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J C Alonso

Publications and source records attributed to J C Alonso.

At least 127 records · Page 7Linked to original sources
PubMed1988

Replication and incompatibility properties of plasmid pUB110 in Bacillus subtilis.

Within plasmid pUB110 we have identified a 1.2 kb segment necessary and sufficient for driving autonomous replication in Rec+ cells at a wild-type copy number. This region can be divided into three functionally discrete segments: a 24 base pair (bp) region that acts as an origin, a 949 bp determinant of an essential replication protein, repU, and a 358 bp incompatibility region, incA, overlapping with the repU gene. The synthesis of the IncA determinant/s proceeds in the direction opposite to that of RepU. The positively (RepU) and negatively (IncA) trans-acting products seem to be involved in the control of plasmid replication. The RepU product has an Mr of 39 kDa, could be overproduced in Escherichia coli, and binds to the pUB110 origin region. Outside the minimal replicon a cis-acting, orientation dependent, 516 bp determinant is required (i) to compete with a coexisting incompatible plasmid and (ii) for segregational stability.

Bacillus subtilis↗
PubMed1988

Construction of a shuttle vector for inducible gene expression in Escherichia coli and Bacillus subtilis.

The construction of a shuttle vector for inducible gene expression allowing fast and easy cloning in Escherichia coli and subsequent transformation of Bacillus subtilis is presented. The expression is based on the regulation of the tac promoter by the Lac repressor which was assayed with the xylE gene from Pseudomonas putida as a marker gene. The lacIq gene, transcribed by the strong spo promoter, allowed full repression of the weak tac promoter.

Bacillus subtilis↗
PubMed1988

Characterization of recombination-deficient mutants of Bacillus subtilis.

An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis.

Bacillus subtilis↗
PubMed1987

Generation of linear multigenome-length plasmid molecules in Bacillus subtilis.

Linear multigenome-length double and single stranded plasmid DNA was identified in a Bacillus subtilis ATP-dependent DNAase mutant strain (addA5) bearing plasmids pC194 or pBD95ts. Plasmid pBC30, a seg mutant of pC194, as well as some pUB110 derivatives with rearrangements external to the minimal replicon, produce high amounts of such a concatemeric DNA, even in Rec+ cells. The synthesis of this type of plasmid DNA occurs in the absence of an active plasmid-encoded Rep protein and is markedly affected in polA5 and recE4 genetic backgrounds. To account for these observations, we propose that the AddAB complex serves to prevent a sigma-type replication of plasmid DNA.

Bacillus subtilis↗
PubMed1987

Initiation of plasmid pC194 replication and its control in Bacillus subtilis.

By deletion analysis we have defined a 1.1 kb segment required for driving autonomous replication of the plasmid pC194. The minimal replicon specifies a positive, RepH, and a negative, Inc8A, trans-acting product and their target sites. The RepH product has a Mr of 34.1 kDa, could be overproduced, and binds specifically to the pC194 origin region. By trans complementation studies we have shown that pC194 replication is indirectly controlled at the level of RepH synthesis by a negative product, IncA, that is transcribed within the repH transcription unit in the opposite direction ("antisense RNA"). The antisense RNA regulates the RepH synthesis by a mechanism that seems to involve RNA/RNA interaction in a manner that interferes with translation. In addition, an autoregulatory control might be operative.

Bacillus subtilis↗
PubMed1987

Plasmid maintenance in Bacillus subtilis recombination-deficient mutants.

An isogenic set of 11 recombination-deficient mutant strains of Bacillus subtilis has been constructed. Whereas plasmid pUB110 is stably maintained in such Rec- cells, the high copy number plasmid pC194 is unstable. Instability in Rec- strains could be mostly attributed to the deleterious effect of the presence of the plasmid on the Rec- cells' growth capability. In part, instability of pC194 derivatives could also be correlated with the presence of an unusually high amount of multimeric DNA molecules.

Bacillus subtilis↗
PubMed1987

A new mutator strain of Bacillus subtilis.

Bacillus subtilis strain SB1207, widely used in our laboratory, was found to be highly temperature-sensitive and to exhibit a strong SOS-independent mutator phenotype at elevated temperatures. Both chromosomal and plasmid-borne genes were affected by the mutator. Lethality and mutator phenotype could not be attributed to a replication shut off or to thymine starvation. Due to the high frequency of base misincorporation, the mutator phenotype probably results from an editing defect rather than from a post-replication defect (mismatch repair).

Bacillus subtilis↗
PubMed1986

Requirements for the formation of plasmid-transducing particles of Bacillus subtilis bacteriophage SPP1.

