Biomedical subjects

M Espinosa

Publications and source records attributed to M Espinosa.

At least 55 records · Page 3Linked to original sources

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

Lagging-strand replication from the ssoA origin of plasmid pMV158 in Streptococcus pneumoniae: in vivo and in vitro influences of mutations in two conserved ssoA regions.

The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RS(B)] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in Streptococcus pneumoniae. Cells containing plasmids with mutations in the RS(B) accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS(B) region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.

Base 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

The maltose/maltodextrin regulon of Streptococcus pneumoniae. Differential promoter regulation by the transcriptional repressor MalR.

The Streptococcus pneumoniae MalR protein regulates the transcription of two divergent operons, malXCD and malMP, involved in maltosaccharide uptake and utilization, respectively. MalR belongs to the LacI-GalR family of transcription repressors. The protein binds specifically to two operator sequences in the intergenic region between these operons. The affinity of MalR for the malMP binding sequence is higher than for the malXCD site. Results obtained in vivo using transcriptional fusions with reporter genes indicate low repression level of malXCD by MalR when compared with malMP. This behavior may be correlated with the existence of separate induction pathways for maltose, maltotriose, and maltotetraose. The similarities found at the operator sequences and binding domains for MalR and enterococcal repressor proteins suggest that the pneumococcal maltosaccharide regulation system is closely related to several Gram-negative metabolic pathways, but not to the structurally similar Escherichia coli maltose regulon.

Amino Acid Sequence↗

PubMed1997

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.

Base Sequence↗

PubMed1997

Determination of specific DNA strand discontinuities with nucleotide resolution in exponentionally growing bacteria harboring rolling circle-replicating plasmids.

Plasmid replication by the rolling circle mechanism and conjugative transfer of plasmids require the generation of a specific strand discontinuity in the DNA. In both processes cleavage at the so-called nic site is catalyzed by plasmid-encoded proteins. The strand discontinuities at the conjugative origins of transfer of plasmid pE194 and pMV158 were determined in Bacillus subtilis and Streptococcus pneumoniae, respectively, with a recently developed runoff DNA synthesis assay. The positions of intracellular cleavage within the respective transfer origins were shown to coincide with the site predicted for pE194 and with the nic site determined in vitro for pMV158. For pMV158, the influence of a mutation in the S. pneumoniae polA gene on the efficiency of replication was investigated. In addition, the nic site within the double-stranded origin of the-rolling circle-replicating plasmid pMV158 in S. pneumoniae as well as that of pFX2 in Escherichia coli was mapped with nucleotide resolution.

Bacillus subtilis↗

PubMed1997

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution.

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

DNA, Bacterial↗

PubMed1997

Initiation of replication of plasmid pMV158: mechanisms of DNA strand-transfer reactions mediated by the initiator RepB protein.

The initiator RepB protein of the rolling circle-replicating plasmid pMV158 has nicking-closing (topoisomerase I-like) activities on supercoiled DNA. RepB is also able to perform a strand-transfer reaction on a single-stranded DNA substrate that contains its target. Several attempts at capturing covalent protein-DNA intermediates were made to identify the mechanism of RepB-mediated activity. Whereas RepB did not generate stable complexes with its target DNA, employment of single-stranded oligonucleotides containing a chiral phosphorothioate in the target DNA allowed us to follow the process of RepB-mediated strand-transfer reaction. This reaction occurred through a number of even steps because the chirality of the phosphorothioate at the reaction site was retained after RepB-mediated strand transfer. This finding suggests the existence of a covalent intermediate during the strand-transfer reaction between the protein and its target DNA. By site-directed mutagenesis at the codon for Tyr99 of RepB, and purification and assay of activity of the mutant protein variants, we showed that the Tyr99 residue is involved in the nucleophilic attack of RepB to its cognate DNA.

Amino Acid Sequence↗

PubMed1997

The mobilization protein, MobM, of the streptococcal plasmid pMV158 specifically cleaves supercoiled DNA at the plasmid oriT.

The streptococcal plasmid pMV158 replicates by the rolling circle mechanism. It encodes a relaxase protein of 494 residues, termed MobM, involved in conjugative mobilization. MobM protein was overproduced, purified, and shown specifically to relax supercoiled pMV158 DNA. The 5'-end and the 3'-end of the nick site introduced by MobM have been determined by sequencing and by primer extension analysis. The nucleophilic attack exerted by MobM is in the 5'-GpT-3' dinucleotide, within the sequence 5'-TAGTGTG/TTA-3'. Upon cleavage, MobM protein remains tightly associated with its target DNA, probably through a covalent bond. The pMV158 oriT did not exhibit homologies with known origins of transfer of plasmids from Gram-negative bacteria. However, several plasmids from Gram-positive hosts have a region identical or very similar to the pMV158 oriT. To our knowledge, this is the first demonstration of a relaxase activity of a mobilization protein from a plasmid replicating by the rolling circle mechanism.

Amino Acid Sequence↗

PubMed1997

Placement with substance-abusing mothers vs. placement with other relatives: infant outcomes.

Evaluating a subset of participants from a larger research project concerning comprehensive drug treatment services for pregnant substance-abusing women and their offspring, this investigation compared those mothers who retained custody of their infants following delivery with those who lost custody and whose infants were placed in the care of other relatives. There were no differences between the groups with respect to maternal demographics, substance abuse (as assessed by self-report prenatally and urine toxicology screens at birth), or mental health status, or with respect to newborn characteristics (including gestational age, birthweight, medical risk status). Further, caregiving behaviors of mothers and relative caregivers showed no significant differences at 6 months. Infants who remained in the care of their biological mothers, however, demonstrated better cognitive development than infants in the care of relatives at 6 months. There was also a discrepancy in terms of safety at 6 months, in that there were three deaths and two reported cases of suspected child abuse/neglect among infants in the care of their mothers, and no deaths and no reports of suspected abuse among infants in relative foster care.

Adult↗

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↗