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J Bacteriol. Jun 2006; 188(12): 4404–4412.
PMCID: PMC1482958

Replication Control of Staphylococcal Multiresistance Plasmid pSK41: an Antisense RNA Mediates Dual-Level Regulation of Rep Expression

Abstract

Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (PrnaI) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when PrnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.

In most plasmids, the first stage of replication requires binding of the replication initiation protein (Rep) to specific DNA sequences found in the origin of replication (oriV). Once this initiation event is achieved, replication will normally proceed to completion. Replication frequency (and hence plasmid copy number) is therefore determined by the availability of active Rep molecules. Effective control of copy number is crucial to the evolutionary viability of plasmids. Without such control, inevitable fluctuations from a steady-state copy number could lead to inefficient vertical inheritance on one hand or the imposition of an unsustainable burden on the host on the other. A number of diverse regulatory mechanisms have been identified for bacterial plasmid replication control systems (reviewed in reference 15). Most commonly, these regulatory systems are negative-feedback loops that often involve a constitutively expressed regulatory molecule, the abundance of which is determined by gene dosage (plasmid copy number), which negatively affects rep expression at the level of transcription or translation. Thus, when a plasmid deviates from its intrinsic copy number, the consequential alteration in the abundance of the negative regulator results in a compensatory adjustment in rep expression, leading to restoration of the steady state.

A number of plasmids utilize antisense RNA molecules to control their rates of replication (reviewed in references 10 and 37). Most of these antisense RNAs modulate plasmid copy number by negatively regulating the synthesis of the replication initiator protein. Antisense RNAs can act to inhibit initiator synthesis at the level of transcription or translation. The antisense RNAs from the gram-positive bacterial plasmids pT181, pIP501, and pAMβ1 invoke hairpin structures in the rep mRNA leader regions that lead to transcription attenuation (12, 28, 31). Alternatively, in other plasmids, antisense RNA regulation is achieved at the level of translation, and several distinct mechanisms have been elucidated in detail. In plasmid R1, antisense RNA (CopA) binding to the target prevents RepA translation by inhibiting translation of a leader peptide (Tap) to which repA is translationally coupled (8). In ColIb-P9 and plasmids of the IncB and IncL/M families, antisense RNA binding prevents formation of a pseudoknot translational activator, which is essential for rep expression (2, 4, 36, 42). In other cases, the corresponding antisense RNAs are believed to interact directly with the rep translation initiation regions, thereby blocking translation (14, 24). Intricate mechanisms of antisense RNA-mediated control have also been elucidated in systems that function in plasmid postsegregational killing, conjugal transfer, transposition, and bacteriophage lysogeny (37).

Antisense RNAs and their target mRNAs characteristically have a high proportion of secondary structure, possessing at least one stem-loop (10, 37). The initial interaction (kissing) between antisense RNA and the target mRNA often occurs through base pairing of complementary single-stranded regions exposed in the loops. In many systems, one of the interacting loop regions possesses a U-turn motif of the sequence 5′-YUNR-3′ that appears to significantly increase antisense RNA pairing kinetics (18, 21). Although over time the initial loop-loop interactions are expected to proceed to a fully annealed antisense/sense RNA duplex, in plasmids R1 and pIP501 full duplex formation is not required for repression of rep expression (11, 29, 38).

Plasmid pSK41 is representative of a family of staphylococcal multiresistance plasmids and confers resistance to aminoglycosides, antiseptics, and disinfectants. pSK41 is also conjugative, capable of promoting the horizontal exchange of genetic material, such as resistance genes, between bacterial strains (17, 32). This is of particular concern in hospital settings where strains of Staphylococcus aureus have evolved and developed resistance to the majority of antimicrobial agents used for patient treatment. Originally found in methicillin-resistant S. aureus isolates in the mid-1970s, pSK41-type plasmids remain prominent in contemporary, multiply resistant clinical strains of S. aureus and indeed have been implicated in the evolution of one vancomycin-resistant S. aureus strain (40). The minimal region required for pSK41 replication contains a single open reading frame that gives rise to the essential replication initiator protein Rep and an upstream region (~0.3 kb) that is termed the rep control region. In vitro DNA binding experiments indicated that the Rep protein bound specifically to four directly repeated sequences that are found centrally within the rep gene (27). The rnaI gene is located within the control region and is transcribed in an orientation divergent from that of rep, giving rise to a transcript complementary to the leader sequence of rep mRNA. A series of deletions into rnaI were found to increase transcription from the rep promoter two- to fourfold, indicating its involvement in rep regulation (27). In this study, we show that RNAI controls expression of the Rep protein by negatively regulating both the amount and the translational efficiency of rep mRNA. A mechanism for RNAI-mediated repression of rep translation initiation is presented, with supporting mutagenesis data.

