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Biomedical subjects

R P Novick

Publications and source records attributed to R P Novick.

At least 91 records · Page 5Linked to original sources
PubMed1987

Translational fusion with a secretory enzyme as an indicator.

A novel type of translational fusion system has been developed by using a secretory protein, staphylococcal beta-lactamase, as an indicator. The beta-lactamase structural gene was modified to provide N-terminal extensions of 13 and 162 amino acids, and in both cases, the fusion protein was processed and the mature active enzyme was secreted; thus, the expression of a particular upstream gene can be analyzed by monitoring the beta-lactamase activity.

Binding Sites↗
PubMed1987

Evaluation of coagulase-negative staphylococci for ability to produce toxic shock syndrome toxin 1.

A large and diverse group of coagulase-negative staphylococci were assayed for the ability to produce toxic shock syndrome toxin 1 (TSST-1) by immunological reactivity, and whole-cell DNAs from 33 of these strains were hybridized with a TSST-1-specific gene probe. None of the strains tested, including isolates that have been reported as TSST-1+, produced the exotoxin, and no DNA homology was found with the gene probe.

Bacterial Toxins↗
PubMed1986

Localization of biologic functions of toxic shock syndrome toxin-1 by use of monoclonal antibodies and cyanogen bromide-generated toxin fragments.

Monoclonal antibodies (mAb) against toxic shock syndrome toxin-1 (TSST-1) were generated that block two of the most important biologic activities of TSST-1, nonspecific T lymphocyte mitogenicity and the suppression of immunoglobulin synthesis. Fourteen hybridomas producing antibody against TSST-1 were isolated independently. The culture supernatant and ascitic fluids from each were analyzed to determine the mAb isotypes. Seven of the mAb were IgG1, and the remaining seven were IgM; all the mAb had kappa light chains. Immunoglobulin was partially purified from hybridoma-generated ascitic fluid by ammonium sulfate precipitation and tested for the ability to block TSST-1-induced mitogenicity and immunosuppression. Three mAb (all IgG1) were shown to block both the toxin-induced mitogenicity and the suppression. None of the mAb tested inhibited just one of the two toxin activities. The neutralizing mAb were then used in Western analysis with previously mapped cyanogen bromide (CNBr)-generated toxin fragments to localize the aforementioned biologic functions. The Western blot analysis showed that the mitogenic and the suppressive functions of TSST-1 were located on a 14,000 dalton internal CNBr fragment.

Antibodies, Monoclonal↗
PubMed1986

The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1.

The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, one-third of the predicted amino acid sequence was confirmed by amino acid sequence analysis of cyanogen bromide-generated TSST-1 protein fragments. The DNA sequencing results identified a 708-base pair open reading frame starting with an ATG, 7 base pairs downstream from a Shine-Dalgarno sequence, and terminating at a UAA stop codon. Amino acid analysis of the intact protein defined the NH2 terminus of the mature protein and located the cleavage point for the signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic structural similarities with other bacterial signal peptides. The coding sequence of the mature protein was 585 base pairs (194 amino acids) in length, and the molecular weight of the predicted protein was 22,049. This is in good agreement with the previously reported molecular weight of TSST-1 (22,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NH2-terminal amino acid sequence analysis performed on isolated TSST-1 CNBr fragments determined the position of the peptides in the TSST-1 sequence and verified the predicted amino acid sequence in those positions. Computer analyses of the amino acid sequence showed that TSST-1 has little or no sequence homology with biologically related toxins, streptococcal pyrogenic exotoxin A, and staphylococcal enterotoxins B and C.

Amino Acid Sequence↗
PubMed1986

Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes.

The molecular processes involved in the transduction of small staphylococcal plasmids by a generalized transducing phage, phi 11, have been analysed. The plasmids are transduced in the form of linear concatemers containing only plasmid DNA; plasmid-initiated replication is required for their generation but additive interplasmid recombination is not. Concatemers are probably generated by the interaction of one or more phage functions with replicating plasmid DNA. Insertion of any restriction fragment of the phage into the plasmid causes an approximately 10(5)-fold increase in transduction frequency, regardless of the size or genetic content of the fragment. The resulting transducing particles (Hft particles) contain mostly pure linear concatemers composed of tandem repeats of the plasmid::phage chimera, and their production requires active plasmid-initiated replication. The high frequency of transduction is a consequence of homologous recombination between the linear chimeric and phage concatemers, which has the effect of introducing an efficient pac site into the former. Following introduction into lysogenic recipient bacteria, the transducing DNA is first converted to the supercoiled form, then processed to monomers by a mechanism that requires the active participation of the plasmid replication system.

