Biomedical subjects

S Bron

Publications and source records attributed to S Bron.

At least 91 records · Page 5Linked to original sources

PubMed1989

Similarity of minus origins of replication and flanking open reading frames of plasmids pUB110, pTB913 and pMV158.

Plasmids pMV158 and pTB913, originating from Streptococcus agalactiae and a thermophilic Bacillus respectively, were sequenced to completion. Both contained a BA3-type minus origin of replication and an RSA-site, believed to constitute a site-specific recombination site. These two regions were more than 99% homologous to the corresponding regions of the Staphylococcus aureus plasmid pUB110. Deleting the BA3-type minus origin resulted in the accumulation of a considerable amount of single-stranded DNA, both in L. lactis subsp. lactis and B. subtilis, indicating that this minus origin was functional in both bacterial species. Like pUB110, both plasmids contained an open reading frame encoding a putative plasmid recombination enzyme (Pre protein), which was located downstream of the RSA-site. On the basis of sequence comparisons between pUB110, pMV158, pTB913, pT181, pE194, pNE131 and pT48 two distinct families of RSA-sites and Pre proteins could be distinguished.

Amino Acid Sequence↗

PubMed1989

Breakage--reunion and copy choice mechanisms of recombination between short homologous sequences.

To study recombination between short homologous sequences in Escherichia coli we constructed plasmids composed of the pBR322 replicon, M13 replication origin and a recombination unit inserted within and inactivating a gene encoding chloramphenicol resistance. The unit was composed of short direct repeats (9, 18 or 27 bp) which flanked inverted repeats (0, 8 or 308 bp) and a gene encoding kanamycin resistance. Recombination between direct repeats restored a functional chloramphenicol resistance gene, and could be detected by a simple phenotype test. The plasmids replicated in a double-stranded form, using the pBR322 replicon, and generated single-stranded DNA when the M13 replication origin was activated. The frequency of chloramphenicol-resistant cells was low (10(-8)-10(-4] when no single-stranded DNA was synthesized but increased greatly (to 100%) after induction of single-stranded DNA synthesis. Recombination between 9 bp direct repeats entailed no transfer of DNA from parental to recombinant plasmids, whereas recombination between 18 or 27 bp repeats entailed massive transfer. The presence or length of inverted repeats did not alter the pattern of DNA transfer. From these results we propose that direct repeats of 9 bp recombine by a copy choice process, while those greater than or equal to 18 bp can recombine by a breakage-reunion process. Genome rearrangements detected in many organisms often occur by recombination between sequences less than 18 bp, which suggests that they may result from copy choice recombination.

Base Sequence↗

PubMed1989

Plasmid deletion formation in recE4 and addB72 mutants of Bacillus subtilis.

Plasmid deletion formation was compared in wild-type, recE4, and addB72 strains of Bacillus subtilis. Deletion frequencies in plasmid pGP1, as monitored with a penP-lacZ fusion, were low in recE4 and high in addB72, in comparison to the wild-type strain. In the wild-type and recE4 strains, deletions between directly repeated sequences were rare. In contrast, about half of the deletions in the addB72 mutant resulted from recombination at direct repeats of 5 bp or more. The sequences at or near the left deletion endpoints showed striking similarities in the three strains. (1) 5'-T-T-T-3', or the complement 5'-A-A-A-3', was frequently located at these sites. (2) 5'-T-G-T-A-3' was found close to most of these termini. (3) Nearly all left termini occurred in a region rich in hyphenated dyad symmetry, which includes the penP transcription/translation regulatory sequences. It is assumed that DNA secondary structures, together with a sequence preference, specify the majority of the left deletion termini, which we speculate to be target sites for topoisomerase I. The right termini of deletions in the wild-type and addB72 mutant were frequently located close to a loose octanucleotide consensus sequence: 5'-G/C-G/C-G/C-G-A/T-A/T-A/T-A/G-3'. In contrast, in the recE4 mutant, the sequence 5'-C-A-G/C-G/C-G/C-G/C-T/G-3' was more frequently found at this position.