MATERIALS AND METHODS

Bacterial strains, growth conditions, plasmids, and primers.

Escherichia coli DH5α (F endA hsdR17 supE44 thi-1 λ recA1 gyrA96 relA1 [var phi]80lacZΔM15; Bethesda Research Laboratories) was used for all cloning procedures, and functional studies were performed using Staphylococcus aureus RN4220 (restrictionless derivative of NCTC 8325-4 [26]). Bacteria were grown at 37°C in Luria-Bertani (LB) media with aeration or on LB agar plates containing 1.5% (wt/vol) agar (Oxoid). When necessary, antibiotics were added to growth media at the following concentrations: ampicillin (Ap), 100 μg/ml; tetracycline (Tc), 10 μg/ml; neomycin (Nm), 15 μg/ml; and chloramphenicol (Cm), 10 μg/ml. The relevant characteristics of plasmids used in this study are listed in Table Table1.1. All oligonucleotides used in this study (Table (Table2)2) were purchased from Geneworks (Australia).

TABLE 1.
Plasmids used in this study
TABLE 2.
Oligonucleotides used in this study

Plasmid pSK5496 was generated by digesting pSK5492 with XbaI and end filling with Klenow fragment prior to self-ligation. This generated an in-frame stop codon 3 nucleotides (nt) down from the rep start codon, ensuring that translation initiation from the rep ribosome binding site (RBS) would not influence cat expression. Thus, in pSK5496, cat expression is independent of the rep RBS, with translation initiating from the native cat RBS, and is therefore indicative of transcription only. Plasmid pSK5497 was constructed by first introducing an XbaI restriction site into codons 2 and 3 of the cat gene of pSK5492 with primers SK41-M05 and SK41-M06. The resulting plasmid was digested with XbaI, and the large fragment was gel purified and self-ligated, resulting in translational fusion between the rep start codon (Met) and codon 2 of the cat gene (Ser). Reporter gene activity in pSK5497 is indicative of the combined transcription and translation rates from the rep control region.

DNA manipulations.

DNA cloning procedures in E. coli were performed by standard methods (34). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. Plasmid DNA was isolated by alkaline lysis (6) or with a Quantum Prep plasmid miniprep kit (Bio-Rad). PCR amplifications and site-directed mutations were generated using Pfu Turbo DNA polymerase (Stratagene). DNA sequencing was performed at the Australian Genome Research Facility.

Plasmids were isolated from S. aureus by using a Quantum Prep plasmid miniprep kit (Bio-Rad). Cells were lysed by addition of 50 μg of lysostaphin to the resuspension solution and incubation at 37°C for 30 to 60 min. Plasmids were introduced into S. aureus by electroporation as previously described (35), using a Bio-Rad gene pulser (1.3 kV). Relative plasmid copy numbers were determined by quantitative image analysis using Quantity One software (Bio-Rad) and/or by the measurement of chloramphenicol acetyltransferase (CAT) enzyme activities in extracts of cells that harbored a plasmid containing a constitutively expressed cat gene (5).

CAT assays.

CAT assays were adapted to a microplate as previously described (27). Lysostaphin, acetyl coenzyme A, and 5-5′-dithio-bis[2-nitrobenzoic acid] were purchased from Sigma Aldrich and bovine serum albumin from New England Biolabs. CAT units presented are the averages of at least five independent assays and are expressed in nanomoles of chloramphenicol acetylated per mg of protein per min at 37°C.

RNA isolation and Northern blotting.

