealed less than 0.3% fimbriated cells under in vitro culture conditions. A 15-30-fold increase in fimbriation was observed when the bacteria were cultivated within J774-A.1 cells. No such increase occurred when the reporter fusions were transformed into TML R66 cured of the virulence plasmid. Insertional inactivation of the spvR gene of the virulence plasmid in Typhimurium TML R66 also abolished induction, whereas corresponding inactivation of spvA or spvB did not reduce induction. No increase in reporter activity was obtained in Typhimurium of line Q1, which is naturally avirulent for mice, although the strain was provided with virulence plasmid pEX102 of line TML R66. We conclude that the intracellular environment of J774-A.1 cells induces the spv genes and that this induction requires gene functions of both the bacterial chromosome and the virulence plasmid.

Animals↗
PubMed1993

Molecular analysis of spv virulence genes of the Salmonella virulence plasmids.

Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium, Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon-poor media, and optimal expression is dependent on the katF locus. The virulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

Gene Expression Regulation, Bacterial↗
PubMed1992

A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.

A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.

Amino Acid Sequence↗
PubMed1992

The virulence plasmid does not contribute to growth of Salmonella in cultured murine macrophages.

The virulence plasmid, characteristic of many serovars of Salmonella sp., and specifically its spv genes, promote intracellular growth of the bacteria in the liver and spleen and are essential for the virulence of these Salmonella serovars in the mouse. In an attempt to establish an in vitro model for studying its function, we evaluated its effect on the intracellular growth of the bacteria in macrophages in culture. We used a number of different macrophage-like cell lines (J774-A.1, IC-21 and PU5-1.8), as well as peritoneal or splenic macrophages from genetically Salmonella-sensitive (Itys, BALB/c) or resistant (Ityr, C3H/HeN) mice, and at different states of activation, stimulated in vivo or in vitro with lipopolysaccharide and/or recombinant gamma interferon. These were found to differ in their ability to suppress or sustain intracellular growth of several Salmonella serovars, but in all cases the growth was independent of the spv genes.

Animals↗
PubMed1992

Plasmid-mediated serum resistance in Salmonella enterica.

The ability of Salmonella enterica to survive an incubation of 2 h in fresh, pooled guinea-pig serum was determined comparing strain sets of serovariants Dublin and Typhimurium, harbouring or lacking the virulence plasmid. All strains showed marked serum resistance, which was slightly decreased among cured strains of Typhimurium. However, when introduced into a rough Escherichia coli strain, all Typhimurium plasmids substantially increased the resistance of the host strain to guinea-pig serum, whereas the Dublin plasmid did not. The traT gene, previously shown to affect serum resistance, was identified on all Typhimurium plasmids, but not on the Dublin plasmid. Mutational inactivation of the traT gene on the Typhimurium plasmids eliminated the serum resistance mediated by the plasmids.

Animals↗
PubMed1991

Amino-terminal sequence analysis of four plasmid-encoded virulence-associated proteins of Salmonella typhimurium.

We have expressed four virulence-associated proteins encoded by the Salmonella typhimurium plasmid pEX102 in Escherichia coli. The genes coding for the proteins MkaA, MkaB, MkaC and MkaD subcloned in the vectors pUC19 or Bluescript KS+ directed substantial production of these proteins in E. coli. The same host harbouring pEX102 did not produce these proteins in detectable amounts. The proteins were exclusively found in the particulate fraction. The amino-terminal sequence analysis showed that the N-termini of these proteins corresponded to the ones predicted from the open reading frames found previously in the DNA sequence of the virulence determinant and that no N-terminal signal sequence processing of the proteins occurred.

Amino Acid Sequence↗
PubMed1991

The mkaC virulence gene of the Salmonella serovar typhimurium 96 kb plasmid encodes a transcriptional activator.

The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four MKa proteins indicated that mkaC enhances the expression of beta-galactosidase from the mkaA-, mkaB- and mkaC-lacZ gene fusions in trans. An mkaD-lacZ fusion or mkaA-lacZ fusion that did not contain DNA proximal to mkaB was not inducible with MkaC, indicating that at least mkaB and mkaA are induced together as an operon. MkaC is thus the first virulence protein whose function has been resolved.