We had previously proposed that the production of concatemeric plasmid DNA in plasmid-transducing SPP1 particles is a consequence of phage-directed rolling-circle-type replication of plasmid DNA. The production of such DNA was greatly enhanced when DNA/DNA homology was provided between phage and plasmid DNAs (facilitation of transduction). Here we present evidence that synthesis of concatemeric plasmid DNA can proceed after phage infection under conditions non-permissive for plasmid replication. We also propose that the naturally occurring homology between plasmid and phage is sufficient to account for the frequency of transduction observed in the absence of facilitating homology. Homology of greater than 47 bp gives the maximal facilitation of plasmid transduction. Recombination is not an essential part in the synthesis of concatemeric plasmid DNA.

Bacillus subtilis↗
PubMed1986

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids.

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.

Acetyltransferases↗
PubMed1985

A gene controlling segregation of the Bacillus subtilis plasmid pC194.

Plasmid pC194-1, a mutant of pC194, and chimeric derivatives of pC194-1 are segregationally unstable in B. subtilis. Such instability could be enhanced by exposure of pC194-1-carrying cells to methyl methanesulfonate. pC194-1 is distinct from pC194 in the addition of two A:T base pairs within the previously defined D region of pC194. Complementation experiments between pC194-1 and other plasmids suggest that the mutation of pC194-1 interferes with the production of a diffusible gene product required for plasmid maintenance.

Bacillus subtilis↗
PubMed1985

Generation of deletions through a cis-acting mutation in plasmid pC194.

When plasmid pC194-1 is ligated to pBR322 to generate plasmid pHV15-1, deletions occur with high frequency within the joined pBR322 DNA. Generation of deletions is recE4 independent, and occurs in B. subtilis with a 1,000-fold higher frequency than in Escherichia coli. In the hybrid plasmid pVH15-1, deletion end-points are not at random, but at defined locations within pBR322. We propose that the base alteration, characterizing pC194-1, has stabilized within the plasmid a stem/loop structure, which acts as a deletion generator.

Bacillus subtilis↗
PubMed1985

Cold sensitivity in the transfer of a plasmid with a deletion hot spot into recombination deficient B. subtilis cells.

Various recombination-deficient mutants of B. subtilis, which are readily transformable by plasmid DNA at 42 degrees C cannot be transformed at 30 degrees C with chimeric plasmid derivatives that contain the deletion hot spot defined previously (Alonso and Trautner 1985a, b). Such interference was also observed in protoplast transformation and SPP1 transduction.

Bacillus subtilis↗
PubMed1985

Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage.

Any SPP1 DNA restriction fragment cloned into Bacillus subtilis plasmid pC194 or pUB110 increased the transduction frequency of the plasmid by SPP1 100- to 1,000-fold over the transduction level of the plasmid alone. This increment was observed irrespective of whether a fragment contained the SPP1 packaging origin (pac). Furthermore, an SPP1 derivative into whose genome pC194 DNA had been integrated transduced pC194 DNA with a greatly enhanced frequency. Transduction enhancement mediated by DNA-DNA homology between plasmid and SPP1 was independent of the extent of homology (size range analyzed, 0.5 to 3.9 kilobases) and the recombination proficiency of donor or recipient.

Bacillus subtilis↗
PubMed1981

DNA gyrase inhibitors block development of Bacillus subtilis bacteriophage SP01.

SP01 development was inhibited by nalidixic acid and novobiocin in the sensitive host Bacillus subtilis 168M. Inhibition by novobiocin was prevented by a Novr mutation in the cellular DNA gyrase gene. Nalidixic acid inhibition persisted in hosts carrying a Nalr gyrase, but could be overcome by phage mutation. We conclude that SP01 requires for its development subunit B of the host DNA gyrase, but replaces or modifies subunit A.

Bacillus subtilis↗
PubMed1980

Novobiocin blocks the shutoff of SPO1 early transcription.

Novobiocin, an inhibitor of bacterial DNA gyrase strongly impairs the development of bacteriophage SPOl. DNA replication seems to be the primary target for the antibiotic in this system, but viral-coded transcription is also affected in several aspects: (a) The level of phage transcription is diminished; (b) the shutoff of the synthesis of early RNA classes is inhibited; (c) RNAs of late class are not synthesized. This last effect is a consequence of the coupling between transcription and replication. The other two results could be taken as an indication that the appropriate secondary structure of the parental phage DNA is a requisite for the recognition of promoters. The introduction of negative turns by DNA gyrase seems to make early genes unavailable for transcription.

Bacillus subtilis↗
PubMedDate not supplied

Characterization of recF suppressors in Bacillus subtilis.

A recF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNA-binding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response.

Bacillus subtilis↗