DNA Replication↗
PubMed1986

Resolution of highly purified toxic-shock syndrome toxin 1 into two distinct proteins by isoelectric focusing.

Highly purified toxic-shock syndrome toxin 1 (TSST-1) was prepared by differential precipitation with ethanol and resolubilization in water followed by successive electrofocusing in pH gradients of 3-10, 6-8, and 6.5-7.5. TSST-1, thus isolated, migrated as two distinct protein bands with isoelectric points of 7.08 (TSST-1a) and 7.22 (TSST-1b). When tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both toxins migrated as homogeneous bands with molecular weights of 22 000. The gel bands were visualized by silver staining. The two toxins have nearly identical amino acid compositions and are immunologically identical as shown by Ouchterlony reactivity against TSST-1 hyperimmune serum. TSST-1a and TSST-1b have the same biological activities as TSST-1: the capacity to induce fever, enhancement of host susceptibility to lethal endotoxin shock, nonspecific T lymphocyte mitogenicity, and suppression of immunoglobulin M synthesis against sheep erythrocytes. These two proteins have been isolated from several different TSS-associated Staphylococcus aureus strains. The data suggest that the differences in isoelectric point result either from the presence of a cofactor or from alternative conformations. Since only two bands appear, microheterogeneity as a result of deamination or acetylation is unlikely.

Amino Acids↗
PubMed1986

Regulation of exoprotein gene expression in Staphylococcus aureus by agar.

Insertion of the erythromycin-resistance transposon Tn551 into the Staphylococcus aureus chromosome at a site which maps between the purB and ilv loci has a pleiotrophic effect on the production of a number of extracellular proteins. Production of alpha, beta and delta hemolysin, toxic shock syndrome toxin (TSST-1) and staphylokinase was depressed about fifty-fold while protein A production was elevated twenty-fold. Hybridization analysis showed that the defect in expression of TSST-1 and alpha hemolysin was at the transcriptional level. Inability of the mutant strain to express either a cloned TSST-1 gene or the chromosomal gene indicates that the transposon has inactivated a trans-active positive control element. This element has been designated agr for accessory gene regulator.

Bacterial Proteins↗
PubMed1986

Incompatibility between plasmids with independent copy control.

Incompatibility between autonomous plasmids has been attributed, for the most part, to interaction between plasmids' negative control systems and/or partitioning systems. In this report it is shown that indirectly regulated plasmids with non-interactive negative control systems are incompatible on the basis of their shared initiator protein. This principle was demonstrated for a family of Staphylococcus aureus plasmids whose copy number is regulated by inhibitory RNAs that control the production of a rate-limiting, trans-active, initiator protein. We have constructed a pair of plasmids that have the same regulation systems and different initiator proteins and another pair with different regulation systems and the same initiators. Both of these pairs of plasmids were shown to be incompatible.

Base 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↗
PubMed1986

RepC is rate limiting for pT181 plasmid replication.

The effect on pT181 plasmid replication of the concentration of the plasmid-coded initiator protein, RepC, has been analyzed. In one type of experiment, plasmid replication was found to stop immediately after the addition of an inhibitory concentration of chloramphenicol (Cm) to growing cultures. Chromosomal replication showed the slow turnoff that is usual for Cm inhibition. Because plasmid replication rate is determined autogenously, no host factor can be rate limiting, suggesting that the specific factor affected is Rep C. In another type of experiment, we constructed a translational fusion between the repC coding sequence and a translationally inducible Cm-acetylase gene, cat-86, using pUB110 as the carrier replicon. The fusion plasmid showed an eightfold amplification of its own copy number and a similar amplification of a co-resident pT181 plasmid upon Cm induction. The amplified plasmids did not show autocatalytic runaway replication but rather established stable elevated copy numbers, indicating the existence of a secondary level of regulation. These results suggest that RepC is rate limiting for pT181 replication and support the hypothesis that pT181 replication is regulated at the level of RepC synthesis. The nature of the secondary regulation is unknown.