S. aureus cultures (50 ml) were grown to an optical density at 600 nm of ~0.9, and cells were pelleted by centrifugation. Cell pellets were resuspended in 1.0 ml of Trizol reagent (GibcoBRL), and cells were lysed mechanically using 100-micron glass beads (Sigma) and a Bio101 Fastprep homogenizer. The RNA isolation procedure was then performed according to the manufacturer's recommendations. An optional centrifugation step was employed to reduce protein after cell lysis and prior to the addition of chloroform. RNA samples were mixed with an equal volume of formamide loading buffer (98% [vol/vol] deionized formamide, 10 mM EDTA, 0.025% xylene cyanol FF, 0.025% bromophenol blue), incubated at 95°C for 2 min, and then loaded onto a 15% denaturing polyacrylamide gel (0.5× Tris-borate-EDTA) that had been prerun for 15 min at 25 W. Samples were electrophoresed at 25 W until the bromophenol blue dye front had reached the bottom of the gel. RNA was transferred to a Hybond N+ nylon membrane by using a semidry transfer apparatus (1 h at 300 mA; Bio-Rad) and fixed to the membrane by UV irradiation. Double-stranded probes were labeled with an AlkPhos direct labeling kit (AP Biotech), and oligonucleotide probes were labeled using a Gene Images 3′-oligolabeling module (AP Biotech). Hybridization and detection were performed with a CDP-Star detection module and Hyperfilm ECL (AP Biotech).

In vitro transcription.

RNA transcripts were synthesized by runoff transcription using T7 RNA polymerase and PCR-generated templates as described previously (11). Primers SK41-R18 and SK41-R19 were used to synthesize RNAI template DNA, and primers SK41-R24 and SK41-R21 were used to generate rep mRNA leader template DNA. Two G residues were added to the 5′ end of the RNAI transcript, and the +1 nucleotide of the Rep mRNA leader was changed to G to help maximize transcription yields (11). The in vitro transcription reaction mixture was incubated at 37°C for 4 h and then treated with 10 U of RNase-free DNase I for an additional 30 min at 37°C.

RNA-RNA binding experiments.

RNA transcripts were dephosphorylated with calf intestinal phosphatase and then 5′ end labeled with [γ-32P]ATP and T4 polynucleotide kinase. Labeled transcripts were gel purified by using a denaturing 15% polyacrylamide gel (1× Tris-borate-EDTA) and eluted by agitating gel slices in 0.4 ml of elution buffer (2 M ammonium acetate, 1% sodium dodecyl sulfate, 25 μg/ml tRNA) at 37°C for 4 h. The gel slices were removed, and RNA was precipitated by addition of 1 ml of 100% ethanol, washed with 80% ethanol, and dissolved in TMN buffer (20 mM Tris-acetate, pH 7.5, 10 mM magnesium acetate, 100 mM NaCl). Purified transcripts were renatured by being heated to 85°C for 3 min and slowly cooled to room temperature over a 1-h period. Binding reactions were performed at 37°C for 30 min in TMN buffer in the presence of a molar excess of tRNA by using a constant amount of labeled RNAI transcript and increasing concentrations of the unlabeled complementary rep mRNA leader transcript. Binding reaction products were then mixed with an equal volume of loading buffer (92% formamide, 17 mM Na2EDTA, 0.025% xylene cyanol, 0.025% bromophenol blue), applied to a semidenaturing 4% polyacrylamide gel, and electrophoresed at 300 V for 2 h.

RESULTS

RNAI negatively regulates plasmid copy number.

The transcriptional start points for pSK41 Rep mRNA and RNAI were recently determined by primer extension mapping, and their respective canonical promoter sequences were predicted (27). To confirm the role of these promoters in expression of each replication transcript, we carried out site-directed mutagenesis with the aim of deviating the promoters further from the consensus such that they were less effective for transcription. The −10 sequence of Prep was changed from 5′-TACTAT-3′ to 5′-TACTGG-3′ (AT12956GG) and the −10 sequence of PrnaI from 5′-TATAAT-3′ to 5′-TATAAG-3′ (A13056C) (base changes are indicated by underlining). The mutations were introduced into the pSK41 minireplicon pSK5487 (Cmr), yielding mutant plasmids pSK6710 and pSK6701, respectively. When these plasmids were introduced into S. aureus RN4220 by electroporation, pSK5487 and the PrnaI mutant derivative pSK6701 gave rise to high numbers of transformants (>1,000 per μg DNA) on plates containing chloramphenicol selection, whereas the Prep mutant pSK6710 failed to produce a single transformant after several electroporation attempts. The requirement for the predicted RBS of Rep was also experimentally determined by mutagenesis. The 5′-AAGGAG-3′ sequence, found 8 nt upstream from the rep start codon, was converted to 5′-AACCAG-3′ (GG13193CC), yielding plasmid pSK6713. Electroporation of RN4220 with pSK6713 consistently failed to give rise to any transformants, indicating that the GG dinucleotide sequence was also essential for replication, most likely by facilitating ribosome binding for Rep translation.

