Biomedical subjects

S Bron

Publications and source records attributed to S Bron.

At least 55 records · Page 3Linked to original sources

PubMed1996

Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I.

Bacillus subtilis contains three chromosomally encoded type I signal peptidases (SipS, SipT and SipU), which remove signal peptides from secretory precursor proteins. In the present study the biological function of SipS and the regulation of its synthesis were analysed. Unlike the type I signal peptidase of Escherichia coli, SipS was essential neither for protein secretion nor viability of the cell. However, in the absence of SipS the rate of processing of several preproteins was reduced, and four of the seven major secreted proteins of B. subtilis were hardly detectable in the growth medium. Surprisingly, the processing of Bacillus amyloliquefaciens alpha-amylase and the secretion of at least two endogenous B. subtilis proteins was improved in the absence of SipS. These findings indicate that the substrate preference of SipS differs from that of SipT and SipU, and that SipS is an important factor determining the efficiency of protein secretion in B. subtilis. SipS is transcribed in a growth phase- and medium-dependent manner. In minimal medium, the growth phase-dependent transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that the expression of sipS is regulated by the same factors that control the expression of most genes for secreted degradative enzymes. Our observations suggest that B. subtilis can modulate its capacity and specificity for protein secretion through the controlled expression of sipS.

Bacillus subtilis↗

PubMed1996

A 22 kb DNA sequence in the cspB-glpPFKD region at 75 degrees on the Bacillus subtilis chromosome.

A 21808 bp nucleotide sequence at 75 degrees on the genetic map of the Bacillus subtilis chromosome was determined. The sequence of this region is adjacent to the glpPFKD operon involved in glycerol utilization. Twenty-six ORFs were identified, one of which corresponds to the cspB gene, encoding a cold-shock protein. Seventeen of the deduced protein sequences of these ORFs displayed significant homology to known proteins in the data banks. One putative operon was identified, consisting of five ORFs, that is probably involved in the uptake and processing of copper. The location of cspB in this sequence does not confirm the genetic mapping data, indicating that the gene is closely linked to comK, which is located at 80 degrees on the B. subtilis chromosome.

Bacillus subtilis↗

PubMed1996

Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20.

We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions. Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B. subtilis chromosomal replication terminator. Here, we demonstrate that this segment is part of a functional replication terminator. This newly identified element, designated TerLS20, is the first replication terminator identified on a theta plasmid from a Gram-positive bacterium. TerLS20 is distinct from other known replication terminators in the sense that it is functional in both orientations. The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.

Bacillus subtilis↗

PubMed1996

The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis.

The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the Escherichia coli beta-galactosidase (lacZ) gene. Two processes affected the accumulation of pGP1 variants with deletions in the penP-lacZ region. First, divergent transcription from genes upstream of penP-lacZ increased pGP1 deletion frequencies up to about 10-fold. Second, the removal of the PenP signal peptide resulted in completely stable plasmids, indicating that the entry of the PenP fragment into the protein export pathway is an important factor in the instability of pGP1. On the basis of these results, we propose a model in which the temporary anchoring of the plasmid to the membrane through the cotranscriptional and cotranslational entry of PenP into the protein export pathway creates domains of local hypersupercoiling, which we assume to be targets for deletion formation.

Amino Acid Sequence↗

PubMed1995

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01.

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 bp region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR I) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage phi X174. This IR was shown to have a partial, RNA polymerase-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerase-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).

Bacteriophage M13↗

PubMed1995

Overproduction of the ATP-dependent nuclease AddAB improves the structural stability of a model plasmid system in Bacillus subtilis.

The effect of the ATP-dependent exonuclease AddAB complex on the structural stability of plasmid pGP1 in Bacillus subtilis was studied. Using deletion mutagenesis and gene amplification techniques, B. subtilis strains were constructed either lacking or overproducing the AddAB complex, a key enzyme in homologous recombination. The deletion mutant possessed no residual ATP-dependent nuclease activity; in contrast, the nuclease activity was up to 30 times higher in lysates of strains carrying multiple copies of the addAB genes in the chromosome. Southern blot analyses of these strains indicated that a linear relationship exists between the number of chromosomal gene copies and the level of AddAB activity. The structural stability of pGP1 was analyzed in the AddAB-deficient and over-producing backgrounds. Frequencies of deletion formation in the plasmid, as monitored by the expression of the pGP1-encoded penP-lacZ fusion on media containing X-gal, were shown to be increased at least 25-fold in the addAB knock-out mutant, whereas the stability of pGP1 was improved up to 15-fold in strains overproducing the AddAB enzyme. A possible explanation for these findings is that interactions between AddAB and plasmid molecules prevent the formation of secondary structures that constitute potential deletion target sites, and thereby enhance the structural stability of plasmids.