Acetyltransferases↗
PubMed1986

cmp, a cis-acting plasmid locus that increases interaction between replication origin and initiator protein.

pT181, a 4.4-kilobase multicopy plasmid of Staphylococcus aureus, encodes a trans-acting initiator protein, RepC, which was rate limiting for replication. Deletions in a 500-base-pair region of the plasmid external to the minimal replicon decreased the ability of the plasmid to compete with a coexisting incompatible plasmid. These deletions, which define a region called cmp (for competition), appeared to affect the interaction of RepC and the plasmid origin of replication. However, in the homoplasmid state the deletions affected neither copy number nor plasmid stability. The Cmp phenotype is orientation independent, and cmp defects could not be complemented in trans.

Bacterial Proteins↗
PubMed1986

Nosocomial transmission of a strain of Staphylococcus aureus causing toxic shock syndrome.

A strain of Staphylococcus aureus producing toxic shock syndrome toxin-1 was repeatedly isolated from the nares of a neurosurgeon. This strain was identical to strains cultured from two of his patients who developed toxic shock syndrome after laminectomy. The relatedness of the isolates was shown by Southern blot hybridization analyses using chromosomal transposons as probes. This approach should be considered, in addition to standard bacteriologic techniques, as an effective method to analyze the relatedness of nosocomial isolates.

Adult↗
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↗
PubMed1985

Plasmid pT181 replication is regulated by two countertranscripts.

A transcription map of the replication control region of the Staphylococcus aureus plasmid pT181 has been constructed. Two major leftward transcripts, RNA III and RNA IV, start at positions 339 and 413, respectively. These two RNAs can serve as mRNAs for a plasmid-specific replication protein RepC. Two short rightward transcripts, RNA I and RNA II, approximately 85 and 150 nucleotides long, respectively, start at position 246. These rightward transcripts (referred to as countertranscripts) do not appear to be translated but act directly as negative regulators of plasmid replication, probably by interfering with translation of the RepC mRNAs. There is no significant base sequence homology among the countertranscripts of pT181, ColE1, and R1/NR1/R6-5, suggesting that the structural parallelism has risen by convergent molecular evolution.

Amino Acid Sequence↗
PubMed1985

MLS-resistance determinants in Staphylococcus aureus and their molecular evolution.

This paper describes the genetic phenomenology of resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr) in Staphylococcus aureus and attempts to place this phenomenology in a broad evolutionary context. As antibiotic resistance in general and MLS resistance in particular are typical variable traits in bacteria of clinical interest, we shall begin by introducing the concept of variable genetic traits, as outlined in Figure 1. Variable traits are those that are expressed by some strains of a given species but not by others--in comparison to constant traits which are always present as part of the standard genetic make-up of the species and have constant chromosomal locations. Variable traits are often associated with variable and mobile genetic elements and it is suggested that, in general, they are not likely to have evolved as such in the species in which they are found. Rather, they will most probably have evolved as constant (chromosomal) traits in other species and acquired genetic mobility much later as a rare occurrence in that species. These rarely occurring mobile variants would then spread horizontally within a range of new species. The MLSr determinants in Gram-positive bacteria would appear to represent a classic example of this process. Their remarkable variability will be described as the extant end-point of the process and a probable evolutionary pathway will be traced back to the streptomycetes which are a likely primary source.

Anti-Bacterial Agents↗
PubMed1985

Expression of the cloned toxic shock syndrome toxin 1 gene (tst) in vivo with a rabbit uterine model.

Toxic shock syndrome (TSS) toxin 1 (TSST1) is produced by strains of Staphylococcus aureus associated with TSS. Purified TSST1 induces in rabbits a shock-like illness with many features similar to TSS in humans. These symptoms were also induced by TSST1-producing bacteria in diffusion chambers implanted in the rabbit uterus. Naturally occurring TSST1+ strains and a TSST1- strain harboring a pE194-derived plasmid carrying the cloned TSST1 determinant tst gave the same symptoms. TSST1- strains and a TSST1- strain carrying a pE194-tst plasmid with a deletion of the tst gene had no effect in rabbits. The results with the plasmid-carrying TSST1+ and TSST1- strains, which were isogenic apart from tst, show that the toxin is responsible for the illness in rabbits and suggest that it is a major factor in the pathogenesis of TSS.

Animals↗