Biomedical subjects

S Bron

Publications and source records attributed to S Bron.

At least 19 recordsLinked to original sources

PubMed1992

Plasmid maintenance in Bacillus stearothermophilus is strain-dependent.

We studied the segregational stability of plasmids based on pTB913, a 4.5-kb rolling-circle plasmid derived from the thermophilic Bacillus plasmid pTB19. In Bacillus stearothermophilus the stability of pTB913 derivatives appeared to be strain-dependent. In strain CU21 large amounts of single-stranded pTB913 DNA were found and the plasmid was highly unstable at 57 degrees C. In strain NUB3621, however, very low amounts of single-stranded plasmid DNA were formed and pTB913-based replicons were only slightly unstable at 57 degrees C. The NUB3621/pTB913 host-vector system seems appropriate for molecular cloning. A RepA-based replicon, also derived from pTB19 but replicating by a theta mechanism, was highly unstable in B. stearothermophilus NUB3621.

DNA Replication↗

PubMed1992

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.

Amino Acid Sequence↗

PubMed1992

The integrated state of the rolling-circle plasmid pTB913 in the composite Bacillus plasmid pTB19.

pTB19, a 27 kb plasmid originating from a thermophilic Bacillus species, contains integrated copies of two rolling-circle type plasmids on a 10.6 kb DNA fragment. In the present study we analysed the part of pTB19 that contains the rolling-circle plasmid pTB913 and the region in between the two rolling-circle plasmids. We show that, in the integrated state, pTB913 was flanked by a 55 bp direct repeat that duplicated part of the replication initiation gene repB. Since repB was interrupted, the integrated pTB913 could not initiate rolling-circle replication. Autonomously replicating pTB913 was produced from pTB19, probably through recombination between the 55 bp direct repeats; this was a rare event. Since the second integrated rolling-circle type plasmid also contained a non-functional replication initiation gene, replication of pTB19 must be controlled by the RepA determinant. Theta-type replication, controlled by RepA is likely to account for the high stability of pTB19. In between the two integrated rolling-circle plasmids was present an open reading frame (447 codons) which could encode a protein of unknown function.

Amino Acid Sequence↗

PubMed1992

Protein export elements from Lactococcus lactis.

Broad-host-range plasmids carrying alpha-amylase or beta-lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the export of the reporter proteins in Escherichia coli. Several of the selected export elements were also active in Bacillus subtilis and L. lactis, although the efficiencies depended strongly on the host organism and reporter gene used. The export elements AL9 and BL1 were highly efficient in L. lactis in the expression and secretion of at least two heterologous proteins (Bacillus licheniformis alpha-amylase and E. coli TEM-beta-lactamase). AL9 even permitted growth of this organism on starch as the sole carbon source. Nucleotide sequence analysis of five selected fragments indicated that these encode oligopeptides with the major characteristics of typical signal peptides. The putative expression signals had a limited similarity to previously described expression signals for E. coli, B. subtilis and L. lactis. Differences in both expression and export efficiency are likely to underlie the host-specific effects.

Amino Acid Sequence↗

PubMed1992

Plasmid pTB913 derivatives are segregationally stable in Bacillus subtilis at elevated temperatures.

We studied the effects of temperature on the segregational stability of derivatives of the rolling-circle-type plasmid pTB913 in Bacillus subtilis. This 4.5-kb plasmid is a deletion derivative of pTB19, which was originally isolated from a thermophilic Bacillus. pTB913 derivatives carrying large inserts or lacking the minus origin for complementary strand synthesis were segregationally unstable at 37 degrees C. In contrast, at 47 degrees C all pTB913 derivatives tested were stably maintained in B. subtilis. The increased stability at 47 degrees C was attributed, at least partly, to increased copy numbers at this temperature. Although considerable amounts of single-stranded and high-molecular-weight plasmid DNA were formed at 47 degrees C, these products did not reduce plasmid stability at this temperature. The increased stability and increased copy number of pTB913 at elevated temperatures extend the use of this plasmid as a cloning vector in B. subtilis and other bacilli.

Bacillus subtilis↗

PubMed1991

Lack of specific hybridization between the lep genes of Salmonella typhimurium and Bacillus licheniformis.

This paper describes an attempt to clone the Bacillus licheniformis lep gene, encoding signal peptidase, using the Salmonella typhimurium lep gene as a hybridization probe. Although a hybridizing fragment was obtained, DNA sequence analysis indicated that it did not contain the lep gene. Instead, the protein encoded by the cloned fragment showed similarity with a variety of L-asparaginases.

