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S J Projan

Publications and source records attributed to S J Projan.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein.

Septicemia due to Staphylococcus aureus often begins as a focal infection (e.g., colonized wounds or catheters) from which the organism gains access to the bloodstream. On the basis of recent data from this laboratory, it is likely that S. aureus colonizes catheters and endothelium by using a fibrinogen-binding protein to mediate adhesion to fibrinogen-coated surfaces. To characterize the fibrinogen-reactive protein, we screened a lambda Zap library of S. aureus DB, a clinical isolate, for clones that were reactive with fibrinogen. Of 100,000 plaques screened, 3 were found to react with fibrinogen on immunoblots. Plasmid DNA prepared from clones 14, 30, and 36, upon digestion with EcoR1, which released the insert, revealed fragments of 4.6, 3.6, and 3.2 kb, respectively. To identify the cloned protein expressed in E. coli, cells were fractionated into periplasmic, membrane, and cytoplasmic fractions. Expression studies of clone 14, which comprised approximately two-thirds of the mature molecule, including the C terminus, revealed a 34-kDa fibrinogen-reactive protein in both the periplasmic and membrane fractions. This protein, designated FbpA, could be partially purified on a fibrinogen column. By using both clones 14 and 36 as templates, the complete DNA sequence of the fibrinogen-binding protein was obtained, yielding a molecule with a predicted size of 69,991 Da. Although sequence analysis revealed a high degree of homology with coagulase, there is a unique sequence of 11 amino acids that is not found in three known coagulases as well as two recently cloned fibrinogen-binding proteins. This unique sequence shares homology with a cell wall anchor motif found in other gram-positive surface proteins.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of sarA of Staphylococcus aureus, a gene required for the expression of agr.

To evaluate the effect of a sar mutation on the agr locus, Northern (RNA) blotting was performed to determine the levels of RNAIII, the agr regulatory molecule, in two isogenic pairs of Staphylococcus aureus strains. Our results demonstrated that RNAIII was either significantly diminished or absent in both sar mutants compared with the parents. The RNAIII level was partially restored in sar mutants complemented with an intact sar gene (designated sarA). Additionally, we were able to complement selected sar phenotypes with a plasmid carrying RNAIII (pRN6735). These studies suggest that the sarA gene is necessary for the optimal expression of agr. The sarA gene of strain RN450 was subsequently cloned and sequenced. Sequence analysis revealed an open reading frame of 372 bp with a predicted molecular size of 14,718 Da and a deduced pI of 8.52. The deduced protein sequence has a predominance of charged residues (33%) and shares sequence similarity with the virF gene of Shigella flexneri.

Amino Acid Sequence↗
PubMed1994

Glycerol monolaurate inhibits the production of beta-lactamase, toxic shock toxin-1, and other staphylococcal exoproteins by interfering with signal transduction.

Glycerol monolaurate (GML) is a naturally occurring surfactant that is used widely as an emulsifier in the food and cosmetics industries and is generally regarded as lacking in important biological activities. The recent observation that it inhibits the production of staphylococcal toxic shock toxin-1 (P. M. Schlievert, J. R. Deringer, M. H. Kim, S. J. Projan, and R. P. Novick, Antimicrob. Agents Chemother. 36:626-631, 1992) is therefore rather surprising and raises the interesting question of how such a compound might interact with cells. In this report, we show that GML inhibits the synthesis of most staphylococcal toxins and other exoproteins and that it does so at the level of transcription. We find that GML blocks the induction but not the constitutive synthesis of beta-lactamase, suggesting that it acts by interfering with signal transduction.

Bacterial Proteins↗
PubMed1994

Plasmids of the pT181 family show replication-specific initiator protein modification.

The rolling circle plasmids of Staphylococcus aureus regulate their replication by controlling initiator (Rep) protein synthesis. It was demonstrated recently that the pT181 initiator protein RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC* (A. Rasooly and R. P. Novick, Science, 262:1048-1050). We establish here that this initiator modification occurs with four other members of the pT181 family and that it occurs in Bacillus subtilis as well as S. aureus. These results suggest that Rep conversion to Rep* is probably universal among plasmids of the pT181 family and is not host dependent.

Amino Acid Sequence↗
PubMed1993

Agr-related sequences in Staphylococcus lugdunensis.

