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S Arvidson

Publications and source records attributed to S Arvidson.

At least 19 recordsLinked to original sources
PubMed1995

Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII.

The synthesis of virulence factors in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, encoded by the agr locus. Transcription of genes coding for secreted toxins and enzymes is stimulated, while transcription of cell-surface protein genes is repressed by RNAIII. In the case of staphylococcal alpha-toxin, RNAIII also seems to stimulate translation by an independent mechanism. In this report we show that in a mutant lacking RNAIII the rate of alpha-toxin (hla) production relative to the cellular concentration of hla mRNA was reduced 10-fold as compared with the wild-type strain. A 75% complementarity between the 5' end of RNAIII and the 5' untranslated region of the hla transcript suggests a direct interaction between the RNAs. A complex of RNAIII and hla mRNA was demonstrated in extracts of total RNA from the wild-type strain, and also with in vitro synthesized RNAs. Ribonuclease T1 digestion experiments revealed that the ribosome binding site of the hla transcript is blocked by intramolecular base-pairing. Hybridization with RNAIII prevents this intramolecular base-pairing and makes the hla mRNA accessible for translation initiation. This is, to our knowledge, the first example of an 'antisense RNA' that stimulates translation of the target mRNA.

Bacterial Toxins↗
PubMed1993

Retarded RNA turnover in Escherichia coli: a means of maintaining gene expression during anaerobiosis.

In this study, we extend earlier observations on the influence of growth rate on mRNA stability and rRNA processing in Escherichia coli during continuous culture, to the effect of anaerobiosis. During slow anaerobic growth (generation time 700 min) both ompA and bla mRNA had a prolonged half-life compared to that during slow aerobic growth and the processing of 9S RNA was even more profoundly retarded, which indicated a general slowing of mRNA turnover. The latter was confirmed by a nearly fourfold increase in the functional half-life of bulk mRNA. In spite of this difference in stability, steady state levels of RNA, as judged by those of the ompA and 9S transcripts, were the same in aerobic and anaerobic cells at a given growth rate. Furthermore, we found that RNA synthesis during anaerobiosis was a fraction of that observed during slow aerobic growth and it is proposed that this offsets the general increase in mRNA stability. Our data therefore suggest that a constant level of RNA is maintained by matching the rate of decay to the level of RNA synthesis.

Anaerobiosis↗
PubMed1992

Decay of ompA mRNA and processing of 9S RNA are immediately affected by shifts in growth rate, but in opposite manners.

By growing Escherichia coli in continuous cultures at various growth rates, we provide definitive evidence that the stability of the ompA mRNA is growth rate dependent. Shifting fast-growing cells into physiological salt buffer led to an immediately increased rate of ompA mRNA decay and to an instantly decreased rate of 9S RNA conversion into 5S rRNA. Shifting slowly growing cells into fresh medium had the opposite effect for each of the two RNA species. The observed regulatory patterns underline the need of cells to adjust the output of ompA and 9S RNAs in response to growth rate changes. At all growth rates and throughout all shift experiments, the half-life of bla mRNA was constant. A stabilization of the ompA transcript was even observed when slowly growing cells were shifted into fresh medium already containing the transcriptional inhibitor rifampicin. A hybrid bla transcript with the 5' untranslated region from the ompA gene behaved similarly to the wild-type ompA messenger in response to a shift in growth rate. In agreement with this result, we found that the same type of 5' cleavages as have been previously shown to initiate the decay of the ompA transcript seem to be involved in stability regulation. In E. coli the degradation of mRNA has been shown to depend on the ams/rne gene. This gene controls the stability-related cleavages in the ompA transcript, catabolic processes, and the cleavages which process the 9S rRNA into 5S RNA, an anabolic process. We discuss these results with respect to the ams/rne gene and the related nuclease activities that control the ompA and 9S RNA cleavages.

Bacterial Outer Membrane Proteins↗
PubMed1990

The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus.

The synthesis of at least 14 extracellular toxins and enzymes in Staphylococcus aureus is regulated by a set of trans-acting elements from the agr (accessory gene regulator) locus. We have shown that the delta-lysin gene (hld) that is transcribed from a promoter immediately upstream of the agr locus, and which is positively controlled by agr, is part of this regulatory system. Deletion replacement mutagenesis of the chromosomal hld gene had the same pleiotropic effect on the synthesis of several virulence factors as agrA mutations. Characteristically, these mutants had an almost complete block in the synthesis of alpha-toxin, serin- and metalloprotease, whereas synthesis of protein A was greater than 10-fold higher than in the parental strain. Corresponding changes in the levels of alpha-toxin and protein A mRNAs were demonstrated by northern blotting experiments. The effects of the hld deletion mutation could be fully complemented by the hld gene on a plasmid. A plasmid insertion mutation in the 3' non-coding region of hld had a similar effect on exoprotein synthesis, indicating a role of the hld transcript in the regulation of exoprotein synthesis. This was confirmed by the finding that the effects of alpha-toxin and protein A synthesis by the hld deletion replacement mutation could be fully complemented by a hld allele in which we had introduced an early stop codon in the delta-lysin structural gene. However, the mutant hld allele could not complement the defect in production of extracellular proteases, indicating that delta-lysin may act in conjunction with its mRNA to regulate the expression of some exoprotein genes.