Bacillus subtilis↗

PubMed1988

Characterization of signal-sequence-coding regions selected from the Bacillus subtilis chromosome.

Signal-sequence-coding regions for protein export were selected from chromosomal Bacillus subtilis DNA. The number of different signals obtained was higher than expected on the basis of known exported proteins in B. subtilis. Most of the selected regions showed the characteristics of typical signal sequences, including a basic N-terminal region followed by a hydrophobic core and a potential signal-peptidase cleavage site. The signal-coding regions were functionally interchangeable between the B. licheniformis alpha-amylase and Escherichia coli TEM beta-lactamase genes. In addition to the signal-sequence-coding regions, the nature of the host cells, and the mature parts of the reporter proteins contributed to the amounts of protein secreted.

Amino Acid Sequence↗

PubMed1988

Structural plasmid instability in Bacillus subtilis: effect of direct and inverted repeats.

Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.

Anti-Bacterial Agents↗

PubMed1988

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation.

The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.

Bacillus subtilis↗

PubMed1988

Synthesis and processing of Escherichia coli TEM-beta-lactamase and Bacillus licheniformis alpha-amylase in E. coli: the role of signal peptidase I.

A mutant of Escherichia coli, in which signal peptidase I synthesis can be regulated, was constructed. The mutant was used to study the effects of signal peptidase I limitation on the synthesis and efficiency of processing of two proteins: the periplasmic E. coli TEM-beta-lactamase and Bacillus licheniformis alpha-amylase, which also accumulates in the periplasm of E. coli. Signal peptidase I limitation resulted in reduced rates of processing of pre-beta-lactamase and in strong inhibition of synthesis of alpha-amylase. The data suggest that beta-lactamase is processed post-translationally and that an intimate relationship exists between the synthesis and processing of alpha-amylase.

Bacillus↗

PubMed1988

Plasmid deletion formation in Bacillus subtilis.

Plasmid pGP1 carrying a penP-lacZ fusion was used to study structural plasmid instability in Bacillus subtilis. In only one of 28 sequenced deletion junction points in the penP-lacZ region short direct repeats (10 bp) and flanking imperfect inverted repeats (12 bp) were associated with endpoints. In 27 deletions no repeated sequences of more than 3 bp were present at the endpoints. In 15 of these the sequence 5'-T-G-T-A-3' was found within 10 bp from the left endpoint. At the left cleavage sites the sequence 5'-T-T-T-3', or the 5'-A-A-A-3' complement thereof, was frequently observed. Most of the left deletion endpoints were located in potential stem-loop structures in the penP transcription/translation regulatory region, which is very rich in hyphenated dyad symmetry. Near the right deletion endpoints a sequence consisting of four G/C residues, followed by three or four A/T residues, was found in 15 cases. It is speculated that DNA topoisomerase I is involved in the formation of the deletions studied.

Bacillus subtilis↗

PubMed1988

Instability of recombinant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts.

Two series of pUB110-derived plasmids were constructed to study segregational stability in Bacillus subtilis. pEB plasmids were based on the entire pUB110, whereas pLB plasmids lack the membrane-binding areas BA3 and BA4. Two kinds of stability defects were observed. The first was characterized by a strong size dependency and occurred with different inserts at various positions in pLB and pEB plasmids. Size-dependent reductions in plasmid copy numbers appeared to underly this phenomenon. This may render pUB110 unsuitable for the cloning of inserts larger than about 3 kb, in particular if no selective conditions can be applied. The second defect, observed with pLB plasmids, was caused by the absence of the membrane-binding areas BA3 and BA4. Deletion of BA3 resulted in the accumulation of single-stranded plasmid DNA, suggesting that BA3 contains the initiation signal for complementary strand synthesis. The BA3 region is very rich in hyphenated dyad symmetry which, in single-stranded DNA, could result in several stable alternative secondary structures. It is speculated that the activity of the BA3-associated initiation signal contributes to the segregational stability of pUB110-derived plasmids in B. subtilis. The absence of the BA3 stability function could not account for all stability defects observed. Additional stability functions seemed to be located on the BA4 fragment.

