Search PubMed⌕ Search

Biomedical subjects

M Rhen

Publications and source records attributed to M Rhen.

At least 19 recordsLinked to original sources
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↗
PubMed1989

Localization by insertion mutagenesis of a virulence-associated region on the Salmonella typhimurium 96 kilobase pair plasmid.

The virulence-associated plasmid pEX102 of Salmonella typhimurium line TML R66 was tagged with the transposon Tn5 and the virulence of the mutants obtained was assayed in the mouse salmonellosis model. Out of 36 independent insertion mutants tested two isolates had clearly reduced virulence in (CBA x C57B1/6)F1 mice. The corresponding Tn5 elements were positioned on the restriction endonuclease map of pEX102 and found to be some 4 kilobases apart (kb) on the 96 kb virulence plasmid.

Animals↗
PubMed1989

Identification and genetic analysis of mkaA--a gene of the Salmonella typhimurium virulence plasmid necessary for intracellular growth.

Salmonella typhimurium, like many other Salmonella serovars, harbours a large plasmid required for mouse virulence and growth of bacteria in host cells. The nature of one virulence-abolishing Tn5 insertion (zzx-2556::Tn5) in the plasmid was characterized. A 3.7 kb insert harbouring this region was cloned in pBR325. Plasmid-directed protein synthesis in minicells indicated that the transposon had eliminated the expression of a 70 kDa protein encoded by the virulence plasmid. Nucleotide sequence analysis of the corresponding wild type DNA region showed a 1773 bp open reading frame (mkaA), encoding a protein of 591 amino acid residues and a predicted molecular mass of 60.6 kDa; zzx-2556::Tn5 was situated within mkaA.

Base Sequence↗
PubMed1988

Mutations in cloned Escherichia coli P fimbriae genes that makes fimbriae-production resistant to suppression by trimethoprim.

The effect of sublethal concentrations of trimethoprim on the expression of P fimbriae was tested in Escherichia coli HB101 recombinant strains. Fimbriation was inhibited at trimethoprim concentrations down to at least 1/64 of the minimal inhibitory concentration. However, the expression of the P fimbrillin by recombinant plasmids containing deletions in front of the fimbrillin gene did not respond to the inhibitory effect of trimethoprim indicating that trimethoprim may act at the level of gene regulation.

Agglutination Tests↗
PubMed1988

Plasmid-associated virulence of Salmonella enteritidis.

Plasmid-associated virulence of Salmonella enteritidis was studied using plasmid-cured and plasmid-reintroduced strains. The plasmidless strain was unable to grow in the liver of mice after intravenous inoculation. Reintroduction of the plasmid pEX106 from the original S. enteritidis fully restored its capacity to grow in the mice. The plasmid pEX102 of Salmonella typhimurium introduced into the plasmid-cured S. enteritidis had a similar effect. The smooth lipopolysaccharide character of the S. enteritidis strains was not affected by the presence or absence of the plasmid, and the same was true of their high resistance to complement in guinea-pig serum as such or with added antibody.

Animals↗
PubMed1988

The role of the traT gene of the Salmonella typhimurium virulence plasmid for serum resistance and growth within liver macrophages.

The role of the traT gene of the Salmonella typhimurium virulence plasmid for the serum resistance of the bacteria and their growth within mouse liver macrophages was investigated. The gene product, the TraT protein, increased the serum resistance in E. coli HB101, which naturally does not carry traT. It also contributed to the serum tolerance of S. typhimurium. The capacity of an isogenic S. typhimurium TML strain triplet, differing in their ability to express TraT and in the quality of the traT gene expressed, to grow in vivo in the mouse liver indicated that, although TraT was dispensable for the net growth of the bacteria within the liver, the expression of a mutated traT gene reduced the growth rate. The traT gene was mapped on the virulence plasmid outside previously defined virulence determinants suggesting that other regions of the virulence plasmid are mainly responsible for the growth within mouse liver macrophages in S. typhimurium.

Animals↗
PubMed1987

Nucleotide sequence analysis of a P fimbrial regulatory element of the uropathogenic Escherichia coli strain KS71 (04:K12).

The nucleotide sequence of a trans-acting P-fimbrial regulatory element obtained from the uropathogenic Escherichia coli strain KS71 (04:K12) was determined. The regulatory element was found to contain an open reading frame of 231 nucleotide residues that showed 95.2% homology with papl, a functionally analogous regulatory gene of E. coli strain J 96.

Amino Acid Sequence↗