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R P Novick

Publications and source records attributed to R P Novick.

At least 55 records · Page 3Linked to original sources
PubMed1993

Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element.

The leading strand replication origin of pT181 plasmids consists of two adjacent inverted repeat elements (IR-II and IR-III), which are involved in origin recognition by the initiator (Rep) protein. The conserved core element, IR-II, which contains the initiation nick site, is induced by Rep to form a cruciform structure, probably the primary substrate for the initiation of rolling circle replication. The divergent repeat, IR-III, constitutes the determinant of origin recognition specificity. We show here that the distal arm of IR-III is not required for sequence-specific recognition, whereas the proximal arm and central region are required. Since the initiator is dimeric, we presume that it binds symmetrically to IR-III. A unique type of DNA-protein interaction is proposed, in which the lack of sequence requirement for the distal arm is a consequence of binding to the adjacent IR-II, which thereby polarizes the stringency of binding to the two arms of IR-III. In addition, genetic evidence indicates that both the spacing and the phasing of IR-II to IR-III are crucial for function and that the central segment of IR-III may serve to position the two flanking half-sites for optimal interaction of Rep with IR-III.

Amino Acid Sequence↗
PubMed1993

Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis.

We have prepared and analyzed two types of gene fusion between the replication initiator gene, repC, and the reporter gene, blaZ, in order to investigate the relationship between pT181 plasmid copy number and RepC initiator protein production. A series of pT181 copy mutant plasmids, with copy numbers ranging from 70 to 800 copies per cell, were analyzed. In one type of gene fusion used in this study, blaZ was translationally coupled to the C-terminal end of the repC coding sequence such that native forms of both proteins were produced. This gene fusion arrangement, which permitted monitoring of RepC production (as BlaZ activity) by plasmids using the protein for their own replication, demonstrated a linear relationship, with one exception, between RepC production and plasmid copy number over a 20-fold range. In the second type of fusion, blaZ was translationally fused to the C-terminal end of repC. As the translational fusion did not produce active RepC protein, the fusion-containing pT181 derivatives were maintained in a strain which provided RepC in trans, and were thus analyzed at constant copy number. In contrast to previous analyses of this type, our translational fusion constructs expressed repC at levels proportional to the copy numbers of the plasmids from which the fusions were prepared. Using these data, we have calculated a minimum figure for the number of RepC molecules synthesized per replication event.

Amino Acid Sequence↗
PubMed1993

Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule.

The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up-regulated by agr whereas surface proteins are down-regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr-null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

Base Sequence↗
PubMed1993

Growth and analysis of crystal forms of toxic shock syndrome toxin 1.

Native toxic shock syndrome toxin 1 (TSST-1) purified from Staphylococcus aureus has been crystallized in four different forms. The highest resolution data (2.05 A) was collected from orthorhombic crystals belonging to the space group C222(1). The unit cell dimensions are a = 108.7 A, b = 177.5 A, c = 97.6 A. Rotation function analysis of this form indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 A. The space group is P2(1) with unit cell parameters of a = 44.4 A, b = 34.0 A, c = 55.2 A, beta = 93.0 degrees.

Bacterial Toxins↗
PubMed1992

Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element.

We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.

Amino Acid Sequence↗
PubMed1992

cis-inhibitory elements in the pT181 replication system.

We report here the existence of a pair of sequence elements in plasmid cointegrates that together block the function of pT181 plasmid replication origins in cis. The study is an outgrowth of the use of plasmid pE194 as a vector for the analysis of the pT181 replication system. We have observed that whereas the isolated pT181 replication origin is fully functional when cloned to pE194, it is inactive when the entire pT181 plasmid genome is cloned. This cis-inhibition is relieved by deletion of all or part of the pE194 palA element or of the pT181 countertranscript promoter. The inhibitory effect of pE194 palA is independent of distance and orientation, whereas the inhibitory effect of the countertranscript promoter is lost when the promoter is moved to a distance of 1.5 kb from the replication origin or inverted in situ. We found that the cis-inhibited pT181 origin expresses origin-specific (Inc3B) incompatibility, which involves competition for the initiator protein. This finding suggests that the cis-inhibited origin binds the initiator protein and therefore that the inhibition affects a step in the initiation process subsequent to initiator binding.

Base Sequence↗
PubMed1992

Nucleotide sequences and biologic properties of toxic shock syndrome toxin 1 from ovine- and bovine-associated Staphylococcus aureus.

