Manuel Espinosa

Conjugative Plasmid Transfer in Gram-Positive Bacteria

SUMMARY Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.

Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1

The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.

Rolling circle-replicating plasmids from Gram-positive and Gram-negative bacteria: a wall falls

Rolling circle‐replicating plasmids constitute a group of small, promiscuous multicopy replicons spread among eubacteria. Until recently, rolling circle replication seemed to be limited to small plasmids from Gram‐positive hosts and to single‐stranded bacteriophages from Gram‐negative bacteria. However, characterization of two small plasmids from Gram‐negative hosts has shown that this replication mechanism is general among eubacteria. This review focuses on a family of highly related promiscuous plasmids that replicate by the rolling circle mechanism, and that have been isolated from various Gram‐positive bacteria and from the Gram‐negative bacterium Helicobacter. They all share homologies at the leading‐strand origins and at the initiator of replication proteins. The plasmids of this family have directly repeated sequences at their plus origin of replication, which is located 5′ from the start point of the mRNA for the initiation of replication protein. Replication is controlled by an antisense RNA and by a transcriptional repressor protein. The features and regulatory circuits of replication of this plasmid family seem to be unique among rolling circle‐replicating plasmids. Members of this family replicate autonomously in Gram‐positive and‐negative hosts.

The structure of plasmid‐encoded transcriptional repressor CopG unliganded and bound to its operator

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon–helix–helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13‐bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19‐bp oligonucleotide, containing the pseudosymmetric element, with two β‐ribbons inserted into the major groove. The DNA is bent 60° by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix–turn–helix regions of unrelated DNA‐binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon–helix–helix and helix–turn–helix proteins.

Plasmid copy number control: an ever‐growing story

Bacterial plasmids maintain their number of copies by negative regulatory systems that adjust the rate of replication per plasmid copy in response to fluctuations in the copy number. Three general classes of regulatory mechanisms have been studied in depth, namely those that involve directly repeated sequences (iterons), those that use only antisense RNAs and those that use a mechanism involving an antisense RNA in combination with a protein. The first class of control mechanism will not be discussed here. Within the second class (the most ‘classical’ one), exciting insights have been obtained on the molecular basis of the inhibition mechanism that prevents the formation of a long‐range RNA structure (pseudoknot), which is an example of an elegant solution reached by some replicons to control their copy number. Among the third class, it is possible to distinguish between (i) cases in which proteins play an auxiliary role; and (ii) cases in which transcriptional repressor proteins play a real regulatory role. This latter type of regulation is relatively new and seems to be widespread among plasmids from Gram‐positive bacteria, at least for the rolling circle‐replicating plasmids of the pMV158 family and the theta‐replicating plasmids of the Inc18 streptococcal family.

The copy number of plasmid pLS1 is regulated by two trans‐acting plasmid products: the antisense RNA II and the repressor protein, RepA

The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and PII From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible replicon did not result in incompatibility towards pLS1. From PII, the 50‐nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers.

Construction of the mobilizable plasmid pMV158GFP, a derivative of pMV158 that carries the gene encoding the green fluorescent protein.

Plasmid pMV158 has been employed to construct cloning non-mobilizable vectors for various Gram-positive organisms. Here we report the construction of a mobilizable pMV158-based plasmid that harbors the gene encoding the green fluorescent protein under the control of a promoter inducible by maltose. The plasmid was mobilized between strains of Streptococcus pneumoniae as well as from S. pneumoniae to Lactococcus lactis or Enterococcus faecalis at the same frequency as its parental. Transconjugant that received the GFP-tagged plasmid could be detected by their fluorescence, which was especially high in E. faecalis cells.

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single‐stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double‐stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell‐free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I‐deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1‐ssoA required the entire DNA polymerase I (polymerase and 5′–3′ exonuclease activities). ssDNA containing the pLS1‐ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase‐directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.

Replication of the promiscuous plasmid pLS1: a region encompassing the minus origin of replication is associated with stable plasmid inheritance.

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RSR.

Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNA II

Two elements, the products of genes copG and rnall, are involved in the copy‐number control of plasmid pLS1. RNA II is synthesized in a dosage‐dependent manner. Mutations in both components have been characterized. To determine the regulatory role of the two genes, we have cloned copG, rnall or both elements at various gene dosages into pLS1‐compatible plasmids. Assays of incompatibility towards wild‐type or mutant pLS1 plasmids showed that: (i) the rnall gene product, rather than the DNA sequence encoding it, is responsible for the incompatibility, and (ii) CopG and RNA II act in trans and are able to correct up fluctuations in pLS1 copy number. A correlation between the gene dosage at which the regulatory elements were supplied and the incompatibility effect on the resident plasmid was observed. The entire copG‐rnall circuit has a synergistic effect when compared with any of its components in the correction of pLS1 copy‐number fluctuations, indicating that, in the homoplasmid steady‐state situation, the control of pLS1 replication is exerted by the co‐ordinate action of CopG and RNA II.