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Biomedical subjects

R P Novick

Publications and source records attributed to R P Novick.

At least 109 records · Page 6Linked to original sources
PubMed1984

Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein.

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.

Bacterial Proteins↗
PubMed1984

Control of pT181 replication II. Mutational analysis.

We describe the isolation and analysis of mutations affecting the regulation of Staphylococcus aureus plasmid pT181 replication. Previous results suggested that regulation is achieved by control of the synthesis of RepC, a plasmid-coded replication protein and that the primary negative control element is CopA RNA, which consists of two transcripts that are complementary to the 5' region of the repC mRNA leader. CopA inhibition probably involves a base pairing interaction with the complementary region of the RepC mRNA leader which would facilitate the formation of a downstream stem-loop in the leader that occludes the repC ribosome binding site. RepC is freely diffusible so that regulation of pT181 replication is indirect. Both CopA RNA-sensitive (recessive) and -insensitive (dominant) mutants were isolated. The recessives have defects in CopA RNA structure or activity, the dominants have defects in the site of action (target) of the inhibitor. Some dominants were located within the copA coding sequence. These therefore affect the structure of CopA RNA as well as that of its target. Other dominant mutations mapped outside of the copA gene and therefore produced wild-type CopA RNA. In contrast to directly regulated plasmids, pT181 copy mutants producing wild-type inhibitor could be co-maintained with the wild-type plasmid and mutational changes in inhibitor-target specificity did not change incompatibility specificity.

Bacillus subtilis↗
PubMed1984

Staphylococcal plasmid cointegrates are formed by host- and phage-mediated general rec systems that act on short regions of homology.

Cointegrates involving pairs of compatible staphylococcal plasmids can be isolated either by co-selection during transduction (Novick et al. 1981) or by selection for survival at the restrictive temperature of a thermosensitive, replication defective plasmid in the presence of a stable one. Cointegrates are formed by recombination at two specific sites, RSA and RSB. RSB is present on each of six plasmids analyzed, namely pT181, pE194, pC194, pS194, pUB110, and pSN2, and RSA is present on two of these, pT181 and pE194. In this communication, it is shown that the RS represent short regions of homology (RSA is some 70 bp in length and RSB is about 30) embedded in largely non-homologous contexts and that the crossovers take place within these homologous regions. The pT181 and pE194 RSA sequences contain several mismatches which permit the localization of the crossover events to several different sites within the overall RS segment. The recombination system involved is therefore general (homology-specific) rather than site-specific (sequence-specific). Mismatches included within the crossover region are always corrected to the pT181 configuration. The cointegrates are therefore formed by a relatively efficient general rec system that recognizes short regions of homology and gives rise to Holliday junctions that probably involve very short heteroduplex overlaps. The sequence results are consistent with asymmetric single-strand invasion of a contralateral gap with nucleotide conversion by copying. It is noted that RSB has substantial homology with the par sequence of plasmid pSC101, suggesting that it may be involved in plasmid partitioning.

Base Composition↗
PubMed1984

Reciprocal intrapool variation in plasmid copy numbers: a characteristic of segregational incompatibility.

An experimental analysis of the concept that incompatible plasmids occupy a common intracellular pool from which copies are drawn at random for replication and assortment is presented. Intrapool variations in an incompatible heteroplasmid strain are inevitable and it is shown that these variations can be exploited by differential selection to amplify one plasmid at the expense of the other. Constant overall copy number is demonstrated for isogenic wild-type replicons and also for isogenic copy mutants whose copy numbers are so great that segregational incompatibility cannot be measured. In the test system used, that of the Staphylococcus aureus plasmid pT181, the rate of replication is probably determined by the availability of a trans-active initiator protein, RepC. In heteroplasmid strains containing wild-type and dominant copy mutant plasmids, although intrapool variation occurs, the total copy number is not constant but varies as a consequence of selection for or against the mutant plasmid. This is because all of the RepC is synthesized from the mutant plasmid (the wild-type is hyper-repressed) and therefore the selection affects the supply of RepC at the same time that it affects the copy number of the plasmid. None of these effects are seen with single plasmids or with compatible pairs.