Sequences related to the Staphylococcus aureus accessory gene regulator (agr) were demonstrated in S. lugdunensis by Southern blot analysis of 13 strains and sequencing of the S. lugdunensis agr-like locus (agr-sl). Northern blot analysis of cellular RNA revealed the presence of a transcript having homology with the agr-P3 transcript (RNAIII) for three of the six strains tested. The three strains containing this transcript produce a hemolysin with phenotypic properties similar to that of S. aureus delta-hemolysin. Nevertheless, unlike agr-P3 from S. aureus, agr-sl does not encode any potential peptide homologous to S. aureus delta-hemolysin, suggesting that the hemolytic activity detected in S. lugdunensis is encoded elsewhere and may be controlled by agr-sl.

Amino Acid Sequence↗
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

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

The effect of a mouthrinse based on nisin, a bacteriocin, on developing plaque and gingivitis in beagle dogs.

The aim of this study was to evaluate the effect of nisin, an antimicrobial peptide, on the development of plaque and gingivitis in beagle dogs when compared with 0.12% chlorhexidine and a placebo. 16 female beagle dogs 1 year of age were brought to optimum gingival health by scaling, root planing and polishing. At the conclusion of the pretreatment phase, the dogs were divided into 4 groups for the application of the test agents and were placed on a plaque promoting diet of Purina Dog Chow softened with water. Test agents included 100 micrograms/ml nisin and 300 micrograms/ml nisin formulated in a vehicle containing 1 mM NaEDTA; negative control comprised of exactly the same formulation but omitting nisin; and 0.12% chlorhexidine as Peridex. Throughout the treatment period, formulations were applied 2x daily to premolar teeth in each quadrant for 1 min using a Monojet syringe. The development of plaque and gingivitis was monitored at 15, 27, 39, 53, 74 and 88 days during the treatment phase using standard measurements of gingival index, plaque index, stain index and bleeding to probing. Throughout the treatment phase, plaque accumulation increased in all groups, but the rate of plaque build-up was less in groups treated with either nisin or chlorhexidine formulations compared with the placebo treated group. The gingival index of dogs in all groups increased throughout the study period. However, from day 27 onward, the groups receiving nisin had lower gingival index scores than did the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1992

Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr.

A single insertion of transposon Tn917LTV1 into the chromosome of a Staphylococcus aureus clinical isolate, strain DB, resulted in a pleiotropic effect on the expression of a number of extracellular and cell-wall-associated proteins. Detailed comparison of phenotypes associated with the mutant, 11D2, and the parent, DB, indicated that the chromosomal locus inactivated as a result of transposon mutagenesis differs from the S. aureus accessory gene regulator locus (agr). In particular, the expression of alpha-hemolysin, which is not detectable in Agr- mutants, was enhanced in mutant 11D2, while it remained at a low level in strain DB. Likewise, protease activity was significantly enhanced in 11D2 compared with DB. In addition, most of the cell-bound proteins were expressed at lower levels in the mutant than the parent strain. This pattern is contrary to that found in switching from Agr+ to Agr- phenotypes. Southern blot hybridization with an agr probe indicated that the inactivated chromosomal locus is distinct from agr. Transduction experiments demonstrated that the phenotypes associated with mutant 11D2 could be transferred to the parental strain DB as well as to RN450, an S. aureus strain with a genetic background similar to strain 8325-4. This locus on the S. aureus chromosome, possibly regulatory in nature, has been designated sar for staphylococcal accessory regulator.

Bacterial Proteins↗
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

A staphylococcal multidrug resistance gene product is a member of a new protein family.

The complete nucleotide sequence (321 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates. The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The smr gene was subcloned and expressed in S. aureus and E. coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes: phosphonium derivatives and quarternary amines. Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations. The deduced polypeptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr). Four transmembrane segments are predicted for smr gene product. Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment. This Glu 13 is conserved in all members of the family of small membrane proteins. We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation. This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylic residues in the transmembrane segments of Smr.

Amino Acid 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

Evaluation of a nisin-based germicidal formulation on teat skin of live cows.

A purified preparation of the nontoxic antimicrobial peptide, nisin (AMBICIN N), was used in the formulation of a germicidal sanitizer suitable for use on cow teats. The germicidal activity of the formulation against mastitis pathogens was measured on teat skin of live cows. The nisin-based formulation gave a mean log reduction of 3.90 against Staphylococcus aureus and 4.22 log reduction against Escherichia coli after exposure for 1 min to the germicide. This activity was comparable with that exhibited by a 1% iodophor teat dip but was significantly greater than that exhibited by the .1 and .5% iodophors and by the .5% chlorhexidine digluconate teat dips. The nisin-based formulation showed little or no potential for skin irritation after multiple application to skin, but iodophor and chlorhexidine digluconate teat dips showed significant potential for skin irritation in comparable studies.

Animals↗
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↗
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↗