Interaction of initiator proteins with the origin of replication of an IncL/M plasmid.

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.

Role of RepA and DnaA proteins in the opening of the origin of DNA replication of an IncB plasmid.

The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5'-TCTTAA-3', which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication.

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.

Pseudoknot-dependent translational coupling in repBA genes of the IncB plasmid pMU720 involves reinitiation.

Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

Molecular and immunological characterization of Mycobacterium avium 65 kDa heat shock protein (Hsp65).

The heat shock protein Hsp65 has been characterized previously in several mycobacterial species. This is the first report of the complete sequence of the coding region of the Mycobacterium avium homologue. The sequence was highly homologous to the Hsp65 of other mycobacterial species, as well as being related closely to the murine and human homologues. Recombinant Hsp65 (rHsp65) was expressed in Escherichia coli to high levels and the recombinant protein tested for its immunogenicity in a murine model of M. avium infection. Although mice infected with M. avium produced antibodies that reacted with rHsp65, they showed low proliferative T-cell responses and no cytokine production in response to the same antigen. However, immunization with rHsp65 in the adjuvant dimethydioctadecylammonium bromide (DDA), induced T cells that responded to native Hsp65 with proliferation and IFN-gamma production, indicating that the recombinant and native forms of the protein were antigenically similar. Therefore, the findings indicate that Hsp65 is not a dominant T-cell antigen during M. avium infection.

Effect of CIS on activity in trans of the replication initiator protein of an IncB plasmid.

RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in cis. The cis activity of RepA is thought to be mediated by CIS, a 166-bp region of DNA separating the coding region of repA from the origin of replication (ori) of pMU720. To investigate the trans activity of RepA, the repA gene, without its cognate ori, was cloned on a multicopy plasmid, pSU39. The ori on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned ori by RepA produced in trans from the pSU39 derivatives. The effect of CIS, when present either on the RepA-producing or the ori plasmid or both, on the efficiency of replication of the ori plasmid in vivo, was determined. The presence of CIS, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of the ori plasmid. This inhibitory activity of CIS was sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA. By contrast, CIS had no effect when present on the ori plasmid. Initiation of replication from the ori in trans was independent of transcription into CIS.

Role of CIS in replication of an IncB plasmid.

Replication of the IncB plasmid pMU720 requires the synthesis of the cis-acting RepA protein and the presence of two DNA elements, ori and CIS. CIS is the 166-bp sequence separating the RepA coding sequence from ori. To investigate how this organization of the pMU720 replicon contributes to the mechanism of initiation of replication, mutations in the sequence and/or the length of CIS were introduced into the CIS region and their effects on the efficiency of replication of the pMU720 replicon in vivo was determined. The CIS region was found to be composed of two domains. The repA-proximal domain, which showed strong transcription termination activity, could be replaced by equivalent sequences from I-complex and IncL/M plasmids, whose replicons are organized in the same fashion as pMU720. Replacement by a trpA transcription terminator afforded only partial replication activity. The repA-distal domain was shown to be a spacer whose role was to position sequence(s) within ori on the correct face of the DNA helix vis-à-vis the repA-proximal portion of CIS. A model for the loading of RepA protein onto ori is discussed.

Analysis of elements involved in pseudoknot-dependent expression and regulation of the repA gene of an IncL/M plasmid.

Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation of repB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of repA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid.

Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA. Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I [SLI]) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA. Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA. Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot. Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different. The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI. Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI. By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules. These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation.

The replication of an IncL/M plasmid is subject to antisense control.

A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnal gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.

Molecular analysis of RNAI control of repB translation in IncB plasmids.

The translation of RepA, the replication initiation protein of the IncB plasmid pMU720, requires that its mRNA (RNAII) folds to form a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. The formation of this pseudoknot is dependent in turn on the translation and correct termination of a leader peptide, RepB. A small countertranscript RNA, RNAI, controls the replication of pMU720 by interacting with RNAII to negatively regulate the expression of repA both directly, by sequestering the proximal bases required for pseudoknot formation, and indirectly, by inhibiting the translation of repB. Inhibition of the translation of repB by RNAI was found to depend on the close proximity of the RNAI-RNAII complex to the translational initiation region of repB, indicating that the primary mechanism of RNAI control involves steric hindrance. Disruption of RNAI control of repB had only a small effect on the copy number of the IncB plasmid, indicating that inhibition of the expression of repA by RNAI is achieved predominantly by inhibition of pseudoknot formation rather than by inhibition of repB translation.

