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J Kornblum

Publications and source records attributed to J Kornblum.

12 recordsLinked to original sources
PubMed1992

Regulation of the protein A-encoding gene in Staphylococcus aureus.

The synthesis of several exoproteins, including protein A (SpA) in Staphylococcus aureus is coordinately regulated by the agr locus. Different constructs of the SpA-encoding gene (spa) were introduced into Agr+ and Agr- derivatives of a spa- strain of S. aureus. Plasmid-located spa with deletions at the 3' end expressed a truncated SpA which was almost exclusively extracellular and which confirmed the role of C-terminal region X in cell-wall binding. In the Agr- host, the production of SpA was elevated severalfold. Transcriptional and translational fusions were constructed to study the agr- mediated regulation of spa gene expression. Translational fusions of a beta-lactamase (Bla)-encoding ApR reporter gene with the spa promoter and N-terminal coding sequences expressed elevated levels of Bla activity in the Agr- host. In contrast, a transcriptional fusion of the spa gene with a promoter of the positively regulated staphylococcal epidermolytic toxin A (ETA)-encoding gene synthesized higher levels of SpA in an Agr+ host, as compared to Agr-. Moreover, the synthesis of SpA in the Agr+ strain was switched on during the transition from the exponential to stationary phase in a similar fashion to ETA itself. These data strongly indicate that the regulation of both SpA and ETA occurs at the transcriptional level in S. aureus. The agr-regulated spa promoter was defined by deletion analysis and by transcript mapping.

Base Sequence↗
PubMed1991

A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus.

Staphylococcus aureus exoprotein expression is controlled by a global regulon known as agr. This system activates transcription of some target genes and represses transcription of others. Target genes expressed postexponentially such as alpha-hemolysin (hla) are activated by agr; target genes expressed during exponential phase such as protein A (spa) are repressed by agr. A unique feature of the agr system is that this transcriptional regulation is mediated by a 517-nucleotide transcript, RNAIII. While it is clear that agr differentially regulates the expression of exponential and postexponential exoproteins, the precise role of agr in the temporal control of these events has not yet been explored. In this report, we examine the effects of expressing RNAIII, the agr regulator, under the control of the inducible beta-lactamase (bla) promoter at different times in the growth cycle. We confirm previous results showing that agr is required for postexponential-phase expression of hla and further show that a separate postexponential-phase signal independent of agr function is also needed for activation of hla transcription. We also show that in an agr mutant transcription of spa occurs throughout the growth cycle, is inhibited immediately upon induction of RNAIII, and is thus indifferent to the postexponential signal required for hla activation.

Bacterial Toxins↗
PubMed1990

Cloning and nucleotide sequence of a chromosomally encoded tetracycline resistance determinant, tetA(M), from a pathogenic, methicillin-resistant strain of Staphylococcus aureus.

This report describes the cloning and sequencing of a chromosomally encoded tetracycline resistance determinant from a clinical isolate of methicillin-resistant Staphylococcus aureus. On the basis of the sequence, the gene is in the tet(M) class, and it was shown that the S. aureus tetA(M) gene is induced at the level of transcription.

Amino Acid Sequence↗
PubMed1989

pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator.

pT181 is the prototype of a family of staphylococcal plasmids that regulate their replication by means of antisense RNAs (countertranscripts) that block expression of the plasmid-coded initiator protein. In this paper, we show that the pT181 countertranscripts induce premature termination (attenuation) of the initiator mRNA by promoting the formation of a termination-causing hairpin just 5' to the initiator start codon. In the absence of the countertranscripts, an upstream sequence, the preemptor, pairs with the proximal arm of the terminator hairpin, preventing termination and permitting transcription of the initiator gene. This system thus differs from the classical attenuators in that attenuation is driven by antisense RNAs rather than by tRNA-induced stalling of ribosomes.

Bacterial Proteins↗
PubMed1988

Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus.

We have previously identified a gene in Staphylococcus aureus, agr, whose activity is required for high-level post-exponential-phase expression of a series of secreted proteins. In this paper, we describe the cloning of this gene in Escherichia coli by using an inserted transposon (Tn551) as a cloning probe. The cloned gene, consisting of a 241-codon open reading frame containing the site of the transposon insertion, was recloned to an S. aureus vector, pSK265, and shown to be functional in S. aureus. Activity was evaluated by determinations of alpha-hemolysin, beta-hemolysin, and toxic shock syndrome toxin-1 production in early-stationary-phase cultures. The cloned gene showed considerable variation with respect to different exoproteins and different host strains compared with the chromosomal agr determinant; this variation could not be attributed to the higher copy number of the cloned gene and probably reflects inapparent subtleties of the regulatory system.

Amino Acid Sequence↗
PubMed1987

A site-specific recombination function in Staphylococcus aureus plasmids.

