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S Iordanescu

Publications and source records attributed to S Iordanescu.

14 recordsLinked to original sources
PubMed1991

The Staphylococcus aureus mutation pcrA3 leads to the accumulation of pT181 replication initiation complexes.

In Staphylococcus aureus cells carrying the pcrA3 chromosomal mutation, plasmid pT181 and its derivatives were maintained at a reduced copy number. A significant proportion of their DNA migrated during agarose gel electrophoresis as nicked DNA. The results obtained in the characterization of this plasmid DNA species show that it represents replication initiation complexes. Such complexes could not be detected in a wild-type host. The replication initiation complexes present in pcrA3 cells could resume replication after a lag. It was concluded from these results that the pcrA3 host mutation affected a step in plasmid pT181 replication immediately following the formation of the replication initiation complex, and that in pcrA3 this step became rate-limiting for plasmid pT181 replication.

Bacterial Proteins↗
PubMed1991

The Staphylococcus aureus chromosomal gene plaC, identified by mutations amplifying plasmid pT181, encodes a sigma factor.

The Staphylococcus aureus chromosomal gene plaC, identified by mutations such as plaC1 that lead to the amplification of plasmid pT181, has been cloned and sequenced. The plaC gene encodes a protein with high similarity (79% identity) with the vegetative sigma factor of Bacillus subtilis, sigA, suggesting that it acts as an RNA polymerase sigma factor in S.aureus. The plaC1 mutation was found to be a C to T transition leading to a proline to serine substitution at amino acid residue 209 of the protein. In other sigma factors this region of the protein is involved in specific recognition of the -10 promoter sequence. The change in sigma factor activity due to this mutation is characterized by its strict specificity for a limited number of promoters and the rather high amplitude of the effect.

Amino Acid Sequence↗
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↗
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↗
PubMed1989

Functional organization of the plasmid pT181 replication origin.

Replication of the staphylococcal plasmid pT181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein RepC. Deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting RepC protein. A shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. Single and double point mutations within these 43 base-pairs were used to determine the sequence requirement for replication within the minimal origin. Deletion mutants and point mutants were tested in replication and competition assays in vivo and in vitro, and in a RepC-mediated nicking assay.

Bacterial Proteins↗
PubMed1989

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221.

pT181 and pC221 are closely related Staphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in the rep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping the rep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1989

Staphylococcus aureus chromosomal mutations that decrease efficiency of Rep utilization in replication of pT181 and related plasmids.

Staphylococcus aureus chromosomal mutants which maintain pT181 and related plasmids at a much reduced copy number but which do not affect the replication of other plasmids have been isolated. The origin of replication and the initiator protein of the affected plasmids are the only elements required for the response to these mutations. The host mutations do not interfere with the pT181 replication control mechanism.

Chromosomes, Bacterial↗
PubMed1989

Staphylococcus aureus chromosomal mutation plaC1 amplifies plasmid pT181 by depressing synthesis of its negative-effector countertranscripts.

A Staphylococcus aureus chromosomal mutation, plaC1, which leads specifically to the amplification of plasmid pT181 has previously been described (S. Iordanescu, Plasmid 10:130-137, 1983). The mechanism by which plaC1 amplifies plasmid pT181 has been approached in two ways: determination of the plasmid region required for the specific response to the plaC1 mutation and evaluation of different parameters of pT181 replication control by using transcriptional and translational fusions with the beta-lactamase gene as an indicator gene. The results obtained indicate that the control region of plasmid pT181 represents the target of the plaC1 effect, which acts primarily by depressing the synthesis of plasmid pT181 countertranscripts, those small, untranslated RNA molecules playing the roles of negative effectors in the replication control mechanism of the plasmid. In turn, the reduction in countertranscript synthesis leads to an increase in the production of the initiator protein RepC, which is limiting for plasmid replication, and a higher plasmid copy number.

Chromosomes, Bacterial↗
PubMed1988

Replication termination for staphylococcal plasmids: plasmids pT181 and pC221 cross-react in the termination process.

We present data which indicate that (i) the origin of replication of plasmids pT181 and pC221 can also function as termination signals; (ii) termination of replication occurs when a round of replication initiated either by RepC at the pT181 origin or by RepD at the pC221 origin reaches either of these origins, proving that the two plasmids cross-react for termination of replication; and (iii) the replication initiated at the origin of another staphylococcal plasmid, pE194, does not terminate at the origin of pT181 or pC221, indicating the existence of a specific relationship between the initiation and termination of a replication event.

Bacterial Proteins↗
PubMed1987

The Inc3B determinant of plasmid pT181. A mutational analysis.

A region encompassing the origin of replication of staphylococcal plasmid pT181 has previously been shown to express an incompatibility effect denoted Inc3B, when cloned into another replicon (Novick et al. 1984). In an attempt to understand the mechanism of this incompatibility effect, and its relationship with the function of the replication origin, mutants deficient in this property were isolated and characterized. The results obtained suggest that the Inc3B effect is due to the competition for replication between the replication origin cloned in a hybrid and the origin of an autonomous plasmid. The Inc3B-deficient mutants isolated expressed different degrees of residual incompatibility. The inc3B mutations which did not express any incompatibility were found also to inactivate the function of the replication origin. All the other mutants which expressed residual Inc3B had a functional origin but presented a significantly reduced ability to use this origin when coexisting with a plasmid using a wild-type pT181 origin. It is suggested that these inc3B mutations represent a new type of origin mutation which affects the ability of the origin to compete with other origins using the same replication system, though the function per se of the origin is not significantly impaired.

DNA Replication↗
PubMed1986

Effect of the deletion of a fragment dispensable for the autonomous maintenance of plasmid pT181 on the competition between incompatible plasmids.

The deletion of the 560-bp HindIII C fragment from pT181 derivatives does not change the stability or copy number of the plasmid but affects its ability to compete with undeleted, incompatible plasmids for maintenance in the host cell. The disadvantage of the deleted plasmids seems to be manifested at the level of replication. It results that for plasmid pT181 a sequence dispensable for autonomous maintenance and replication control could affect the outcome of the competition between autonomous, incompatible plasmids.

Base Sequence↗
PubMed1976

Two restriction and modification systems in Staphylococcus aureus NCTC8325.

The presence of two distinct host specificities in Staphylococcus aureus strain NCTC8325 was revealed by the isolation of restriction- and modification-deficient mutants. The two host specificity systems, designated S1 and S2, are both active on phage 80mualpha but are not additive in their restricting activity. Restriction-deficient, modification-proficient mutants were invariably affected in both restriction systems. The functional relationship between these two systems is discussed.

DNA, Bacterial↗