Bacillus subtilis↗

PubMed1987

The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg.

Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied. In a restriction-deficient but modification-proficient mutant of B. subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E. coli (10(4) clones per microgram target DNA). Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb). In the restriction-proficient B. subtilis strain, the class of large inserts was underrepresented. Transformation of B. subtilis with E. coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways. First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host. The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb). Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid. In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0-16 kb), and no structural instability was observed. It is concluded that for shotgun cloning in B. subtilis, the use of restriction-deficient strains is highly preferable. Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction. It is postulated that AsuII sites are additional target sites for BsuM restriction.

Bacillus subtilis↗

PubMed1987

Structural plasmid instability in recombination- and repair-deficient strains of Bacillus subtilis.

Plasmid pGP1, containing a fusion between the penicillinase gene of Bacillus licheniformis and the beta-galactosidase gene of Escherichia coli, was constructed. This plasmid enabled a study of structural plasmid instability in Bacillus subtilis wild-type cells and a variety of B. subtilis strains, defective in recombination- and DNA-repair functions. Large differences with respect to the level of stability of this plasmid were observed in the various genetic backgrounds.

Bacillus subtilis↗

PubMed1987

Stability function in the Bacillus subtilis plasmid pTA 1060.

Plasmid pBB2 (11.3 kb) was constructed by genetically labeling the cryptic Bacillus subtilis plasmid pTA 1060 with the pC194-derived CmR and the pUB110-derived KmR markers. In nonselective media pBB2 was segregationally almost completely stable (loss rates less than or equal to 0.02% per cell generation). In contrast, pBB3, obtained by deleting from pBB2 a region consisting of two ClaI fragments (1.45 and 0.20 kb, respectively), was unstable (loss rates greater than or equal to 0.5% per cell generation). This indicates that a genetic element required for stability is located on one or both of these fragments. In pBB3 cop, a mutant with a two- to threefold increased copy number, the rate of plasmid loss was reduced compared to that of pBB3. The insertion of a 4.2-kb Escherichia coli DNA fragment reduced the stability of pBB2 only slightly, suggesting that this vector may be useful for the cloning of relatively large fragments.

Bacillus subtilis↗

PubMed1987

Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis.

To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.

Bacillus subtilis↗

PubMed1985

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis.

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.

Bacillus subtilis↗

PubMed1985

Transformation in Bacillus subtilis: further characterization of a 75,000-dalton protein complex involved in binding and entry of donor DNA.

A 75,000-dalton protein complex purified from membranes of competent Bacillus subtilis cells was previously shown to be involved in both binding and entry of donor DNA during transformation. The complex, consisting of two polypeptides, a and b, in approximately equal amounts, showed strong DNA binding as well as nuclease activity (H. Smith, K. Wiersma, S. Bron, and G. Venema, J. Bacteriol. 156:101-108, 1983). In the present experiments, peptide mapping indicated that the two polypeptides are not related. Chromatography on benzoylated, naphthoylated DEAE-cellulose showed that polypeptide b generated single-stranded regions in double-stranded DNA. A considerable amount of the DNA was rendered acid soluble by polypeptide b. The nuclease activity of polypeptide b was reduced in the presence of polypeptide a. This resulted in an increased fraction of high-molecular-weight double-stranded DNA containing single-stranded regions. The acid-soluble DNA degradation products formed by polypeptide b consisted exclusively of oligonucleotides. In contrast to its nuclease activity, which was specifically directed toward double-stranded DNA, the DNA binding of the native 75,000-dalton complex to single-stranded DNA was at least as efficient as to double-stranded DNA.