DNA Replication↗
PubMed1984

Staphylococcal enterotoxin A gene is associated with a variable genetic element.

The genetic determinant of Staphylococcus aureus enterotoxin A (SEA) has been cloned in pBR322 in Escherichia coli and found to be expressed and secreted into the periplasmic space in that organism. The SEA gene (entA) is within a 2.5-kilobase-pair HindIII fragment that is part of a discrete genetic element 8-12 kilobase pairs in length. This entA element has a standard chromosomal location [between the purine (pur) and isoleucine-valine (ilv) markers] in most S. aureus strains. In some strains it is unlinked to pur-ilv. However, its internal structure is conserved at different locations. Some naturally occurring SEA-nonproducer (EntA-) strains lack the entire entA element, and one instance of its spontaneous loss is reported. Other naturally occurring strains have EntA- structural variants of the element at the same pur-ilv location at which the intact element is most commonly found. Some of these strains are EntA-, others are EntA+; the latter have a second, unlinked copy of the element containing their functional entA gene. These results suggest that entA is associated with a structurally unstable, possibly mobile, discrete genetic element.

DNA Restriction Enzymes↗
PubMed1983

Determination of plasmid copy number by fluorescence densitometry.

A simple and reliable method for the determination of plasmid copy numbers by direct fluorescence densitometry of ethidium bromide-stained electrophoretic gels was developed. In developing the method, the following parameters were evaluated and controlled: plasmid DNA trapping in the linear chromosomal DNA, staining-destaining kinetics for ethidium bromide, linearity of the fluorescence response, and the effect of the molecular topology of DNA on ethidium bromide binding to DNA in agarose.

DNA, Bacterial↗
PubMed1983

Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus.

pT181 is a naturally occurring Staphylococcus aureus plasmid, encoding inducible resistance to tetracycline. The plasmid has a copy number of about 20 per cell, and belongs to the incompatibility group inc3. The complete nucleotide sequence of pT181 has been determined and consists of 4437 bp. The nucleotide sequence contains 69.8% A-T and 30.2% G-C pairs. pT181 was found to contain four open reading frames capable of coding for polypeptides containing more than 50 amino acids. All the putative polypeptides are coded by one strand. The molecular weights of the four putative polypeptides are (in daltons): A, 37,500; B, 35,000; C, 23,000, and D, 18,000. Polypeptide A corresponds to the repC protein, earlier shown to be specifically required for pT181 replication. Polypeptide B (and possibly polypeptide D) are involved in tetracycline resistance. No role has yet been established for polypeptide C; deletion of the coding sequence for the C polypeptide has no detectable effect on any property of the pT181 plasmid. A region consisting of about 1200 bp contains information for the replication and copy number control of this plasmid. The sequencing results are discussed in relation to the replication properties and tetracycline resistance associated with the pT181 plasmid.

Base Sequence↗
PubMed1983

Effects of tylosin feeding on the antibiotic resistance of selected gram-positive bacteria in pigs.

The effect of tylosin on macrolide resistance of gram-positive bacteria of pigs was determined. After an initial base-line period during which the pigs were given antibiotic-free feed, 1 group of 8 pigs was given tylosin feed (100 g/US ton of feed), and a 2nd group of 7 was given antibiotic-free feed. Samples were taken at 2- to 3-week intervals. For each pig, rectal, skin, and nasal swab samples were collected for enumeration of fecal streptococci and skin and nasal staphylococci. Percentages of macrolide resistant organisms of each group were tabulated on the basis of colony counts from antibiotic free and erythromycin-containing plates. After the introduction of tylosin into the feed of 1 group, a clear difference between the 2 groups with respect to the macrolide resistance of their gram-positive microflora was observed. The data indicate that tylosin feeding results in an increase in macrolide resistance of the bacterial flora of pigs.