Toxic shock syndrome toxin (TSST) 1 was purified from ovine (TSST-ovine) and bovine (TSST-bovine) mastitis-associated Staphylococcus aureus. These toxins were previously reported to have molecular weights identical to that of human TSST-1. However, TSST-ovine was reported as having an isoelectric point (pI) of 8.5, whereas TSST-bovine has the same pI (7.2) as TSST-1. Nucleotide sequence analysis revealed that TSST-bovine was identical to TSST-1 and that TSST-ovine had 14 nucleotide differences that changed 9 amino acid residues. Only 1 nucleotide difference, at position 514, was predicted to cause an amino acid charge difference, as glutamic acid at position 132 of TSST-1 was changed to lysine in TSST-ovine. Like TSST-1, TSST-ovine was mitogenic, but unlike TSST-1, it was not pyrogenic, was unable to enhance endotoxic shock, and was unable to induce TSS in a rabbit model. Also, TSST-ovine was less reactive to certain monoclonal antibodies raised against TSST-1.

Amino Acid Sequence↗
PubMed1992

Effect of glycerol monolaurate on bacterial growth and toxin production.

Glycerol monolaurate (GML) is a naturally occurring surfactant that has potential use as an additive to tampons and wound dressings to reduce the incidence of certain bacterial toxin-mediated illnesses. In vitro studies were undertaken to evaluate the effect of GML on the growth of and toxin production by potentially pathogenic bacteria. GML inhibited the growth of clinical isolates of group A, B, F, and G streptococci at concentrations of 10 to 20 micrograms/ml. Exotoxin production, including that of pyrogenic exotoxins and hemolysins, was reduced by concentrations of GML that were below those inhibitory for growth as well as growth inhibitory. The growth of Staphylococcus aureus strains from patients with toxic shock syndrome and scalded skin syndrome was inhibited or delayed in the presence of 100 to 300 micrograms of GML per ml. Growth inhibition by GML could be overcome by the production of lipase. S. aureus elaboration of hemolysin, toxic shock syndrome toxin 1, and exfoliative toxin A was inhibited at GML concentrations below those necessary to inhibit growth. Results similar to those for S. aureus were obtained in tests of S. hominis. Escherichia coli growth and Salmonella minnesota growth were unaffected by GML, but an S. minnesota Re mutant was susceptible to growth-inhibitory activity. Endotoxin release into the medium from E. coli cells was also unaffected by GML, but the release or activity of E. coli hemolysin was increased by GML. Streptococcal pyrogenic endotoxin A production by an E. coli clone was not affectd by GML. These studies indicate that GML is effective in blocking or delaying the production of exotoxins by pathogenic gram-positive bacteria.

Glycerides↗
PubMed1992

Glucose and nonmaintained pH decrease expression of the accessory gene regulator (agr) in Staphylococcus aureus.

The effect of glucose on accessory gene regulator (agr) expression in Staphylococcus aureus was examined. agr is a global regulator that affects the expression of numerous genes, including those for some factors implicated in virulence, such as toxic shock syndrome toxin 1, alpha-hemolysin, and protein A. The agr locus determines two divergent transcripts, designated RNAII and RNAIII. RNAII contains four open reading frames (agrABCD), and RNAIII encodes delta-hemolysin. The mechanisms responsible for agr-mediated regulation are not well understood, but it appears that the RNAIII transcript plays a central role in the regulation of a number of target genes, including those for alpha-hemolysin (hla), beta-hemolysin (hlb), protein A (spa), and staphylococcal enterotoxin B (seb+). In this study, S. aureus cultures were grown either in a shake flask system with a complex medium or in a fermentor system with a completely defined medium in which the pH and glucose concentration were maintained. Northern (RNA) blot analysis revealed that a dramatic reduction in agr expression was apparent only when the cultures contained glucose and when the pH was 5.5 or was not maintained. The effect of glucose on two agr target genes, sec+ and hla, was also studied. Glucose-containing cultures produced less sec+ and hla mRNAs at maintained pH (6.5). In addition, the glucose effect on sec+ and hla was enhanced under conditions that inhibited agr expression (i.e., pH 5.5 or a nonmaintained pH).

Fermentation↗
PubMed1991

The effect of plasmid copy number mutations on pT181 replication initiator protein expression.

Previous studies have shown that plasmid pT181 controls its replication by countertranscript-mediated regulation of the rate of synthesis of the pT181 initiator, RepC. In this study, the relation has been studied between plasmid copy number and RepC synthesis for a series of pT181 copy number mutants. For each mutant plasmid, the repC coding sequence along with its 5' regulatory region was translationally fused to the beta-lactamase structural gene on a vector plasmid unrelated to pT181. By means of these constructs, the effect of regulatory mutations on the initiator synthesis could be measured at constant copy number. With one exception, the mutant control regions showed elevated beta-lactamase activity in comparison to the wild-type. However, the relative increase was not very well correlated with the copy number of the corresponding mutant plasmid. The possibility is considered that factors such as DNA secondary structure may have important ancillary effects on the regulation mechanism.

Bacterial Proteins↗
PubMed1991

Fluid replacement protection of rabbits challenged subcutaneous with toxic shock syndrome toxins.

