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J C Alonso

Publications and source records attributed to J C Alonso.

At least 91 records · Page 5Linked to original sources
PubMed1994

Genetic analysis of the DnaA-dependent priming in the initiation of lagging strand DNA synthesis of plasmid pUB110 in Bacillus subtilis.

Derivatives of Bacillus subtilis plasmid pUB110 lacking the major lagging strand replication origin (ssoU-) accumulate intracellular single-strand circular (SS(c)) DNA intermediates and are unable to propagate in dnaB and dnaD hosts. DnaA-dependent priming requires a DnaA box in a stable hairpin form; a higher copy number of a DnaA box is not sufficient as a signal for the conversion of the SS(c) into its dsDNA form. The introduction into the plasmid of a hairpin structure, whose stem carries a DnaA box, mediates conversion of SS(c) into dsDNA and makes plasmid replication independent of the B. subtilis dnaB function. This conversion signal has been termed ssoA.

Bacillus subtilis↗
PubMed1994

The beta recombinase from the Streptococcal plasmid pSM 19035 represses its own transcription by holding the RNA polymerase at the promoter region.

The beta protein encoded by the Streptococcus pyogenes plasmid pSM19035 is a site-specific recombinase involved in both resolution of plasmid multimers into monomers and DNA inversion. It has been proposed that the DNA region to which the beta recombinase binds to mediate recombination includes a promoter from which orf alpha and the beta gene are transcribed. We have determined the sites at which transcription of the orf alpha and the beta gene initiates in vitro and we have demonstrated that highly purified beta recombinase acts as a repressor of its own synthesis. The promoters are located within the beta recombinase binding site, which we have defined previously. The binding of the beta recombinase to its target site does not seem to exclude RNA polymerase from the promoter, despite the overlapping of their binding sites. Therefore, it is likely that the beta recombinase does not repress transcription by a mere steric hindrance on RNA polymerase binding.

Base Sequence↗
PubMed1994

A novel site-specific recombinase encoded by the Streptococcus pyogenes plasmid pSM19035.

Genetic evidence suggests that the beta protein encoded by the Streptococcus pyogenes plasmid pSM19035 is involved both in the resolution of plasmid multimers into monomers and in DNA inversion. In this report we show that the highly purified beta protein is unable to mediate DNA recombination unless a host factor(s) is provided. In the presence of the host factor(s), the beta protein is able to catalyze in vitra intramolecular recombination between two specific sites on supercoiled templates: DNA resolution was obtained when the two recombination sites were directly oriented, whereas DNA inversion was the product if the recombination sites were in inverse orientation. In the absence of the host factor(s) the beta protein forms a specific complex with its target site. The beta protein binding site has been localized by DNase I footprinting to an 85 bp region that can be divided into two discrete sites (I and II). These sites are about 34 bp in length, they are separated by about 16 bp, and contain two 12 to 13 bp imperfectly conserved sequences (half-sites) with dyad axis symmetry. The protein binds co-operatively to sites I and II; between 3.6 and 4.2 beta protein protomers are required to saturate the DNA substrate. These data, together with gel retardation assays, suggest that the protein binds to DNA as two dimers, one to each discrete site, and that the dimers probably interact with each other. The beta protein binding site, though it resembles that of other DNA resolvases of the Tn3/gamma delta (Tn1000) family, differs in that only two adjacent sites are found (sites I and II), while those DNA resolvases normally bind to three adjacent sites. The results presented here suggest that a host factor(s) could work as an accessory effector to compensate for the absence of the third binding site.

Bacterial Proteins↗
PubMed1994

Analysis of cis and trans acting elements required for the initiation of DNA replication in the Bacillus subtilis bacteriophage SPP1.