Molecular analysis and characterization of a broad-host-range plasmid, pEP2.

Plasmid pEP2 was found to encode a protein, RepA, which is essential and rate limiting for its replication in Escherichia coli and Corynebacterium pseudotuberculosis. Mutations which altered the rate of synthesis of this protein in E. coli affected the copy number and segregational stability of pEP2 in the two hosts. RepA contains 483 amino acid residues and has the calculated molecular weight of 53,925. It shows 45% amino acid residue identity with open reading frame ORF2 of pSR1, a plasmid isolated from Corynebacterium glutamicum (J. A. C. Archer and A. J. Sinskey, J. Gen. Microbiol. 139:1753-1759, 1993). Plasmid pEP2 was shown to accumulate single-stranded DNA corresponding to the RepA coding strand during its replication in E. coli and C. pseudotuberculosis, suggesting that it may replicate by a rolling circle mechanism. However, RepA has no significant sequence homology with the replication initiator proteins of plasmids known to use this mode of replication.

Mechanism of binding of the antisense and target RNAs involved in the regulation of IncB plasmid replication.

The replication frequency of the IncB miniplasmid pMU720 is dependent upon the expression of the repA gene. Binding of a small, highly structured, antisense RNA (RNA I) to its complementary target in the RepA mRNA (RNA II) inhibits repA expression and thus regulates replication. Analyses of binding of RNA I to RNA II indicated that the reaction consists of three major steps. The first step, initial kissing complex formation, involves base pairing between complementary sequences in the hairpin loops of RNA I and RNA II. The second step is facilitated by interior loop structures in the upper stems of RNA I and RNA II and involves intrastand melting and interstrand pairing of the upper stem regions to form an extended kissing complex. This complex was shown to be sufficient for inhibition of repA expression. The third step involves stabilization of the extended kissing complex by pairing between complementary single-stranded tail regions of RNA I and RNA II. Thus, the final product of RNA I-RNA II binding is not a full duplex between the two molecules.

Mutations affecting pseudoknot control of the replication of B group plasmids.

The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure. Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major effects on the translation of repA.

Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication.

Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop. Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II). Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II. From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed.

Mutations affecting translational coupling between the rep genes of an IncB miniplasmid.

The nature of translational coupling between repB and repA, the overlapping rep genes of the IncB plasmid pMU720, was examined. Mutations in the start codon of the promoter proximal gene, repB, reduced the efficiency of translation of both rep genes. Moreover, there was no independent initiation of repA translation in the absence of repB translation. The position of the repB stop codon was crucial for the efficient expression of repA, with the wild-type positioning being optimal. Translational coupling was found to be totally dependent on the formation of a pseudoknot structure. A model which invokes formation of a pseudoknot to facilitate initiation of repA is proposed.

Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids.

Minireplicons from the I-complex plasmids R387 (IncK) and pIE545 (IncZ) were constructed, and the nucleotide sequences of their replication regions were compared with that of the B plasmid, pMU720. The coding sequence of the putative replication protein, RepA, of each plasmid was located. RepA of K and B plasmids were homologous, whereas RepA of Z resembled RepA1 of FII plasmid. Sequences upstream of RepA were conserved in the three I-complex plasmids. Group B and Z plasmids were incompatible.

Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.

Role of countertranscript RNA in the copy number control system of an IncB miniplasmid.

Transcriptional mapping studies of the IncB minireplicon pMU720 demonstrated the existence of a long RNA molecule, RNA II, whose 5' portion is complementary to the product of the incompatibility gene RNA I. By using gene fusion and transcriptional fusion plasmids, it was shown that RNA I regulated the expression of the RNA II gene product and that it did so primarily at the level of translation. The target of RNA I was mapped to lie within a 216-base region of RNA II containing the sequence complementary to RNA I. Introduction of the target for RNA I in trans increased the copy number of an IncB minireplicon, indicating that RNA I and RNA II form the basis of the copy number control system of IncB plasmids.

Analysis of the incompatibility determinants of I-complex plasmids.

The isolation and characterization of minireplicons corresponding to group B and I-complex plasmids are reported. The molecular structures of the small RNAs that may play a major role in the replication control and incompatibility reactions of the plasmids are compared. A mutant plasmid with changed copy number and incompatibility specificity is described. This mutant had a single-base-pair substitution in the DNA region that codes for the small RNA.