Escherichia coli↗
PubMed1990

Nucleotide sequence of mkaD, a virulence-associated gene of Salmonella typhimurium containing variable and constant regions.

We have identified the nucleotide (nt) sequence of mkaD, a virulence-associated gene of the Salmonella typhimurium virulence plasmid, pEX102. The gene shows 98% homology on nt sequence level to mkfA, a corresponding gene of the S. typhimurium virulence plasmid pIP1350. The few nt changes, however, caused more extensive changes on the amino-acid level. The differences between mkaD and mkfA were clustered in distinct variable regions rather than being randomly scattered along the sequence. A third salmonellar virulence plasmid, pLT2, contained an mkaD gene identical to that of pEX102. Our observation suggests that the conserved virulence determinant on the plasmids of Salmonellae may contain different alleles of the same gene.

Amino Acid Sequence↗
PubMed1990

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid.

The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.

Amino Acid Sequence↗
PubMed1989

rfaP mutants of Salmonella typhimurium.

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.

Anti-Bacterial Agents↗
PubMed1989

Isolation, cloning, and primary structure of a cationic 16-kDa outer membrane protein of Salmonella typhimurium.

By using acid-urea polyacrylamide gel electrophoresis, two cationic proteins were found in the isolated outer membranes of Salmonella typhimurium SH5014. Also, all the other enterobacterial strains studied (five additional strains of S. typhimurium, one strain of Salmonella minnesota, and three strains of Escherichia coli K12) had those proteins. The most abundant (OMB2) was purified in preparative acid-urea polyacrylamide gel electrophoresis and reversed-phase high pressure liquid chromatography (HPLC). It had a molecular mass of 16 kDa, a pI above 10.0, and was rich in arginine and lysine. 72% of the total amino acid sequence was determined by sequencing several HPLC-purified proteolytic fragments and 55 amino acids from the NH2 terminus. Furthermore, we isolated by molecular cloning the corresponding gene, named it ompH, and determined its nucleotide sequence. By combining protein and nucleotide sequence data, we determined the primary structure of the entire OmpH protein. It consists of 141 amino acids, possesses regions very rich in basic amino acids, and has a molecular mass of 15,862 kDa.

Amino Acid Sequence↗
PubMed1989

Molecular organization of genes constituting the virulence determinant on the Salmonella typhimurium 96 kilobase pair plasmid.

The ability of intracellular growth is plasmid-dependent in Salmonella typhimurium. Only a small portion of this 96 kilobase pair plasmid appears essential for intracellular growth. The genetic organization of this region (the essential virulence determinant) was resolved. Fragments of the virulence determinant were cloned from the 96-kb plasmid pEX102 and transformed into minicell-producing E. coli. Plasmid-directed protein synthesis was investigated in metabolically labeled minicells. This analysis indicated the presence of at least four genes, mkaA, mkaB, mkaC and mkaD, within the virulence determinant encoding proteins of 70, 31, 30 and 29 kDa, respectively. The genes were positioned on the restriction map of the 96-kb virulence plasmid and the map locations confirmed by nucleotide sequence analysis of two new virulence genes (mkaB and mkaC).

Amino Acid Sequence↗
PubMed1989

Occurrence of Salmonella typhimurium virulence plasmid-specific sequences in different serovars of Salmonella.

We have subcloned the 96-kilobasepair (kb) virulence plasmid, pLT2, of Salmonella typhimurium line LT2 into 7 subfragments. Using these subclones as probes, 35 independent Salmonella isolates were tested for complementary DNA sequences Sequences homologous to pLT2 were present in 15 of the isolates. All of these contained sequences homologous to at least one specific probe representing 15 kb of pLT2. The traT gene from pLT2 was absent in 6 of these 15 isolates. Three strains reported to be cured of the plasmid were shown to harbour some sequences with homology to the pLT2 plasmid. Seven isolates were shown to contain homologous sequences with pBR322 but not with the pLT2 plasmid.

Cloning, Molecular↗