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S J Projan

Publications and source records attributed to S J Projan.

At least 55 records · Page 3Linked to original sources
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

Mobilization of the relaxable Staphylococcus aureus plasmid pC221 by the conjugative plasmid pGO1 involves three pC221 loci.

The Staphylococcus aureus plasmid pC221, a 4.6-kilobase multicopy chloramphenicol resistance plasmid that forms plasmid-protein relaxation complexes, was mobilized for transfer by the conjugative plasmid pGO1. Two open reading frames on the pC221 genome, now designated mobA and mobB, as well as a cis-acting locus, the putative oriT, were shown to be in involved in pC221 mobilization. The mobA (but not mobB) and oriT loci were required for pC221 relaxation, and relaxation was necessary but not sufficient for pC221 mobilization by pGO1. oriT was cloned onto a pE194 derivative and complemented in trans for both relaxation and mobilization. Mobilization of relaxable plasmids in S. aureus appears to be analogous to mobilization by donation observed in gram-negative bacteria.

Conjugation, Genetic↗
PubMed1988

Nucleotide sequence of pS194, a streptomycin-resistance plasmid from Staphylococcus aureus.

pS194 is a naturally occurring Staphylococcus aureus plasmid encoding streptomycin resistance. The plasmid has a copy number of about 25 per cell, and belongs to the inc5 incompatibility group. The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames potentially encoding proteins of greater than 100 amino acids. All four of these reading frames are on the same coding strand. The first reading frame, repE, encodes a 38 kd protein specifically required for pS194 replication. The second open reading frame, str, encodes a 34 kd polypeptide required for streptomycin resistance, probably a streptomycin adenylyltransferase. The third potential polypeptide, rlx, would be 37 kd and is probably required for relaxation complex formation and plasmid mobilization by conjugative plasmids. The fourth, orfD, overlapping the rlx reading frame, is potentially 27 kd, and may also be involved in mobilization.

Amino Acid Sequence↗
PubMed1988

Comparative analysis of five related Staphylococcal plasmids.

The genomic organization of five small multicopy staphylococcal plasmids comprising the pT181 family has been analyzed. In addition to pT181, the family presently includes the streptomycin resistance plasmid pS194 and the chloramphenicol resistance plasmids pC221, pC223, and pUB112. Although they belong to five different incompatibility groups, the five plasmids have similar basic replicons, use the same basic copy control mechanism, and have a common structural organization. It has been demonstrated previously that pT181 and pC221 encode trans-active replication proteins (RepC and RepD, respectively) which specifically recognize the respective plasmid's origin of replication in both cases is initiated by site-specific nicking and 3' extension. The other three plasmids in this family encode similar replication proteins; 63% of the predicted amino acid residues are identical for all five and the least similar pair shows 75% identity at the amino acid level. However, despite this homology, the replication proteins and origins of replication of different members in this family did not show cross complementation in vivo. Outside of the basic replicon, which comprises about one-third of each plasmid's genome, functional organization is also conserved. The resistance determinants are all located in the same position, immediately downstream of the replication protein coding sequence, and all are transcribed in the same direction. The three chloramphenicol resistance determinants encode highly homologous chloramphenicol transacetylases which are unrelated to the tet and str gene products. Three of the five plasmids form relaxation complexes and the involved genome segments are closely related. The other two are not homologous to these three in the corresponding region, but are homologous to each other and encode a site-specific recombinase, Pre. It is suggested that the replication, resistance, and relaxation complex regions of these plasmids can be regarded as conserved segments ("cassettes") assembled in various combinations, but always with the same spatial arrangement.

Amino Acid Sequence↗
PubMed1988

A rapid method to quantitate non-labeled RNA species in bacterial cells.

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.

Bacillus subtilis↗
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

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.

Amino Acid Sequence↗
PubMed1987

Translational fusion with a secretory enzyme as an indicator.

A novel type of translational fusion system has been developed by using a secretory protein, staphylococcal beta-lactamase, as an indicator. The beta-lactamase structural gene was modified to provide N-terminal extensions of 13 and 162 amino acids, and in both cases, the fusion protein was processed and the mature active enzyme was secreted; thus, the expression of a particular upstream gene can be analyzed by monitoring the beta-lactamase activity.

Binding Sites↗
PubMed1986

The DNA cleavage mechanism of iron-bleomycin. Kinetic resolution of strand scission from base propenal release.

The action of iron-bleomycin and O2 in cleaving DNA has been resolved into two kinetic events following the initial attack on DNA by the kinetically competent drug species, "activated bleomycin." At 4 degrees C, DNA strand scission, monitored both viscometrically and fluorimetrically (t1/2 = 2.5-5 min), precedes the release from DNA of nucleic base propenals, which is half complete in about 40 min. Therefore, a moderately stable intermediate consisting of cleaved DNA bearing a base propenal precursor is formed. The release of tritium from deoxyribose carbon-2 occurs at the time of DNA scission, which is consistent with the base propenal precursor retaining the deoxyribose-3'-phosphate bond. Specific mechanistic proposals are discussed.

Bleomycin↗
PubMed1986

Incompatibility between plasmids with independent copy control.