Bacillus subtilis↗

PubMed1995

Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon.

A 3.1 kb fragment of the large (approximately 55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced. In contrast to the parental plasmid, derived minireplicons were unstably maintained. Using deletion analysis the fragment essential and sufficient for replication was delineated to 1.1 kb. This 1.1 kb fragment is located between two divergently transcribed genes, denoted orfA and orfB, neither of which is required for replication. orfA shows homology to the B.subtilis chromosomal genes rapA (spoOL, gsiA) and rapB (spoOP). The 1.1 kb fragment, which is characterized by the presence of several regions of dyad symmetry, contains no open reading frames of more than 85 codons and shows no similarity with other known plasmid replicons. The structural organization of the pLS20 minimal replicon is entirely different from that of typical rolling circle plasmids from Gram-positive bacteria. The pLS20 minireplicons replicate in polA5 and recA4 B.subtilis strains. Taken together, these results strongly suggest that pLS20 belongs to a new class of theta replicons.

Amino Acid Sequence↗

PubMed1995

Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis.

In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication. The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060. The SSO of pTA1015 was isolated by shotgun cloning in a specially designed vector, pWM100, which has no SSO of its own. Sequence analysis revealed that the SSO of pTA1015 is almost identical to formerly described palT type SSOs. Also pTA1020 and pTA1060 were shown to contain SSOs highly homologous to palT. Using Southern hybridization with the palT of pTA1015 as a probe, the SSO of pTA1040 was cloned. Sequence analysis revealed a region of 200 bp which is 77% identical to the palT of pTA1015. The plasmids pTA1030 and pTA1050 contain an SSO which is highly homologous to the SSO of pTA1040. The majority of the SSOs of rolling-circle plasmids from B.subtilis seem to belong to two related families which we denote as palT1 (present on pTA1015, pTA1020 and pTA1060) and palT2 (present on pTA1030, pTA1040 and pTA1050). Both families of SSOs are highly efficient single-strand-conversion signals in B.subtilis.

Bacillus subtilis↗

PubMed1995

Identification of the potential active site of the signal peptidase SipS of Bacillus subtilis. Structural and functional similarities with LexA-like proteases.

Signal peptidases remove signal peptides from secretory proteins. By comparing the type I signal peptidase, SipS, of Bacillus subtilis with signal peptidases from prokaryotes, mitochondria, and the endoplasmic reticular membrane, patterns of conserved amino acids were discovered. The conserved residues of SipS were altered by site-directed mutagenesis. Replacement of methionine 44 by alanine yielded an enzyme with increased activity. Two residues (aspartic acid 146 and arginine 84) appeared to be conformational determinants; three other residues (serine 43, lysine 83, and aspartic acid 153) were critical for activity. Comparison of SipS with other proteases requiring serine, lysine, or aspartic acid residues in catalysis revealed sequence similarity between the region of SipS around serine 43 and lysine 83 and the active-site region of LexA-like proteases. Furthermore, self-cleavage sites of LexA-like proteases closely resembled signal peptidase cleavage sites. Together with the finding that serine and lysine residues are critical for activity of the signal peptidase of Escherichia coli (Tschantz, W.R., Sung, M., Delgado-Partin, V.M., and Dalbey, R.E. (1993) J. Biol. Chem. 268, 27349-27354), our data indicate that type I signal peptidases and LexA-like proteases are structurally and functionally related serine proteases. A model envisaging a catalytic serine-lysine dyad in prokaryotic type I signal peptidases is proposed to accommodate our observations.

Amino Acid Sequence↗

PubMed1995

Use of continuous culture for the selection of plasmids with improved segregational stability.

