Double or triple sets of replication functions as inverted and direct repeats on in vitro reconstructed streptococcal MLS resistance plasmids

In vitro rearrangement of plasmid pDB102 together with comparative studies of other streptococcal plasmids allowed the localization of replication and copy control functions on sequences which were present on pDB102 and its naturally occurring ancestor pSM19035 as duplicates in inverted orientation. Evidence is presented that neither the presence of duplicate replication regions nor their arrangement in inverted orientation was essential for plasmid survival. Among the in vitro reconstructed plasmids were several that stably carried two or three sets of replication and copy control functions either as inverted or direct repeats or both. A copy control mutation is described which led to a tenfold increase of copy number over that of the naturally occurring plasmid pSM19035.     

Functioning of the TA cassette of streptococcal plasmid pSM19035 in various Gram-positive bacteria

Toxin-antitoxin (TA) systems are common in microorganisms and are frequently found in the chromosomes and low-copy number plasmids of bacterial pathogens. One such system is carried by the low copy number plasmid pSM19035 of the pathogenic bacterium Streptococcus pyogenes. This plasmid encodes an omega-epsilon-zeta cassette that ensures its stable maintenance by post-segregational killing of plasmid-free cells. In this study, the activity of the ω-ε-ζ cassette was examined in various Gram-positive bacteria with a low G/C content in their DNA. The broad host range of pSM19035 was confirmed and the copy number of a truncated derivative in transformed strains was determined by real-time qPCR.     

The toxin-antitoxin system of the streptococcal plasmid pSM19035

pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the omega-epsilon-zeta operon of this plasmid constitutes a novel proteic plasmid addiction system in which the epsilon and zeta genes encode an antitoxin and toxin, respectively, while omega plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of zeta gene expression in both bacterial species are counteracted by proper expression of epsilon. The epsilon-zeta toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.     

Involvement of the ftsA gene product in late stages of the Escherichia coli cell cycle.

The erythromycin resistance determinant of plasmid pDB102, a derivative of plasmid pSM19035, was cloned into the single HindIII site of the 3.6-megadalton cryptic Streptococcus mutans plasmid pVA318 and introduced into Streptococcus sanguis strain Challis by transformation. Plasmid pDB201, which was isolated from one of the transformants, consisted of the vector plasmid and the 1.15-megadalton HindIII fragment D of pSM19035. HindIII fragment D contained within it one of the two unique "spacer" sequences of pSM19035. Electron micrographs of self-annealed molecules of the recombinant plasmid revealed classical stem-loop structures, and the resistance determinant of pSM19035 appeared as a transposon-like structure. No differences were observed in either the type or the level of erythromycin resistance by pSM19035 or pDB201. The availability of a cloned erythromycin resistance determinant should be useful for future comparative studies of macrolide, lincosamide, and streptogramin B resistance plasmids in streptococci.     

Role of the N-terminal region and of beta-sheet residue Thr29 on the activity of the omega2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035

The dimeric regulatory protein wild-type omega (wt omega2) binds to arrays of 7-bp sequences (heptads) present in the operator DNA region of copy control and partition functions of plasmid pSM19035. Each omega2 protein probably binds with an antiparallel beta-sheet structure in the major groove of the 7-bp subsite of the operator DNA. Exchange of threonine at position 29 to alanine (T29A) drastically affects the activity of variant protein omega2T29A both in vivo and in vitro, and reduces the thermodynamic stability deltaG(o)u, but does not change the conformation. Likewise, the binding affinity to DNA is reduced and the association of the two monomeric subunits of the omega2T29A dimer is weakened, as manifested by an increase in the dissociation constant from 3.2 microM for wt omega2 to 6.3 microM for omega2T29A. Denatured dimers are formed upon thermal unfolding of wt omega2 and omega2T29A at ca. 45 microM (D(n)<-->D(u)). Removal of 8 (omega2deltaN8), or even 18 (omega2deltaN18) N-terminal amino acids has no obvious effect either on the core structure or on the activity in comparison to wt omega2. The stability of variants omega2deltaN8 and omega2deltaN18 is similar to that of wt omega2, and their binding to operator DNA is not impaired.     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Site-specific recombination by the beta protein from the streptococcal plasmid pSM19035: minimal recombination sequences and crossing over site.