The development of SPP1 has been studied in several B. subtilis mutants conditionally defective in initiation of DNA replication. Initiation of SPP1 replication is independent of the host DnaA (replisome organizer), DnaB, DnaC and DnaI products, but requires the DnaG (DNA primase) and the DNA gyrase. Furthermore, SPP1 replication is independent of the DnaK (heat shock) protein. The phage-encoded products required for initiation of SPP1 replication have been genetically characterized. Analysis of the nucleotide sequence (3.292 kilobases) of the region where SPP1 initiation replication mutants map, revealed five open reading frames (orf). We have assigned genes 38, 39 and 40 to three of these orfs, which have the successive order gene 38-gene 39-orf39,1-gene 40-orf41. The direction of transcription of the reading frames, the lengths of the mRNAs as well as the transcription start point, upstream of gene 38 (PE2), were identified. Proteins of 29.9, 14.6 and 46.6 kDa were anticipated from translation of gene 38, gene 39 and gene 40, respectively. The purified G38P and G39P have estimated molecular masses of 31 and 15 kDa. G38P and G39P do not share significant identity with primary protein sequences currently available in protein databases, whereas G40P shares substantial homology with a family of DNA primase-associated DNA helicases. G38P binds specifically to two discrete SPP1 DNA restriction fragments (EcoRI-4 and EcoRI-3). The G38P binding site on EcoRI-4 was localized on a 393 bp DNA segment, which lies within the coding sequence of gene 38. The putative binding site on EcoRI-3 was inferred by DNA sequence homology, it maps in a non-coding segment. G39P, which does not bind to DNA, is able to form a complex with G38P. The organization of the SPP1 genes in the gene 38 to gene 40 interval resembles that one found in the replication origin regions of different Escherichia coli double-stranded DNA phages (lambda, phi 80 and P22). We propose that the conserved gene organization is representative of the replication origin region of a primordial phage.

Amino Acid Sequence↗
PubMed1993

Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis.

The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis. The complete nucleotide (nt) sequence of pBT233 has been determined. Analysis of the nt sequence revealed nine major open reading frames (orfs). The repS, erm1 and erm2 genes have been assigned to three of these orfs, and given the gene order, repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1. The organization of genes of the repS-orf gamma region resembles the organization of genes in the repE-orfI region of pAM beta 1. Messenger RNA species of molecular weights corresponding to repS, orf alpha + orf beta, orf gamma, orf delta and orf epsilon + orf zeta were detected by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs beta, gamma, delta, epsilon and zeta, respectively. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system. The orf beta and orf gamma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs, delta, epsilon and zeta, encode proteins with unknown activity. Deletion of a 1.5-kb segment (nt 2999-4552) with coding capacity for orf beta, orf gamma and orf delta does not seem to affect plasmid maintenance. Removal of a 3.0-kb fragment (nt 4598-7689) with coding capacity for orf epsilon and orf zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Purification of the beta product encoded by the Streptococcus pyogenes plasmid pSM19035. A putative DNA recombinase required to resolve plasmid oligomers.

Genetic evidence suggests that the gene beta product of Streptococcus pyogenes plasmid pSM19035 is required for converting plasmid multimers into monomers. The beta protein was purified from cells overexpressing the cloned gene. N-terminal protein sequence analysis demonstrated that the purified protein had the predicted sequence, except that the N-terminal initiator methionine was not present. Native beta protein consists of a dimer of two identical subunits with a molecular mass of 23.8 kDa (25 kDa in SDS-PAGE). The beta protein (isoelectric point of 9.7) binds specifically to a DNA fragment (312 bp in length) which contains the promoter region of the orf alpha-gene beta operon and two regions (sites I and II) that show dyad axes of symmetry. It is proposed that protein beta binds to sites I and II to mediate resolution of plasmid oligomers.

Bacterial Proteins↗
PubMed1993

Sequence analysis of the left end of the Bacillus subtilis bacteriophage SPP1 genome.

The left end of the genome of Bacillus subtilis bacteriophage SPP1 is represented by EcoRI DNA fragments 12 and 1 (EcoRI-12 and EcoRI-1). A number of different deletions were identified in EcoRI-1. A detailed physical and genetic map of EcoRI-1 from wild-type (wt) phage and SPP1 deletion mutants was constructed. Genes encoding essential products involved in late and early stages of phage DNA metabolism were mapped at the left and right ends of the 8.5-kb EcoRI-1, respectively. Deletions fell within the internal 5157-bp DNA segment of EcoRI-1. The nucleotide (nt) sequence of this region and of the endpoints of two deletions, delta X and delta L, were determined. The nt sequence of the junctions in SPP1 delta X and SPP1 delta L showed that, in these deletions, a segment of DNA between short directly repeated sequences of 10 and 13 bp, located 3427 and 4562 bp apart in the wt sequence, had been eliminated. In both cases, the copy of the repeated sequence was retained in the deletion mutant, consistent with the hypothesis that the deletions originated by homologous intramolecular recombination. The corresponding region in wt phage had fifteen presumptive open reading frames (orfs) and the previously identified SPP1 early promoters (PE1). The poor growth phenotype associated with the SPP1 deletion mutants was attributed to premature transcriptional read through from promoter(s) of the early region into late operon brought into close vicinity of the late genes due to the deletion event.