Amino Acid Sequence↗

PubMed1991

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli.

The effects of 25-fold overproduction of Escherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from the Bacillus subtilis chromosome and the mature part of TEM-beta-lactamase, were studied in E. coli. One precursor (pre[A2d]-beta-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[A13i]-beta-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature beta-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-beta-lactamase and pre(A2)-beta-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export in E. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.

Bacillus subtilis↗

PubMed1991

Plasmid deletion formation between short direct repeats in Bacillus subtilis is stimulated by single-stranded rolling-circle replication intermediates.

The effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.

Bacillus subtilis↗

PubMed1991

The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions.

Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species. It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913. Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline. The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism. This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913. A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region. The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

Amino Acid Sequence↗

PubMed1991

Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis.

The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half that produced in wild-type E. coli cells. The production of E. coli SPase I in B. subtilis was increased approximately fivefold by cloning the lep gene into a high-copy-number plasmid. The expression of E. coli SPase I in B. subtilis did not appear to increase the rate of processing of two hybrid secretory precursor proteins. Two observations may explain the failure of E. coli SPase I to stimulate processing of exported proteins in B. subtilis. First, the E. coli SPase I was apparently not exposed on the outside of the B. subtilis cytoplasmic membrane, indicating its incorrect insertion into the membrane. Second, in vitro processing studies, using cell-free extracts of B. subtilis producing E. coli SPase I, suggested that the enzyme was not active. A further outcome of this study was that conditions favouring processing of precursors by SPase I in cell-free extracts of E. coli did not favour processing by the corresponding enzyme in B. subtilis cell-free extracts. This suggests that significant differences exist between the two enzymes. The observation that antibodies directed against E. coli SPase I did not cross-react with B. subtilis membrane proteins supports this idea.

Amino Acid Sequence↗

PubMed1990

An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis.

The recently described beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis [Haima et al., Gene 86 (1990) 63-69]was optimized in several ways. First, the efficiency of translation of the lac Z delta M15 gene was improved. Second, the plasmid-borne lacZ delta M15 gene was segregationally stabilized by integration into the B. subtilis chromosome. Third, a new lacZ alpha complementing cloning vector was constructed, containing more unique target sites. It was shown that large heterologous DNA fragments (up to at least 29 kb) could be cloned with lacZ alpha-complementing vectors carrying the replication functions of the cryptic B. subtilis plasmid pTA1060, and that these inserts were structurally stably maintained for at least 100 generations of growth.

Bacillus subtilis↗

PubMed1990

Development of a beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis.

A versatile beta-galactosidase alpha-complementation system for Bacillus subtilis was developed, which can be used for molecular cloning in this Gram+ organism. The cloning system, which is based on the highly efficient host-vector system 6GM-pHP13, offers several advantages over previously described systems: (1) convenient direct selection of recombinants; (2) the cloning of large heterologous DNA fragments with high efficiency; and (3) the availability of six unique target sites: SphI, NdeI, NheI, BamHI, SmaI and EcoRI.

Bacillus subtilis↗

PubMed1990

Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: use of AUA as a restart codon.

An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the beta-galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active beta-galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the beta-galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.

Amino Acid Sequence↗

PubMed1990

Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants.

A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of beta-galactosidase alpha-complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.

Bacillus subtilis↗

PubMed1990

Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis.

pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcus faecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAM beta 1 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.

Conjugation, Genetic↗

PubMed1975

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPPI, SPO2 and phi105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo- or endonuclease activity. Only Mg2+ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5 x 10(7)) have an average molecular weight of about 3 x 10(5).

Adenosine Triphosphate↗

PubMed1975

Restriction and modification in B. subtilis. Nucleotide sequence recognised by restriction endonuclease R. Bsu R from strain R.

Restriction endonuclease R from Bacillus subtilis strain R cleaves nonmodified SPP 1 DNA in approximately 80, and lambda DNA in about 200 different sites. DNA digests with this endonuclease and with endonuclease Hae III from Haemophilus aegyptius show identical fragmentation patterns on gel electrophoresis, indicating that the two enzymes recognise the same nucleotide sequence. The polynucleotide kinase reaction was used in conjunction with two-dimensional ionophoretic nucleotide mapping methods to identify the 5'-nucleotide sequences at the sites of cleavage by the B. subtilis restriction endonuclease. The results show that the recognition sequence is (see article) where arrows indicate the points of strand scission. Each of the four possible nucleotides can occur in the positions flanking the recognition site.

Bacillus subtilis↗