Quantitative image analysis of restricted plasmid DNA isolated from S. aureus RN4220 cells harboring plasmid pSK5487 (wild-type minireplicon) or pSK6701 (PrnaI mutant) indicated that the copy number of the latter plasmid was 20- to 40-fold higher than that of the former (Fig. (Fig.1).1). A comparable value of approximately 35-fold was determined using CAT enzyme assays to quantitate expression of the constitutively expressed cat gene carried by these plasmids in S. aureus RN4220 cell extracts (Table (Table3).3). A second mutation, this time in the −35 sequence of PrnaI, was generated for comparison (refer to Fig. Fig.44 for positions of mutations in rep mRNA). The PrnaI −35 sequence was changed from 5′-TTGTAA-3′ to 5′-CCTTAA-3′ (CAA13082AGG), giving rise to the mutant derivative pSK6746, which was found to have a copy number approximately 39-fold higher than that of the wild-type parent plasmid pSK5487 (Fig. (Fig.1;1; Table Table3).3). An equivalent plasmid carrying mutations in both PrnaI sequences, pSK6749 (A13056C and CAA13082AGG), exhibited an approximately 40-fold elevation of its copy number (Fig. (Fig.1;1; Table Table3).3). Northern blot analysis revealed that the RNAI transcript was abundant in S. aureus RN4220 cells harboring pSK5487 but was undetectable in cells carrying the PrnaI mutant plasmids pSK6701, pSK6746, and pSK6749 (Fig. (Fig.1),1), indicating that both the −35 and the −10 PrnaI sequence mutation largely abolish RNAI synthesis, concomitant with a dramatic increase in plasmid copy number.

FIG. 1.
Copy number analysis of PrnaI mutant plasmids and detection of antisense transcripts. The PrnaI sequences of plasmids are listed, with the −35 and −10 consensus sequences for canonical promoters boxed and base changes from the wild-type ...
FIG. 4.
Predicted secondary structures of pSK41 RNAI antisense molecule and Rep mRNA leader. The RNAI antisense molecule forms three stem-loop structures, designated SLRNA-I, -II, and -III, and has an overall minimum free energy (ΔG) of −14.6 ...
TABLE 3.
Summary of rep and rnaI promoter mutants

To confirm that the derepression observed was due to reduced levels of RNAI resulting from the PrnaI mutation, the ability of RNAI supplied in trans from a second plasmid to complement the defect was investigated. Since we have been unable to clone the rnaI gene and its promoter in E. coli by using a variety of cloning vectors, restriction sites, and fragment orientations, the pSK41 minireplicon pSK5413, containing the entire pSK41 rep region (16), was used as a source of trans-acting RNAI; it should be noted that we have shown previously that pSK41 Rep protein has no effect on Prep transcription (27). When RNAI was provided in trans, the high copy number of the PrnaI mutant minireplicon pSK6701 was reduced to wild-type levels (Table (Table3).3). Taken together, these results clearly implicate RNAI as a negative regulator in pSK41 copy number control.

RNAI acts on both the transcriptional and translational levels of rep expression.

We have previously shown that 3′ deletions of the rep control region, which removed PrnaI but left Prep intact, resulted in a two- to fourfold increase in transcription from Prep (27). Such a moderate level of RNAI-mediated transcriptional repression is inconsistent with the far greater effect of RNAI on plasmid copy described above, hinting that expression of rep might additionally be modulated at a posttranscriptional level. To allow the effects of RNAI regulation on rep transcription and translation to be evaluated independently, two plasmids were constructed with the pSK41 replication control region fused to the cat reporter gene (Fig. (Fig.2).2). The first construct, pSK5496, is a transcriptional fusion and hence reveals only regulation that affects the amount of rep transcription. The second construct, pSK5497, is a translational fusion that reports the amount of rep translation and hence reveals regulation imposed at the levels of transcription and/or translation. Thus, repression of rep transcription should reduce activities measured using both fusion constructs whereas inhibition of rep translation should reduce only the activity measured using pSK5497. The rnaI gene is carried by both of these constructs. S. aureus RN4220 cells harboring pSK5496 were found to express 45 ± 9 U of CAT activity (Table (Table3),3), indicating moderate levels of transcription from Prep in the wild-type situation. In comparison, CAT activity detected for the equivalent plasmid with the AT12956GG Prep mutation (pSK6711) (Table (Table3)3) was found to be minimal, at or beyond the sensitivity limits of the assay (<2 U); similarly, no CAT activity was detected for the Prep mutant translational fusion construct (pSK6712) (Table (Table3).3). Notably, the corresponding wild-type translational fusion plasmid, pSK5497, also encoded no detectable CAT activity (Table (Table3),3), demonstrating that that there is significant regulation of rep at the posttranscriptional level.

