Biomedical subjects

G del Solar

Publications and source records attributed to G del Solar.

At least 19 recordsLinked to original sources

PubMed2000

A functional lagging strand origin does not stabilize plasmid pMV158 inheritance in Escherichia coli.

Plasmid rolling circle replication generates single-stranded DNA intermediates. The intracellular amount of these molecules depends upon the efficiency of the conversion of single-stranded into double-stranded plasmid forms, that is, the functionality of the lagging strand origin (sso). The broad-host-range streptococcal plasmid pMV158 harbors two different ssos, both of which function efficiently in Streptococcus pneumoniae but poorly in Escherichia coli. Plasmid pMV158 is stably inherited in the pneumococcal host, but it is unstable in E. coli. A pMV158 derivative lacking its two ssos is unstable in both strains. We have cloned into this derivative the coliphage f1 lagging strand origin. Whereas the f1 sso was fully functional in E. coli, it did not show any activity in S. pneumoniae, a bacteria closely related to the pMV158 natural host. The presence of the f1 sso did not stabilize pMV158 inheritance in either the gram-positive or the gram-negative host.

Cloning, Molecular↗

PubMed2000

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.

DNA Replication↗

PubMed1998

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 beta-ribbons inserted into the major groove. The DNA is bent 60 degrees 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.

Amino Acid Sequence↗

PubMed1998

Structural features of the plasmid pMV158-encoded transcriptional repressor CopG, a protein sharing similarities with both helix-turn-helix and beta-sheet DNA binding proteins.

The small transcriptional repressor CopG protein (45 amino acids) encoded by the streptococcal plasmid pMV158 was purified to near homogeneity. Gel filtration chromatography and analytical ultracentrifugation showed that the native protein is a spherical dimer of identical subunits. Circular dichroism measurements of CopG indicated a consensus average content of more than 50% alpha-helix and 10-35% beta-strand and turns, which is compatible with the predicted secondary structure of the protein. CopG exhibited a prolonged intracellular half-life, but deletions in regions other than the C-terminal affected the global structure of the protein, severely reducing the half-lives of the CopG variants. This indicates that CopG has a compact structure, perhaps constituted by a single domain. Molecular modeling of CopG showed a good fitting between the helix-turn-helix motifs of well-known repressor proteins and a bihelical unit of CopG. However, modeling of CopG with ribbon-helix-helix class of DNA binding proteins also exhibited an excellent fit. Eleven out of the 12 replicons belonging to the pMV158 plasmid family could also encode Cop proteins, which share features with both helix-turn-helix and beta-sheet DNA binding proteins.

Amino Acid Sequence↗

PubMed1998

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the pMV158-encoded plasmid transcriptional repressor protein CopG.

Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control. A new procedure for overproduction and purification of the protein has been developed. The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter. Purified CopG protein has been crystallized using the sitting-drop vapor diffusion method. The crystals, belonging to orthorhombic space group C222(1) (cell constants a = 67.2 A, b = 102.5 A, c = 40.2 A), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7. Complete diffraction data up to 1.6 A resolution have been collected. Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 A3/Da).

Amino Acid Sequence↗

PubMed1998

Identification of a new gene in the streptococcal plasmid pLS1: the rnaI gene.

The streptococcal plasmid pMV158 has been reported to harbor five genes: three involved in initiation of rolling circle replication and its control (copG, repB, and maII), one involved in conjugative mobilization (mobM), and the fifth one specifying constitutive resistance to tetracycline (tet). The mobM gene was removed in the construction of the pMV158-derivative plasmid pLS1, which was used in this study. By in vitro transcription assays, primer extension experiments, and construction of mutations, here we demonstrate the presence of another gene (the sixth of pMV158), termed maI, which is transcribed in opposite orientation with respect to the plasmid mRNAs, to render RNA I. The 5'-end of RNA I has an 8-nt sequence which is complementary to a region of the lagging-strand origin (ssoA) comprising a 6-nt consensus sequence involved in lagging strand synthesis. This suggested that RNA I could influence, positively or negatively, initiation of lagging strand synthesis from the pLS1-ssoA. However, plasmids defective in RNA I synthesis exhibited a phenotype similar to the wild type in terms of efficiency of replication from the ssoA and copy number. When the maI gene was cloned into a compatible plasmid, the resulting recombinants did not exhibit incompatibility toward plasmids with the pLS1 replicon. Thus, RNA I does not seem to be a true copy number control element. We postulate that transcription from the maI promoter may facilitate extrusion of the hairpin of the plasmid double-strand origin, which is the target of the initiator of replication protein.

Base Sequence↗

PubMed1998

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2.

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place.

Amino Acid Sequence↗

PubMed1998

Replication and control of circular bacterial plasmids.

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3'-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can "sense" and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.

Base Sequence↗

PubMed1997

Replication control of plasmid pLS1: the antisense RNA II and the compact rnaII region are involved in translational regulation of the initiator RepB synthesis.