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.

Amino Acid Sequence↗
PubMed1986

Conversion of a homogeneously methicillin-resistant strain of Staphylococcus aureus to heterogeneous resistance by Tn551-mediated insertional inactivation.

Plasmid pRN3208, thermosensitive for replication, and carrying the erythromycin transposon Tn551, was used for insertional inactivation of methicillin resistance in a highly and homogeneously resistant strain of Staphylococcus aureus. Two kinds of insertionally inactivated cells were obtained. Cultures of the major class contained highly methicillin resistant cells with a frequency of about 10(-3) to 10(-4), produced DNA with methicillin resistance transforming activity, and also produced penicillin binding protein 2a, the 78 kd low affinity penicillin binding protein characteristic of methicillin resistant Staphylococcus aureus, in apparently normal quantities. The single member of class B had no detectable methicillin resistant cells (less than 10(-8)) with an MIC greater than 1 micrograms/ml, contained no DNA with methicillin resistant transforming activity and no penicillin binding protein 2a. The data suggest that in the class A cells insertional inactivation did not affect the structural gene(s) of methicillin resistance but a regulatory locus or loci needed for the homogeneous expression of resistance.

Bacterial Proteins↗
PubMed1985

Replication control for pT181, an indirectly regulated plasmid.

PT181 is a fully sequenced Staphylococcus aureus plasmid whose size is 4,437 bp. It specifies tetracycline resistance and has a copy number of about 22 per cell in exponentially growing cultures. The functional organization of the pT181 replicon is centered around the coding sequence for a 35-kd protein, RepC, that is absolutely required for replication of the plasmid. The replication origin is contained within the repC coding sequence and the region immediately 5' to the RepC start is involved in control of the plasmid replication rate. PT181 replication is controlled at the level of RepC synthesis by a negative regulatory system that is functionally similar to that of the Co1E1 and IncFII plasmids of Escherichia coli. The pT181 control circuit involves 2 short transcripts, RNA I and RNA II, that are transcribed from the region specifying the 5' end of the untranslated repC mRNA leader and in the opposite direction. These are referred to as countertranscripts. The countertranscripts regulate RepC synthesis by a mechanism that probably involves interaction with the repC mRNA leader in a manner that interferes with translation. Both of the countertranscripts seem to be necessary for normal replication control; their separate roles remain unclear. Unlike plasmids of the Co1E1 and IncFII groups, plasmids such as Co1E1 are considered to have direct regulation of replication because the inhibitory element of the copy control circuit directly inhibits the initiation of replication. Plasmids such as pT181 are considered to have indirect regulation of replication because the product of the regulated step, RepC, is trans-active. Plasmids of the IncFII type are considered to have direct regulation of replication because the product of the regulated step, RepA is cis-active The analysis of pT181 replication physiology has illustrated 2 important differences between directly and indirectly regulated plasmids: a) for directly regulated plasmids, copy mutants specifying a normal inhibitor substance but an inactive target site exclude the wild-type or recessive mutants by directly interfering with their replication. Analogous mutants of indirectly regulated plasmids coexist readily with the wild-type and all mutants (although they do manifest segregational incompatibility) because the Rep protein is always shared by all plasmids in the cell, regardless of its source. b) Mutations of directly regulated plasmids in the region where target transcript and countertranscript overlap may give rise to totally new incompatibility groups because they engender independently self-correcting copy pools.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1985

Comparative sequence and functional analysis of pT181 and pC221, cognate plasmid replicons from Staphylococcus aureus.

The nucleotide sequence of pC221, a 4.6 kb Staphylococcus aureus plasmid is presented. The replication region of the plasmid is identified and compared with the corresponding region of pT181, a compatible but related plasmid. Both plasmids encode trans-active replicon-specific initiator proteins, RepC for pT181 and RepD for pC221. Plasmid replication rate is controlled by regulation of the rate of synthesis of the initiator protein by means of inhibitory 5' countertranscripts. Key elements of the control system are closely conserved between the two plasmids whereas less critical elements show extensive divergence. Overall architecture is also conserved, suggesting functional parallelism. The replication origin for both plasmids is contained within the N-terminal region of the initiator protein coding sequence; the two coding sequences are highly homologous but have two important areas of divergence, one within the origin region, the other near the C-terminus. In vivo recombinants between the two plasmids isolated previously (Iordanescu 1979) have crossover points within the initiator gene, between the two divergent regions. The recombinant plasmids have hybrid initiator proteins and are defective for replication, requiring the simultaneous presence of the parental plasmid from which their origin is derived. They are able to complement replication-defective mutants of the other parental plasmid, suggesting that the recognition specificity of the hybrid initiator protein resides in its C-terminal end and that the specific recognition site for the protein corresponds to the divergent region within the origin.

Amino Acid Sequence↗