Amino Acid Sequence↗
PubMed1990

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase.

We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion.

Amino Acid Sequence↗
PubMed1989

Identification and nucleotide sequence of the delta-lysin gene, hld, adjacent to the accessory gene regulator (agr) of Staphylococcus aureus.

A Tn551 insertional mutation in the accessory gene regulator (agr) locus of the Staphylococcus aureus chromosome resulted in the decreased production of at least seven extracellular toxins and enzymes and a simultaneous increase in the production of protein A and coagulase (Recsei et al. 1986). Adjacent to this locus we have now identified another gene, hld, transcribed into a 0.5 kb RNA which codes for the staphylococcal delta-lysin. The expression of hld was totally repressed in a strain carrying the agr insertional mutation. Hybridization with strand-specific probes and primer extension analysis revealed that hld and agr are transcribed in opposite directions, starting 188 nucleotides apart. The hld gene is mainly expressed during the post-exponential growth phase and is totally repressed during early exponential growth. Determination of hld mRNA half-life in different growth phases indicated that this regulation is at the level of transcription.

Amino Acid Sequence↗
PubMed1988

Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus.

Insertion of the erythromycin resistance transposon Tn551 into a single site of the Staphylococcus aureus chromosome resulted in decreased production of alpha-toxin, serine and metallo-proteinases and several other extracellular proteins and a simultaneous increase in the production of protein A. The site of insertion, designated exp, was separate from the structural gene for alpha-toxin and protein A. Hybridization analysis showed that the effect of the insertional mutation on the expression of the alpha-toxin and protein A was at the level of transcription. The chromosomal DNA flanking the transposon and the corresponding DNA of the wild-type strain was cloned in Escherichia coli. Northern blot hybridization experiments revealed that the exp locus codes for a major RNA of approximately 3.5 kb. This RNA was not found in the insertional mutant nor in a spontaneous exp mutant. A map of the exp locus constructed by Northern blot and restriction enzyme analysis showed that the insertional mutation was located in the middle of the coding sequence of the 3.5 kb RNA. The insertional mutant was reverted to wild type by inserting a recombinant plasmid containing most of the coding sequence of the 3.5 kb RNA.

Bacterial Proteins↗
PubMed1988

Cloning and expression in Escherichia coli of genes encoding a multiprotein complex involved in secretion of proteins from Staphylococcus aureus.

The genes encoding the multiprotein membrane-bound ribosomal protein (MBRP) complex (mrp genes), associated with membrane-bound ribosomes in Staphylococcus aureus, were cloned in Escherichia coli. All four components (molecular sizes 71, 60, 46, and 41 kilodaltons) of the MBRP complex were expressed from an 8.5-kilobase DNA fragment as judged by Western blot (immunoblot) analysis. The order of the individual genes within the cloned DNA fragment was determined by deletion mutagenesis and subcloning of various restriction fragments. Three RNAs, transcribed from the same DNA strand, were identified within the MBRP-coding region: one large RNA of approximately 5.9 kilobases, presumably coding for all four MBRP components, and two minor RNAs, coding for MBRP-71 and MBRP-60. The two minor RNAs seemed to be transcribed from promoters within the large transcription unit. Attempts to make insertional inactivations of the mrp genes with an internal 600-base-pair DNA fragment of the MBRP-coding region as a target were unsuccessful, presumably because such insertions are lethal.

Blotting, Northern↗
PubMed1987

Correlation between the rate of exoprotein synthesis and the amount of the multiprotein complex on membrane-bound ribosomes (MBRP-complex) in Staphylococcus aureus.

The membrane-bound ribosome protein (MBRP)-complex of Staphylococcus aureus was studied using antibodies to its individual components. The four polypeptides of the complex were firmly held together, and none were present in large excess. The membrane-bound fraction of the MBRP-complex was accessible to trypsin only after removal of the membrane-bound ribosomes; it also remained associated with the membrane-bound ribosomes even after solubilization of the membranes with Triton X-100. Furthermore, the amount of MBRP-complex in the membrane was proportional to the rate of exoprotein synthesis. These results strongly suggest a role for the MBRP-complex in protein secretion.

Autoradiography↗
PubMed1984

Detection of a membrane-associated protein on detached membrane ribosomes in Staphylococcus aureus.

Membrane ribosomes from Staphylococcus aureus which were detached from the membrane by extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a molecular weight of 60 000, which was absent from cytoplasmic ribosomes. MBRP was detected and quantified by immunological methods. When membrane ribosomes were dissociated into 50S and 30S subunits, MBRP remained associated with the 50S particle. MBRP was found both on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71 000, 46 000 and 41 000. Four proteins with the same molecular weights as those of the MBRP complex were found associated with membrane ribosomes. The proteins of molecular weight 71 000, 60 000, 46 000 and 41 000 seemed to be present in stoichiometrically equivalent amounts in the complex.

Autoradiography↗