Amino Acid Sequence↗
PubMed1993

Functional analysis of pSM19035-derived replicons in Bacillus subtilis.

We determined the effect of various Bacillus subtilis dna(Ts) mutations on pSM19035 replication. At non-permissive temperature, plasmid replication depends on the dnaB, dnaC, dnaG and dnaI initiation replication products, but does not require the dnaA function. DNA elongation is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in dna(Ts) mutants impaired in different subunits of the enzyme. Single-stranded plasmid replication intermediates, corresponding to the leading strand, accumulate in the dnaD strain. On the basis of electron microscopic analysis of replication intermediates, we confirmed that the plasmid replicates unidirectionally by a theta mechanism.

Bacillus subtilis↗
PubMed1993

Purification and properties of the RecR protein from Bacillus subtilis 168.

Genetic evidence suggests that the Bacillus subtilis recR gene product is involved in DNA repair and recombination. To assign a biochemical function to the recR gene product, the RecR protein was labeled and purified by monitoring the radioactive label. NH2-terminal protein sequence analysis of RecR was consistent with the deduced amino acid sequence of the recR gene. The RecR protein (molecular mass of 25 kDa, isoelectric point 5.4) bound single- and double-stranded DNA in a filter binding assay. RecR-DNA complex formation is enhanced by the presence of a damage in the DNA substrate. The RecR-DNA complex formation proceeds in the absence of divalent cations and nucleotide cofactors, but is markedly stimulated by ATP and divalent cations. In our experimental conditions the apparent equilibrium constants of the optimized RecR-DNA complexes are 3 x 10(-7) M and 9 x 10(-7) M for damaged and undamaged DNA, respectively. The binding reaction is cooperative. Electron microscopy studies show that the presence of divalent cations increases the rate of RecR protein self-assembly. Addition of ATP or dATP promotes the organization of discrete series of quaternary structures on DNA, but ATP gamma S inhibits the DNA binding activity. A possible mechanism for the RecR function in DNA repair is discussed.

Bacillus subtilis↗
PubMed1993

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis.

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (orf) [alpha and beta]. Inactivation or deletion of orf beta results in SegA- plasmids. Better than random segregation requires an active segB region. The segB region contains two orfs (orf epsilon and orf zeta). Inactivation of either of the orfs does not lead to an increase in cell death, but orf zeta- plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.

Bacillus subtilis↗
PubMed1993

Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination.

A recN- (recN1) strain of Bacillus subtilis was constructed. The effects of this and recF, recH and addAB mutations on recombination proficiency were tested. Mutations in the recN, recF, recH and addAB genes, when present in an otherwise Rec+ B. subtilis strain, did not affect genetic exchange. Strains carrying different combinations of mutations in these genes were constructed and examined for their sensitivity to 4-nitroquinoline-1-oxide (4NQO) and recombination proficiency. The recH mutation did not affect the 4NQO sensitivity of recN and recF cells and it only marginally affected that of addA addB cells. However, it reduced genetic recombination in these cells 10(2)- to 10(4)-fold. The addA addB mutations increased the 4NQO sensitivity of recF and recN cells, but completely blocked genetic recombination of recF cells and marginally affected recombination in recN cells. The recN mutation did not affect the recombinational capacity of recF cells. These data indicate that the recN gene product is required for DNA repair and recombination and that the recF, recH and addAB genes provide overlapping activities that compensate for the effects of single mutants proficiency. We proposed that the recF, recH, recB and addA gene products define four different epistatic groups.

Bacillus subtilis↗
PubMed1992

Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing genes 1 to 6. The products of gene 1 and gene 2 are required for pac cleavage.