Toxic shock syndrome toxin 1 (TSST-1) and streptococcal pyrogenic exotoxin A (SPE A) belong to a family of pyrogenic toxins produced by Staphylococcus aureus and Streptococcus pyogenes, respectively. Both toxins are responsible for causing toxic shock syndrome (TSS) and related illnesses, clinically characterized by multiorgan involvement. The most severe TSS symptom is acute hypotension and shock after the initial febrile response. In this study, we examined possible mechanisms of shock development in TSS, particularly the role of T-cell proliferation, endotoxin enhancement by toxins, and capillary leakage. American Dutch belted rabbits, with subcutaneously implanted miniosmotic pumps filled with either TSST-1 or SPE A, served as the animal model. For both TSST-1 and SPE A-treated rabbits, administration of cyclosporin A prevented toxin-induced T-cell proliferation but failed to protect the rabbits. Polymyxin B treatment of rabbits, to neutralize endogenous endotoxin, partially protected rabbits from challenge with either exotoxin; two of six rabbits survived on day 2 when treated with only TSST-1, whereas six of six animals survived after challenge with TSST-1 and polymyxin B. Similarly, with SPE A-treated rabbits, only 1 of 10 animals without polymyxin B treatment survived on day 8, but 4 of 6 rabbits survived on day 8 when given polymyxin B. Fluid replacement was successful in preventing lethality. Twelve of 14 rabbits survived when given TSST-1 with fluid, and all rabbits treated with SPE A and fluid survived. Finally, by using miniosmotic pumps, staphylococcal exfoliative toxin A and concanavalin A were administered to rabbits in an attempt to induce lethality. These two T-cell mitogens caused T-cell proliferation but failed to induce lethality in rabbits. The data suggest that toxin interactions causing vascular leakage and to some extent endotoxin enhancement are of major importance in development of hypotension and shock in TSS. It appears that T-cell proliferation may not contribute significantly to the induction of shock and death.

Animals↗
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

Molecular requirements for T cell activation by the staphylococcal toxic shock syndrome toxin-1.

The activation of Ag-specific, Ia molecule-restricted, TCR V beta 3+ T cell clones by staphylococcal toxic shock syndrome toxin-1 (TSST-1), was investigated. The results show that although Ag- and TSST-1-induced activation of T cell clones both require TCR expression and similar biologic activation signals, the Ia molecule requirement for TSST-1 recognition was much less stringent than that observed for antigenic peptide recognition. In addition, T cell clones recognized TSST-1 without processing by APC. These results suggest that the ability of TSST-1 to polyclonally activate T cells is dependent on TCR recognition of the intact toxin molecule bound to a nonpolymorphic region(s) of the Ia molecule resulting in the same activation events induced by Ag recognition.

Animals↗
PubMed1990

Mutational and physiological analyses of plasmid pT181 functions expressing incompatibility.

Plasmid pT181 is a small multicopy plasmid from Staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, Inc3A and Inc3B. Inc3A incompatibility is expressed by the primary replication control determinant, copA, which specifies two small transcripts, RNA I and RNA II, that jointly inhibit the synthesis of the rate-limiting initiator protein, RepC. Inc3B incompatibility is expressed by the leading strand replication origin and is due to competition for RepC. The copA region from each of 11 different pT181 copy number mutants was cloned onto the pT181-compatible vector, pE194, and tested for its ability to inhibit the replication of pT181 and its copy number mutants. The pT181 replication origin was also cloned and tested for its ability to inhibit the replication of the same plasmids. In general copA mutations that alter the production or sequence of RNA I and RNA II greatly reduced or completely eliminated Inc3A activity. Unlike the wild-type, all of the copy mutants were resistant to Inc3B inhibition. The separately cloned wild-type copA and ori regions each reduced the copy number of pT181 in proportion to their gene dosage, but neither blocked replication completely. It is proposed that the cloned Inc determinants cause incompatibility by interfering with the plasmid's copy correction mechanism; this interference destabilizes the plasmid even under conditions where its average copy number is not greatly reduced.

Bacterial Proteins↗
PubMed1990

Initiation of rolling-circle replication in pT181 plasmid: initiator protein enhances cruciform extrusion at the origin.

Plasmid pT181 DNA secondary structures have been analyzed in vitro by nuclease S1 digestion and in vivo by bromoacetaldehyde treatment. A cruciform structure occurring at the pT181 replication origin in vitro is greatly enhanced by the binding of the plasmid-encoded initiator protein RepC. In vivo a DNA secondary structure also existed in the replication origin. Its frequency of formation was correlated with efficiency of RepC utilization. These data suggest that cruciform extrusion at the origin is involved in initiation of pT181 rolling-circle replication. A neighboring DNA structure influences the conformation of the origin in vivo.

Acetaldehyde↗