Animals↗
PubMed1982

Coding sequence for the pT181 repC product: a plasmid-coded protein uniquely required for replication.

pT181 is a 4.4-kilobase plasmid from Staphylococcus aureus specifying tetracycline resistance and present in about 20 copies per cell. The existence of a diffusible pT181 product required for plasmid replication has been proposed on the basis of trans-complementable thermosensitive mutants defective in plasmid maintenance (phenotype Tsr). In this report, the Tsr mutants are shown to have primary replication defects, and the genetic complementation data are confirmed biochemically. All of five mutations are in a single cistron, the repC cistron; interruption of the plasmid DNA molecule at any of three neighboring restriction sites inactivates repC function. Analysis of the DNA sequence in this region reveals an open reading frame of 939 base pairs which encodes the repC product, a 313-amino acid protein. pT181 replication has been demonstrated in cell-free extracts to require specifically a pT181-coded protein of approximately the same size, and it is proposed that this protein is, indeed, the repC product. Preliminary evidence is discussed suggesting that the pT181 replication rate is controlled at the level of synthesis of the repC protein.

Bacterial Proteins↗
PubMed1982

Functional origin of replication of pT181 plasmid DNA is contained within a 168-base-pair segment.

We have used a recently developed in vitro replication system from Staphylococcus aureus to determine the origin and direction of replication of pT181 plasmid DNA. The origin was located to within 168 base pairs by two methods: (i) sequential labeling of restriction endonuclease fragments after synchronous initiation in vitro in the presence of various amounts of dideoxy-TTP and (ii) by constructing in vitro deletions of pT181 DNA close to the origin of replication and testing for their ability to replicate in vitro pT181 plasmid was found to replicate unidirectionally and anticlockwise, as the map is conventionally drawn. The nucleotide sequence of the region containing the origin of replication has been determined and found to be partially or entirely contained within the coding sequence for the repC protein, which is uniquely required for pT181 plasmid replication. Preliminary evidence suggesting that pT181 replicates by a rolling circle mechanism is discussed.

Bacterial Proteins↗
PubMed1982

Structural analysis of plasmid pSN2 in Staphylococcus aureus: no involvement in enterotoxin B production.

Earlier studies have suggested the involvement of a small 1.3-kilobase plasmid, pSN2, in the production of enterotoxin D by certain Staphylococcus aureus strains. On the basis of extensive biochemical studies on pSN2, including the determination of its coding properties and its primary nucleotide sequence, we conclude that this plasmid is not in act involved in enterotoxin B production in S. aureus: although the toxin genes are apparently chromosomal, it is probable that they are part of a special genetic system such as a hitchhiking transposon.

Bacterial Proteins↗
PubMed1982

Genetic studies on Staphylococcal strains from patients with toxic shock syndrome.

Thirteen isolates of Staphylococcus aureus that produce the toxic shock syndrome exotoxin were screened to identify and characterize this specific determinant and understand its role in pathogenicity. These stains belong to phage group I, are sensitive to phage 29, and are similar with respect to their resistance to cadmium, arsenate, and penicillin. These three resistances, commonly found on plasmids in many strains of S. aureus, were not plasmid-associated in 13 toxic shock strains. The cadmium and arsenate resistances were cotransferred both in transduction and in protoplast fusion; penicillin resistance was unlinked. The toxic syndrome exotoxin gene was not linked to any of these three traits. We suggest that the trait is borne by a special genetic element that acts as a heterologous chromosomal insertion and is independent of the cadmium-arsenate linkage group or the penicillinase-determinant. The genetic properties of extracellular proteins in S. aureus are reviewed as possible models for the acquisition or expression of this toxic shock antigen.

Arsenates↗
PubMed1981

Replication of plasmid pT181 DNA in vitro: requirement for a plasmid-encoded product.

PT181 is a naturally occurring 4.5-kilobase Staphylococcus aureus plasmid encoding resistance to tetracycline. The plasmid has a copy number of about 20 per cell; a mutant, cop-608, that has a copy number of 800-1000 has been isolated. A cell-free extract has been developed that carries out complete replication of this plasmid. Extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pT181. In an extract from which endogenous DNA has been removed, DNA synthesis is dependent upon the addition of exogenous plasmid DNA. The replication system is specific for pT181 and related plasmids but it is inactive with other S. aureus plasmids. Furthermore, pT181 DNA does not replicate in extracts made from plasmid-negative strains or strains containing other plasmids. The results suggest that a specific plasmid-encoded substance is required for the replication of pT181 DNA.

Cell-Free System↗