FIG. 2.
Transcriptional and translational reporter gene constructs used in this study. The cat reporter gene (hatched box) was transcriptionally fused to the rep control region to generate plasmid pSK5496 and translationally fused to the control region to generate ...

To determine the influence of RNAI on rep transcription and translation, the PrnaI A13056C mutation was introduced into pSK5496 and pSK5497, generating pSK6702 and pSK6703, respectively. Reporter gene assays revealed that the PrnaI mutation caused an approximately threefold increase in the CAT activities measured using the transcriptional fusions pSK5496 and pSK6702 (Table (Table3),3), suggesting that RNAI reduces the amount of rep transcript by approximately 64%. Assays using the translational fusions pSK5497 and pSK6703 revealed that the PrnaI mutation caused a greater-than-24-fold increase in CAT activity, which indicates that RNAI inhibits the production of Rep protein by at least 96%, of which more than 88% can be attributed to inhibition at the level of translation. The CAT assay data from the other reporter plasmids containing PrnaI mutations (pSK6747, pSK6748, pSK6750, and pSK6751) (Table (Table3)3) led to very similar RNAI repression values for both transcription and translation. Reporter assays revealed that carriage of pSK5413 coresident with pSK6702 or pSK6703 restored rep transcription and translation, respectively, to wild-type levels, consistent with the restorative effect on the copy number of the PrnaI minireplicon pSK6701 when coresident with pSK5413 (Table (Table3).3). Together, these results indicate that RNAI acts in trans and negatively regulates rep expression at the levels of both transcription and translation (Table (Table3).3). Furthermore, analysis of Prep up mutants demonstrated that increases in the amount of rep mRNA relative to RNAI concomitantly reduced the level of repression observed (pSK6752 to pSK6757) (Table (Table3),3), as expected for an antisense RNA control system. These studies also indicated that S. aureus RN4220 could not tolerate dramatic increases in rep expression (pSK6755 to pSK6763) (Table (Table3),3), possibly due to excessive metabolic burden, titration of host replication factors, and/or runaway plasmid replication.

Interaction of rep mRNA with RNAI: structural predictions.

To obtain direct evidence for the existence of an antisense RNA control system, the interaction between RNAI and rep leader RNA was investigated using molecules synthesized in vitro. As shown in Fig. Fig.3,3, the electrophoretic mobility of RNAI was retarded after incubation with rep leader RNA (nt +1 to +243), confirming an interaction between the two molecules.

FIG. 3.
Complex formation between RNAI antisense and rep mRNA leader transcripts. End-labeled RNAI was incubated with increasing concentrations (doubling; lanes 1 to 7) of unlabeled rep mRNA leader transcript for 30 min at 37°C and fractionated using ...

The secondary structures of the RNAI antisense molecule and the rep message were predicted using a free energy minimization algorithm for RNA folding (30, 44) and are shown in Fig. Fig.4.4. To gain insight into structures that might form in the rep leader following interaction with the RNAI antisense transcript, the analysis was redone with sequences complementary to RNAI deleted (hence precluding the formation of SLRep-I and SLRep-II [where SL is stem-loop]). The resulting prediction indicated that interaction with RNAI could result in the formation of a more thermodynamically stable extended SLRep-III structure (ΔG = −9.9 kcal/mol). Such a transition of SLRep-III between two alternative structures, induced by RNAI binding, might represent the basis of a molecular switch for turning off rep expression, since the extended SLRep-III configuration both resembles a rho-independent terminator and sequesters the rep RBS within its stem (Fig. (Fig.4).4). However, alteration of the poly(T) sequence found immediately downstream of SLRep-III (pSK6704, pSK6705, and pSK6706) (Table (Table1;1; Fig. Fig.4)4) showed that the structure does not function in transcription attenuation, since transcription, translation, and plasmid copy number were not affected (data not shown).