Packaging of Bacillus subtilis phage SPP1 DNA into viral capsids is initiated at a specific DNA site termed pac. Using an in vivo assay for pac cleavage, we show that initiation of DNA synthesis and DNA packaging are uncoupled. When the DNA products of pac cleavage were analyzed, we could detect the pac end that was destined to be packaged, but we failed to detect the other end of the cleavage reaction. SPP1 conditional lethal mutants, which map adjacent to pac, were analyzed with our assay. This revealed that the products of gene 1 and gene 2 are essential for pac cleavage. SPP1 mutants that are affected in the genes necessary for viral capsid formation (gene 41) or involved in headful cleavage (gene 6) remain proficient in pac site cleavage. Analysis of the nucleotide sequence (2.769 x 10(3) base-pairs) of the region of the genes required for pac cleavage revealed five presumptive genes. We have assigned gene 1 and gene 2 to two of these open reading frames (orf), giving the gene order gene 1-gene 2-orf 3-orf 4-orf 5. The direction of transcription of the gene 1 to orf 5 operon and the length of the mRNAs was determined. We have identified, upstream from gene 1, the major transcriptional start point (P1). Transcription originating from P1 requires a phage-encoded factor for activity. The organization of gene 1 and gene 2 of SPP1 resembles the organization of genes in the pac/cos region of different Escherichia coli double-stranded DNA phages. We propose that the conserved gene organization is representative of the packaging machinery of a primordial packaging system.

Amino Acid Sequence↗
PubMed1992

Intramolecular homologous recombination in Bacillus subtilis 168.

Plasmid resolution from a phage::plasmid chimera was used to measure directly intramolecular recombination in Bacillus subtilis. The system is based on a sigma-replicating plasmid (pC194) cloned into a dispensable region of the lytic bacteriophage SPP1. The plasmid, which confers chloramphenicol resistance, is resolved when SPP1::pC194 phages infect B. subtilis cells, provided the chimera carries a functional, intact copy of the plasmid repH gene. Intramolecular homologous recombination was independent of the RecA and RecL-RecR functions, but dependent on RecF, RecB, RecG, RecP, RecH and AddAB functions. These results are consistent with the hypothesis that B. subtilis has multiple pathways for genetic recombination and allow us to tentatively place the recB and recG genes into a new epistatic group epsilon.

Bacillus subtilis↗
PubMed1991

Parameters affecting plasmid stability in Bacillus subtilis.

For the analysis of parameters affecting plasmid stability in Bacillus subtilis, we used a pUB 110-derived shuttle plasmid containing direct and inverted nucleotide repeats (DRs and IRs). Deletions of up to 6 kb were found to occur between DRs of 7 to 16 bp. IRs as small as 43 or 58 bp were shown to stimulate the formation of these deletions in their neighbourhood. However, these structural features (DRs and IRs) per se were not responsible for plasmid instability. The unstable recombinant plasmids, but not their deletion-carrying (delta) derivatives, were found to impair the growth of the host and to accumulate high amounts of linear plasmid multimers [high mol. wt. (hmw) DNA]. We propose that the accumulation of hmw DNA may be the major reason for the selective pressure against recombinant plasmids, and the enrichment of delta-plasmids. Host mutations and other parameters increasing the stability of recombinant plasmids in B. subtilis are described.

Bacillus subtilis↗
PubMed1991

Physical and biochemical characterization of recombination-dependent synthesis of linear plasmid multimers in Bacillus subtilis.

The synthesis and structure of linear multimeric plasmid molecules (hmw DNA) in Bacillus subtilis were investigated. The replication of covalently-closed-circular supercoiled (form I) DNA requires the rate-limiting plasmid-encoded replication initiation protein. Unlike form I, hmw DNA synthesis is partially resistant to inhibition of cellular transcription or translation and requires the host DnaB protein. In addition, hmw DNA synthesis involves host recombination and repair functions (RecE and Poll). Analysis of hmw DNA by electron microscopy displayed linear DNA molecules up to 100 kb in size, which were either single-stranded, double-stranded or double-stranded with single-stranded ends. Structural features of hmw DNA molecules were mapped by means of heteroduplex studies using defined strand-specific probes. The results suggest that a recombination intermediate, but not plasmid-encoded replication, is involved in the initiation of hmw DNA synthesis.

Bacillus subtilis↗
PubMed1991

Molecular analysis of the Bacillus subtilis recF function.

recF resides between the dnaN and gyrB genes of Bacillus subtilis. The recF15 mutation results in replacement of a glutamate residue in the wild type with a lysine residue in the mutant RecF protein. We investigated the in vivo regulation of recF using a transcriptional fusion to the xylE gene and assaying mRNA production. We found that novobiocin leads to a four-fold induction in recF gene expression, but this is not observed in a gyrB mutant strain. Enhancement of expression of the recF gene in the presence of novobiocin is unrelated to the SOS response. The RecF protein, which has a predicted molecular mass of 42.2 kDa, does not seem to be involved in its own regulation.

Bacillus subtilis↗
PubMed1991

Characterization of Bacillus subtilis recombinational pathways.

Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.

Alleles↗