Replication of the streptococcal plasmid pLS1 is controlled by two plasmid-encoded gene products: the repressor protein CopG and the antisense RNA, RNA II. Two different mutants in rnaII have been isolated. The 5'-end and the levels of RNA II synthesized by pneumococcal cells harbouring the wild-type pLS1 or mutant plasmids (affected in either genes copG or rnaII) were analysed. One of the rnaII mutants exhibited a high-copy-number phenotype, whereas an in vitro-constructed mutation, which affects the -10 region of the rnaII promoter, resulted in plasmids lacking copy-number phenotype. The latter mutation had a pleiotropic effect: It abolished RNA II synthesis, but it also affected the initiation of translation signals of the gene encoding the RepB initiator protein. Transcriptional and translational fusions, together with in vitro inhibition of RepB synthesis by specific oligonucleotides, showed translational inhibition of RepB synthesis by RNA II, perhaps by directly blocking the accessibility of the ribosomes to the repB initiation of translation signals.

Bacterial Proteins↗

PubMed1996

Isolation and characterization of pLS1 plasmid mutants with increased copy numbers.

Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.

Amino Acid Sequence↗

PubMed1996

Broad-host-range plasmid replication: an open question.

Many factors can influence the ability of plasmids to colonize different hosts, efficient replication probably being the most critical one. Two major strategies seem to facilitate promiscuous plasmid replication: (i) initiation independent of host initiation factors; and (ii) versatile communication between plasmid and host initiation factors. Appropriate communication between a replicon and the different hosts, which becomes crucial at the initation of plasmid replication, plays a major role in plasmid promiscuity. Fused replicons or mechanisms that rescue collapsed replication forks may increase the efficiency of plasmid propagation. However, their contribution to plasmid promiscuous replication remains to be fully evaluated. Several examples of host-specific adaptation of promiscuous plasmids point to an enormous flexibility of these replicons.

DNA Replication↗

PubMed1995

Plasmid rolling circle replication and its control.

This review summarises current information on rolling circle replicating plasmids originally isolated from Gram-positive bacteria with a low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of these small, high copy number plasmids that replicate via an asymmetric RC mechanism. The regulation of plasmid copy number is also discussed.

Bacterial Proteins↗

PubMed1995

Specific nicking-closing activity of the initiator of replication protein RepB of plasmid pMV158 on supercoiled or single-stranded DNA.

Asymmetric rolling circle replication of the promiscuous replicon pMV158 is initiated by the plasmid-encoded RepB protein. In vitro, purified RepB protein introduces a nick within the leading strand origin of replication by a nucleophylic attack on the phosphodiester bond at the dinucleotide GpA. Some changes within and around this dinucleotide were recognized by the protein. RepB nicked and closed supercoiled pMV158 DNA, having an optimum activity at 60 degrees C. We have imitated, in vitro, a process of rolling circle replication, since RepB was able to nick (initiation) and to covalently close (termination) single-stranded oligonucleotides containing the protein cleavage sequence. Covalent DNA-protein complexes were not found, indicating that RepB has unique features among plasmid-encoded proteins involved in rolling-circle replication or conjugative mobilization.

Bacterial Proteins↗

PubMed1995

Lagging-strand origins of the promiscuous plasmid pMV158: physical and functional characterization.

The streptococcal plasmid pMV158 replicates by a rolling circle mechanism, which involves the generation of single-stranded plasmid DNA intermediates. This plasmid has the unique feature of having two lagging-strand origins of replication. One of these origins, termed ssoU, is functional in Streptococcus pneumoniae and in Bacillus subtilis in an orientation-dependent manner. The other origin, ssoA, is only functional in the former host. RNA polymerase seems to be involved in the initiation of the conversion of single- to double-stranded plasmid DNA from both ssoA and ssoU. Mutational and deletion analyses have allowed us to define ssoA as being within a highly structured, non-coding 199 bp region. Within this region, two elements which are conserved in several rolling-circle replicating plasmids are located, the recombination site RSB and a 6 base consensus sequence. Both elements may play a role in the conversion of single- to double-stranded plasmid DNA.

Bacillus subtilis↗

PubMed1995

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 rnaII, 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, rnaII 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 rnaII 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-rnaII 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.

Base Sequence↗

PubMed1995

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein.

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.

Amino Acid Sequence↗

PubMed1994

Chemical synthesis of a fully active transcriptional repressor protein.

Plasmid pLS1-encoded 45-amino acid transcriptional repressor CopG (formerly RepA) has been chemically synthesized. A one-step purification of the synthetic protein has been developed, which yields high levels of pure protein with low or no contamination of truncated products. We have compared some properties of the chemical CopG protein with those of the biologically purified CopG. The two proteins were indistinguishable in (i) their ability to generate specific protein-DNA complexes, (ii) their capacity to protect a restriction site included within the CopG DNA target, and (iii) in their in vitro capacity to specifically repress synthesis of copG mRNA.

Amino Acid Sequence↗

PubMed1993

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 RSB.

Bacillus subtilis↗