Regulated ectopic expression and allelic-replacement mutagenesis as a method for gene essentiality testing in Staphylococcus aureus.

Conditional expression systems were utilized for the ectopic induction of essential genes in Staphylococcus aureus. Resulting strains were then subjected to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. This strategy produced test strains whereby cellular viability was uniquely dependent on the presence of inducer and provided a direct and absolute confirmation of genetic essentiality for each locus. The procedure is particularly useful for genes that are difficult to analyze by conventional inactivation strategies due to either small size or complex genomic organization.

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

Rapid method for the identification of essential genes in Staphylococcus aureus.

A strategy based on a vector host-dependent for autonomous replication, pSA3182, was utilized both for the rapid screening for Staphylococcus aureus genes essential for cell viability and for the introduction of specific polarity-neutral deletions in nonessential genes. The results obtained support the use of pSA3182 for both purposes.

Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy.

The replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC to the pT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild-type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram-positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.

Replication enhancer requirement for recognition of heterologous replication origin by an initiator protein.

In the pT181 plasmid family, the replication initiation protein (Rep) encoded by each plasmid recognizes only its cognate origin, unless the Rep protein is expressed at abnormally high levels. Heterologous recognition of the origin of the pC221 plasmid by the RepC protein of the pT181 plasmid requires that cmp, the pT181 replication enhancer, be present on the same plasmid as the origin of replication. These findings indicate that cmp has a role in the specificity of Rep-ori recognition and support the model that cmp facilitates the formation/stabilization of the RepC-origin complex.

Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.

Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level.

Characterization of the Staphylococcus aureus chromosomal gene pcrA, identified by mutations affecting plasmid pT181 replication.

The Staphylococcus aureus chromosomal gene pcrA, identified by mutations, such as pcrA3, that affect plasmid pT181 replication, has been cloned and sequenced. The pcrA gene encodes a protein with significant similarity (40% identity) to two Escherichia coli helicases: the helicase II encoded by the uvrD gene and the Rep helicase. The pcrA3 mutation was found to be a C to T transition leading to a threonine to isoleucine substitution at amino acid residue 61 of the protein. The pcrA gene seems to belong to an operon containing at least one other gene, tentatively named pcrB, upstream from pcrA. The PcrA protein was shown to be essential for cell viability and overproduction has deleterious effects on the host and plasmid replication.

Plasmid pT181-linked suppressors of the Staphylococcus aureus pcrA3 chromosomal mutation.

Plasmid pT181 replication is affected in hosts carrying the chromosomal pcrA3 mutation, resulting in significantly lower plasmid copy numbers. Mutations suppressing this effect have been isolated and characterized. The suppressor mutations were found to map in the plasmid repC gene and manifested pcrA allele specificity, suggesting the existence of a direct RepC-PcrA interaction.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

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.