Bacillus subtilis↗

PubMed1984

Restriction of hemimethylated DNA by the Bacillus subtilis R system.

The effects of restriction by the BsuR system on hemimethylated SPP1 DNA were investigated. In vitro, single-stranded nicks were introduced in the nonmodified strand of the hemimethylated DNA at the same sites as recognized in nonmodified homoduplex DNA. Transfection with BsuR-treated hemimethylated DNA was severely reduced. In vivo, transfection with hemimethylated DNA was also severely reduced in competent B. subtilis R cells. In contrast, transfection of protoplasts of the R strain with this DNA was not affected. The apparent restriction by competent cells was attributed to the special mode of processing of transfecting DNA.

Bacillus subtilis↗

PubMed1984

Genetic manipulation of the restricted facultative methylotroph Hyphomicrobium X by the R-plasmid-mediated introduction of the Escherichia coli pdh genes.

The inability of Hyphomicrobium X to grow on compounds such as pyruvate and succinate is most likely due to the absence of a functional pyruvate dehydrogenase (PDH) complex. Further support for this was sought by studying the effect of the introduction of the Escherichia coli pdh genes in Hyphomicrobium X on the pattern of substrate utilization by the latter organism. These genes were cloned by in vivo techniques using the broad-host range conjugative plasmid RP4::Mucts. Plasmid RP4 derivatives containing pdh genes were selected by their ability to complement a pyruvate dehydrogenase deletion mutant of E. coli, strain JRG746 recA (ace-1pd) delta 18. The plasmids thus obtained could be transferred through an intermediary host (C600 recA), selecting only for an antibiotic resistance coded for by RP4 and back into JRG746 or other E. coli pdh mutants, upon which they still conferred the wild type phenotype. Enzyme assays showed that the latter strains, when carrying plasmid RP4'pdh1 also possessed PDH complex activity. Conjugation between the auxotrophic E. coli JRG746 (RP4'pdh1) strain and Hyphomicrobium X on pyruvate minimal agar gave rise to progeny which, on the basis of its morphology (stalked bacteria), their ability to grow on C1-compounds and to denitrify (now also with pyruvate) were identified as hyphomicrobia. This Hyphomicrobium X transconjugant was also able to grow in minimal medium with succinate, but no other novel growth substrates have been identified so far.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗

PubMed1984

Transformation in Bacillus subtilis: a 75,000-dalton protein complex is involved in binding and entry of donor DNA.

A 75,000-dalton protein complex involved in DNA binding during transformation was purified from membranes of competent Bacillus subtilis cells. Previous results (Smith et al., J. Bacteriol. 156:101-108, 1983) showed that the complex contained two polypeptides, polypeptide a (molecular weight, 18,000; isoelectric point, 5.0) and polypeptide b (molecular weight, 17,000; isoelectric point, 4.7) in approximately equal amounts. In the present experiments the two polypeptides were extracted from two-dimensional gels and studied separately and in combination with respect to DNA binding and nuclease activities. For DNA binding the interaction of both polypeptides was required. DNA binding occurred efficiently in the presence of EDTA. Nuclease activity was restricted to polypeptide b. The nucleolytic properties of b were identical to those of the native 75,000-dalton complex. Polypeptide a affected b by reducing its nuclease activity. Analysis of the nuclease subunit b on DNA-containing polyacrylamide gels revealed nuclease activities at four different molecular weight positions. These activities were identical to the major competence-specific nuclease activities which were previously implicated in the entry of donor DNA during transformation (Mulder and Venema, J. Bacteriol. 152:166-174, 1982). These results indicate that the 75,000-dalton protein complex is composed of two different competence-specific polypeptides involved in both binding and entry of donor DNA. The possible roles of the two polypeptides in the transformation of B. subtilis are discussed.

Bacillus subtilis↗