The beta recombinase from the broad host range Grampositive plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented recombination sites in the presence of a chromatin-associated protein (Hbsu). The recombination site had been localized to a 447 bp DNA segment from pSM19035. This segment includes a 90 bp region that contains two adjacent binding sites (I and II) for beta protein dimers. Using in vitro recombination assays, we show that this 90 bp region is necessary and sufficient for beta protein-mediated recombination; this defines the six site as the region required for beta protein binding. The point of crossing over has been localized to the center of site I. Hbsu has a strong binding affinity for an unknown site located within the 447 bp segment containing the six site. We discuss the possibility that Hbsu recognizes an altered DNA structure, rather than a specific sequence, generated in the synaptic complex.     

Cloning and expression in Bacillus subtilis of the gene for neutral protease of Bacillus brevis

Plasmids pCB20 and pCB22 were used for cloning and expression of the Bac brevis 7882 neutral protease gene in Bac. subtilis cells. The protease-containing fragments of 13 and 14 kb were cloned in pCB20 plasmid based on replication region of Streptococci plasmid pSM19035. Expression of the gene was shown to take place in Bac. subtilis. Application of vegetative promoters of the previously identified expression unit EU19035 greatly increases the expression of the protease in Bac. subtilis. Bac. subtilis cells, expressing the gene of Bac. brevis neutral protease, do not sporulate, are considerably larger than the cells which do not contain the gene and form multicellular structures.     

Mapping of RNA polymerase binding sites in R12 derived plasmids carrying the replication-incompatibility region and the insertion element IS1.

Interactions between Escherichia coli RNA polymerase holoenzyme and three small plasmid DNAs (pSM1, pSM2, and pSM15) derived from the drug resistant factor R12 have been studied. These plasmids carry the copy number and incompatibility determinants, the origin of DNA replication and the rep gene(s) necessary for plasmid replication. They also contain the insertion element IS1 and the putative finO cistron. Thirteen DNA segments within the largest of the three plasmids (pSM2) were able to form either a binary and/or ternary complex with RNA polymerase. A unique strong binding site was mapped within the left end of IS1. Five binding sites were found within the rep-cop-inc region. Four of these are weak binding sites whereas the fifth does not form a stable binary complex and was detected by ternary complex formation. A strong binding site was located in the putative finO region whereas the remaining six binding sites are located in regions with unidentified genetic functions.     

Expression unit in the region of replication initiation in the streptococcal plasmid pSM19035

Several sequences, resembling vegetative promoters and ribosome-binding sites of Bacilli were found in the primary structure of the replication region of Streptococci plasmid pSM19035. Promoterless alpha-amylase gene of Bac. amyloliquefaciens and lambda cI857 gene, supplied with BamHI site upstream of the initiator ATG-codon, were used for functional characterization of the structures. As a result, Bac. subtilis synthesized alpha-amylase up to 0.5 g/l, and lambda-repressor up to 3% of the intracellular water-soluble protein. The repressor, synthesized in Bac. subtilis, regulates lambda PR promoter in the cells. Plasmid pCB22 is constructed for the convenience of usage of the found expression unit, called EU19035. The plasmid has BamHI and BgIII sites on different distances from the ribosome-binding site.     

Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.     

Characterization of Bacillus subtilis clones surviving overproduction of Zeta, a pSM19035 plasmid-encoded toxin

The postsegregational killing system of pSM19035 plasmid consists of the proteins Zeta and Epsilon, a toxin and an antidote, respectively. Zeta mutants were isolated with the use of Bacillus subtilis strain with the zeta gene under control of an inducible promoter integrated into the chromosome. Results of mutant analysis point to the amino terminal part of the Zeta protein as being responsible for the toxicity.     

Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12.

The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.     

Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids of Escherichia coli

Plasmid Rsc13, a small derivative of the plasmid R1, contains a region necessary for replication as well as a complete copy (4957 bp) of the ampicillin resistance transposon, Tn3. We determined the nucleotide sequence of the replication region of Rsc13 to be 2937 bp and then compared this region (designated the 2.9-kb region) to the analogous region of pSM1, a small derivative of the plasmid R100 which has common ancestry with R1. Rsc13 and pSM1 were 96% homologous in this 2.9-kb region except for a discrete region of about 250 bp which showed only 44% homology. The sequence and distribution of nucleotide substitutions between Rsc13 and pSM1 supported a map of possible genes and sites which have previously been seen in the replication region of Rsc13 and pSM1 which showed only 44% homology. Analysis of the amino acid sequence and predicted conformation of the two RepA2 polypeptides, however, suggested that they were very similar. We proposed that the repA2 region of R1 and R100 was replaced by a substitution of a short DNA segment from another plasmid which was evolutionarily related to R1 and R100 but had more divergence. This event may have been mediated by a mechanism similar to that of gene conversion as described in eukaryotic systems.