Mutations support the proposed mechanism of translational control.

Mutations were generated to destabilize the extended SLRep-III secondary structure (Fig. (Fig.4).4). The first of these mutations changed the bases complementary to the rep RBS in extended SLRep-III from 5′-CUCCUU-3′ to 5′-CUGGUU-3′ (CC13174GG) and reduced the predicted free energy of the extended stem-loop from −9.9 kcal/mol to −3.3 kcal/mol. This double point mutation increased the combined transcription/translation from <2 U (pSK5497) to 5.2 ± 2.2 U of CAT activity (pSK6766) but did not significantly alter the transcription/translation rate of the equivalent plasmid that already contained the PrnaI A13056C mutation (pSK6703), which remained at approximately 50 U of CAT activity (pSK6769; 51 ± 11 CAT U). The CC13174GG mutation had no effect on transcription alone, either in the presence or in the absence of RNAI (pSK6765 and pSK6768, respectively), and it resulted in an approximately fivefold increase to the copy number of the pSK41 minireplicon (pSK6764; 4,922 ± 997 U of CAT activity [compared to 1,012 ± 181 U for the pSK5487 wild type]). These observations suggest that the extended SLRep-III structure forms only in the presence of RNAI and not in its absence and that formation of the extended structure inhibits rep at the level of translation.

A second mutation, this time deleting part of the sequence complementary to the rep RBS in SLRep-III, was also generated. Deletion of the 4-nt sequence, 5′-CUCC-3′, found at positions +208 to +211 of the rep mRNA (Fig. (Fig.4)4) (CTCC13172Δ) was expected to have an even greater impact on formation of the extended SLRep-III structure and was found to increase the combined transcription/translation rate nearly twice as much as the double point mutation (CC13174GG), to 9.2 ± 2.3 U of CAT activity (pSK6772). In this case, however, in the absence of RNAI, the 4-nt deletion (CTCC13172Δ) resulted in a twofold increase in transcription/translation (pSK6775; 113 ± 15 CAT U), indicating that the accessibility of the rep RBS is increased by the mutation and implying that it is normally partially obscured by the unextended SLRep-III structure. The unextended SLRep-III structure could be destabilized by the deletion, since several nucleotides normally present at its base might instead be incorporated into the base of a variant SLRep-II structure.

DISCUSSION

The results outlined above indicate that pSK41 copy number control is mediated via an interaction between RNAI and the rep mRNA leader that negatively regulates both the amount of rep transcript and the efficiency with which it is translated. The rep translation control mechanism proposed here is consistent with many of the established principles of antisense RNA gene regulation. The antisense RNA (RNAI) is small and untranslated, and both the antisense and target molecules are predicted to be highly structured and to contain multiple stem-loops (Fig. (Fig.4).4). The stems of the Rep mRNA are predicted to have bulged-out residues that, in the case of plasmid R1, have been shown to promote antisense/target binding rates and prevent degradation of transcripts by RNases (22, 23, 25). The loops of SLRNAI-III and SLRep-I are complementary and GC rich, and SLRep-I contains a potential U-turn motif (5′-YUNR-3′) (Fig. (Fig.4)4) that could promote initial antisense/target base pairing (3, 18, 21). We have also previously shown by Northern blotting that the abundance of RNAI is high in comparison to the abundance of the rep message (27). A fundamental principle of antisense-mediated replication control requires that the antisense RNA be expressed constitutively and in excess of the target mRNA, so that deviations in plasmid copy number (gene dosage) lead to concomitant alteration in abundance of the inhibitory antisense RNA, thus allowing the control system to detect and react to the copy number deviation.