Incompatibility between autonomous plasmids has been attributed, for the most part, to interaction between plasmids' negative control systems and/or partitioning systems. In this report it is shown that indirectly regulated plasmids with non-interactive negative control systems are incompatible on the basis of their shared initiator protein. This principle was demonstrated for a family of Staphylococcus aureus plasmids whose copy number is regulated by inhibitory RNAs that control the production of a rate-limiting, trans-active, initiator protein. We have constructed a pair of plasmids that have the same regulation systems and different initiator proteins and another pair with different regulation systems and the same initiators. Both of these pairs of plasmids were shown to be incompatible.

Base Sequence↗
PubMed1986

A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.

In the process of randomly mutagenizing a recombinant pBR322 clone with transposon Tn5, a high copy number plasmid mutant, pLO88, has been isolated. The copy number phenotype of pLO88 is observed only at elevated temperatures, greater than or equal to 37 degrees C, and is due to the precise position of a Tn5 insertion. Nucleotide sequence of the Tn5-pBR322 junction reveals that Tn5-88 has inserted into an open reading frame that codes for a 63 amino acid protein previously shown to negatively regulate pBR322 plasmid copy number. By deleting portions of the Tn5 it is shown that the copy number phenotype is due not only to the insertion of Tn5 in pBR322 but also to the requirement that some Tn5 sequences remain intact. It appears that an outwardly directed Tn5 promoter initiates the synthesis of a transcript (RNA X) that interferes with the normal repressor RNA (RNA I)-primer RNA (RNA II) interaction at elevated temperatures.

DNA Replication↗
PubMed1985

Replication control for pT181, an indirectly regulated plasmid.

PT181 is a fully sequenced Staphylococcus aureus plasmid whose size is 4,437 bp. It specifies tetracycline resistance and has a copy number of about 22 per cell in exponentially growing cultures. The functional organization of the pT181 replicon is centered around the coding sequence for a 35-kd protein, RepC, that is absolutely required for replication of the plasmid. The replication origin is contained within the repC coding sequence and the region immediately 5' to the RepC start is involved in control of the plasmid replication rate. PT181 replication is controlled at the level of RepC synthesis by a negative regulatory system that is functionally similar to that of the Co1E1 and IncFII plasmids of Escherichia coli. The pT181 control circuit involves 2 short transcripts, RNA I and RNA II, that are transcribed from the region specifying the 5' end of the untranslated repC mRNA leader and in the opposite direction. These are referred to as countertranscripts. The countertranscripts regulate RepC synthesis by a mechanism that probably involves interaction with the repC mRNA leader in a manner that interferes with translation. Both of the countertranscripts seem to be necessary for normal replication control; their separate roles remain unclear. Unlike plasmids of the Co1E1 and IncFII groups, plasmids such as Co1E1 are considered to have direct regulation of replication because the inhibitory element of the copy control circuit directly inhibits the initiation of replication. Plasmids such as pT181 are considered to have indirect regulation of replication because the product of the regulated step, RepC, is trans-active. Plasmids of the IncFII type are considered to have direct regulation of replication because the product of the regulated step, RepA is cis-active The analysis of pT181 replication physiology has illustrated 2 important differences between directly and indirectly regulated plasmids: a) for directly regulated plasmids, copy mutants specifying a normal inhibitor substance but an inactive target site exclude the wild-type or recessive mutants by directly interfering with their replication. Analogous mutants of indirectly regulated plasmids coexist readily with the wild-type and all mutants (although they do manifest segregational incompatibility) because the Rep protein is always shared by all plasmids in the cell, regardless of its source. b) Mutations of directly regulated plasmids in the region where target transcript and countertranscript overlap may give rise to totally new incompatibility groups because they engender independently self-correcting copy pools.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1985

Comparative sequence and functional analysis of pT181 and pC221, cognate plasmid replicons from Staphylococcus aureus.

The nucleotide sequence of pC221, a 4.6 kb Staphylococcus aureus plasmid is presented. The replication region of the plasmid is identified and compared with the corresponding region of pT181, a compatible but related plasmid. Both plasmids encode trans-active replicon-specific initiator proteins, RepC for pT181 and RepD for pC221. Plasmid replication rate is controlled by regulation of the rate of synthesis of the initiator protein by means of inhibitory 5' countertranscripts. Key elements of the control system are closely conserved between the two plasmids whereas less critical elements show extensive divergence. Overall architecture is also conserved, suggesting functional parallelism. The replication origin for both plasmids is contained within the N-terminal region of the initiator protein coding sequence; the two coding sequences are highly homologous but have two important areas of divergence, one within the origin region, the other near the C-terminus. In vivo recombinants between the two plasmids isolated previously (Iordanescu 1979) have crossover points within the initiator gene, between the two divergent regions. The recombinant plasmids have hybrid initiator proteins and are defective for replication, requiring the simultaneous presence of the parental plasmid from which their origin is derived. They are able to complement replication-defective mutants of the other parental plasmid, suggesting that the recognition specificity of the hybrid initiator protein resides in its C-terminal end and that the specific recognition site for the protein corresponds to the divergent region within the origin.

Amino Acid Sequence↗
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↗