The translational control system of pSK41 appears to be distinct from other plasmid antisense-mediated translation control systems. In replication of R1 and related plasmids, the antisense RNA (CopA) binds to the leader region of the repA mRNA, sterically blocking translation of a leader peptide reading frame (tap) that is translationally coupled to, and essential for, repA translation (7, 8, 43). The repA RBS is contained within a stem-loop structure that prevents its translation (7), and the tap and repA reading frames marginally overlap. Translation of tap leads to disruption of the stem-loop, allowing the rep RBS to become active for translation. The ColIb-P9 (IncIα) and related IncB and IncL/M plasmids are also, to various degrees, dependent upon translation of a leader peptide that functions in an analogous manner (20, 33, 42). The antisense RNAs bind to a distal region in the rep leader; however, here the major effect of the RNA duplex is to prevent formation of a pseudoknot activator that is essential for repZ expression (2, 41). The formation of the pseudoknot activator requires a number of base pairings (7-10) between a GC-rich loop found towards the 5′ end of the rep message and a complementary sequence found in a stem-loop immediately upstream of repZ that houses its RBS (1, 4). Like these plasmids, the pSK41 antisense RNAI is complementary to a region in the rep mRNA leader at a distance from the rep RBS, but no leader peptide is located in the vicinity of the RBS of the rep gene; nor are there any sequences with the potential to form a pseudoknot activator as with the IncIα, IncB, or IncL/M plasmids. Instead, we propose a different mechanism for pSK41 whereby binding of the antisense RNA to a region of the rep mRNA upstream of rep induces formation of a thermodynamically stable stem-loop that encompasses the rep RBS, leading to strong inhibition of rep translation. In the absence of RNAI, the rep RBS is expected to be in a single-stranded state and hence be accessible for ribosomes to translate the rep coding region. The mechanism for RNAI-mediated transcriptional control of pSK41 replication remains to be elucidated. As indicated by the transcriptional reporter gene fusions, the amount of Prep-derived mRNA increased by approximately threefold when PrnaI was inactivated by mutagenesis. Antisense RNA-mediated transcriptional control has been described previously in detail for the replication of three gram-positive plasmids, pT181 (31), pIP501 (12), and pAMβ1 (28). pT181 replicates via a rolling-circle mechanism, whereas replication in the related pIP501 and pAMβ1 plasmids proceeds through a theta-mechanism. However, in all three plasmids the antisense-mediated transcription attenuation mechanisms appear very similar in both the organization of control elements and the molecular mechanisms. The key control element in each of the attenuation mechanisms is a sequence found immediately upstream of the replication initiator gene that resembles a rho-independent terminator: a stem-loop structure immediately followed by a poly(U) sequence. The terminator structure forms and has activity only when the respective antisense RNAs interact, and in the absence of the antisense RNA, read-through transcription occurs (12, 28, 31). In each system, deletion or alteration of the poly(U) sequences resulted in the inability of the antisense RNAs to induce transcription termination (12, 28, 31). In pSK41, the position and secondary structure of inverted repeat IV (IR-IV) strongly resemble those of the attenuators of pT181, pIP501, and pAMβ1; however, deletion of the entire structure did not result in an increase in transcription from Prep (27). In this study, we altered the poly(T) sequence from 5′-TTTTT-3′ to 5′-TCACT-3′ and found that it also had no significant effect on transcription or plasmid copy number. Furthermore, some of our other mutations (CC13174GG and CTCC13172Δ) that affected stem pairings in IR-IV were not found to have a significant effect on transcription and indicate that IR-IV does not function as an attenuator. This evidence does not completely rule out the possibility of an attenuation mechanism but does suggest that RNAI-mediated transcriptional control in pSK41 is unusual.

The observed negative effect of RNAI on rep transcription cannot be attributed to transcriptional interference resulting from the proximity of the convergent promoters, since a PrnaI mutation could be complemented by RNAI supplied in trans. An as yet unexplored possibility is that pSK41 RNAI binding may accelerate degradation of the rep mRNA. In a number of antisense RNA-regulated systems, the antisense/target RNA duplexes are known to be specifically processed by RNases (9, 13, 19, 39) and this and/or possible subsequent degradation could have the potential to reduce the number of translatable transcripts.

In summary, our studies show that pSK41 employs an antisense RNA, RNAI, that negatively regulates expression of the replication initiator, Rep. Wild-type cellular concentrations of RNAI reduce the number of Rep transcripts by approximately 64% and, more significantly, provide a molecular switch that greatly inhibits translation of the Rep message by more than 88%. The mechanism that we have presented here provides a new and distinct example of plasmid copy number control and further illustrates the great diversity of antisense RNA-mediated control mechanisms that exist in nature.

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia, project grant 307620.

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