In this report a method that enables the selection of stable plasmid variants and the isolation of DNA sequences that improve plasmid maintenance is described. The method is based on the principle that in populations of cells carrying derivatives of a plasmid that differ only in the level of segregational stability, when grown in a chemostat under conditions with selective pressure on the plasmid, cells that carry more stable plasmid variants will be enriched. We developed the system for Lactococcus lactis using segregationally unstable derivatives of the gram-positive theta plasmid pAM beta 1 as selection vectors. The results showed that the method is suitable for the enrichment, and subsequent purification, of three classes of plasmids with improved maintenance properties. The first class involved mutations in the pAM beta 1-derived selection plasmid. These mutations resulted in increased copy numbers, thereby rendering the plasmid segregationally more stable. The other two classes were based on the insertion of additional, stability promoting, sequences in the selection plasmids. We showed that these sequences can constitute either replication functions derived from another plasmid or functions directly involved in plasmid maintenance. The site-specific resolution function of pAM beta 1 was used as an example of the latter functions. We anticipate that the method described should have general applicability for the development of stable host-vector systems in bacteria.

Bacteriological Techniques↗

PubMed1995

Effects of the generation of single-stranded DNA on the maintenance of plasmid pMV158 and derivatives in different Bacillus subtilis strains.

The effects of the single-strand origins (SSOs) of plasmid pMV158 on (i) the conversion of its single-stranded (ss) replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed. The rolling-circle plasmid pMV158, which replicates via ssDNA intermediates, contains two single-strand origins (SSOs) of replication, palA and palU. In this paper the results obtained with Bacillus subtilis are described; complementary studies with Lactococcus lactis are presented in the accompanying paper (Meijer et al., 1995). While in L. lactis both SSOs are functional as ssDNA conversion signal, only palU appeared to be active B. subtilis. Similar to the situation in L. lactis, the accumulation of large amounts of ssDNA resulted in a severe decrease in plasmid maintenance in B. subtilis. In the latter bacterium large amounts of ssDNA were only accumulated, however, when plasmids lacking a functional SSO were propagated in RecA mutant strains. In wild-type RecA strains these plasmids accumulated only modest amounts of ssDNA and they were maintained at fairly stable levels. The results suggest that in B. subtilis a RecA-mediated alternative pathway exists for the conversion of ssDNA which can improve plasmid maintenance. In addition to ssDNA accumulation and the antagonizing role of RecA therein, two other plasmid regions were shown to affect pMV158 maintenance in B. subtilis. One was the mob gene region, which had a negative effect on plasmid maintenance, and the other the palA type SSO. Although palA was not functional as an ssDNA conversion signal in B. subtilis, its presence had a positive effect on pMV158 maintenance.

Bacillus subtilis↗

PubMed1995

Effects of the generation of single-stranded DNA on the maintenance of plasmid pMV158 and derivatives in Lactococcus lactis.

The effects of the single-strand origins (SSOs) of the broad-host-range streptococcal plasmid pMV158 on (i) the conversion of its single-stranded (ss) DNA replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed. pMV158 is distinguished from most other plasmids that replicate by the rolling-circle mechanism by the presence of two single-strand origins of replication, palA and palU. In this paper the results obtained with Lactococcus lactis are presented; complementary studies with Bacillus subtilis are presented in the accompanying paper (Meijer et al., 1995). In the presence of both SSOs, no ss plasmid DNA was observed in L. lactis. The removal of either palA or palU resulted in the appearance of low amounts of ssDNA. High amounts of ssDNA were detected, however, when both SSOs were deleted. The results indicated that both SSOs were active, albeit that palU was the most effective of the two. In the presence of both SSOs, the plasmid was stably maintained in L. lactis under nonselective growth conditions. Also, the derivatives containing only one of the two SSOs were maintained rather stably. In contrast, the derivative devoid of both SSOs was poorly maintained. It was concluded that, in the absence of a functional SSO, the generation of large amounts of ssDNA drastically reduces the maintenance of pMV158 in L. lactis. The results also showed that the presence of the plasmid-located mob gene, required for conjugative mobilization, was involved neither in the accumulation of ssDNA nor in the maintenance of pMV158.

Bacterial Proteins↗

PubMed1995

The endogenous Bacillus subtilis (natto) plasmids pTA1015 and pTA1040 contain signal peptidase-encoding genes: identification of a new structural module on cryptic plasmids.

Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis plasmids pTA1015 and pTA1040. It is composed of two genes: one specifies an unidentified protein with a putative signal peptide; and the other (sipP) specifies a functional type 1 signal peptidase (SPase). The homologous, but non-identical, sipP genes of the two plasmids are the first identified plasmid-specific SPase-encoding genes. With respect to structure and activity, the corresponding enzymes (denoted SipP) are highly similar to the chromosomally encoded SPase, SipP, of B. subtillis and several newly identified SPases of other bacilli. Our findings suggest that plasmid-encoded SPases have evolved because, of under certain conditions, SPase can be a limiting factor for protein secretion in B. subtilis.

Amino Acid Sequence↗

PubMed1994

The majority of lactococcal plasmids carry a highly related replicon.

DNA sequence analysis and Southern hybridizations, together with complementation experiments, were used to study relationships between lactococcal plasmid replicons. pWVO2, pWVO4 and pWVO5, which co-exist in Lactococcus lactis subsp. cremoris Wg2, and pIL7 (isolated from another strain) all contained a functional replication region which appeared to be very similar to that of some known lactococcal plasmids. They contain a gene encoding a highly conserved RepB protein (60-80% amino acid identity between pWVO2, pWVO4 and pWVO5), which is essential for replication. When supplied in trans, repB of pWVO2 complemented a repB deficiency of pWVO5. Upstream of the repB gene, all these plasmids contain a strongly conserved region including a 22 bp sequence tandemly repeated three-and-a-half times, and an A/T-rich region. The similarity with pWVO2, which is known to replicate via a theta mechanism, suggests that all plasmids of this family are capable of theta replication. Southern hybridizations revealed that many lactococcal strains contain plasmids of this family.

Amino Acid Sequence↗

PubMed1994

Design, synthesis, and preliminary characterization of tyrosine-containing polyarylates: new biomaterials for medical applications.

Five structurally related, aliphatic polyarylates were synthesized from tyrosine-derived diphenols and diacids. The diphenols were a homologous series of three desaminotyrosyl-tyrosine alkyl esters (ethyl, hexyl, octyl) which had previously been used in the synthesis of mechanically strong and tissue-compatible polycarbonates. The diacids (succinic acid, adipic acid, sebacic acid) were selected among compounds that were known to be of low systemic toxicity. By using different diacids as comonomers, the flexibility of the polymer backbone could be varied while the desaminotyrosyl-tyrosine alkyl esters provided pendent chains of various length. Some of the thermal and mechanical properties of the five polymers could be correlated to their chemical structure: the glass transition temperature decreased from 53 to 13 degrees C, and the tensile modulus (measured at room temperature) decreased from 1500 to about 3 MPa when the length of the aliphatic diacid in the polymer backbone and/or the length of the alkyl ester pendent chain was increased. The presence of an arylate bond in the polymer backbone introduced a hydrolytically labile linkage into the polymer structure. Under physiological conditions in vitro all polymers degraded: thin films retained only about 30-40% of their initial molecular weight (Mw) after 26 weeks of storage in phosphate buffer solutions (pH 7.4) at 37 degrees C. Release studies with p-nitroaniline as a model drug indicated that a diffusion controlled release process occurred. The rate of p-nitroaniline release could be correlated with the glass transition temperature of the polymer.

Biocompatible Materials↗

PubMed1993

In vitro assay for the Bacillus subtilis signal peptidase SipS: systems for efficient in vitro transcription-translation and processing of precursors of secreted proteins.

The signal peptidase (SPase) SipS of Bacillus subtilis is responsible for the processing of precursors of secreted proteins. It differs from the SPases of Gram-negative bacteria in structure and specificity. To assay the activity of SipS in vitro, two efficient transcription-translation systems for the synthesis of radio-labelled precursors were developed. The systems were completely derived from B. subtilis. Post-translational in vitro processing of pre-staphylokinase by SipS was demonstrated. SipS activity was stimulated in vitro by several non-ionic detergents, whereas it was not affected by a large variety of proteinase inhibitors. SipS shares the latter property with other SPases.

Bacillus subtilis↗

PubMed1993

The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in Lactococcus lactis.

The effects of the rolling-circle and theta modes of replication on the maintenance of recombinant plasmids in Lactococcus lactis were studied. Heterologous Escherichia coli or bacteriophage lambda DNA fragments of various sizes were inserted into vectors based on either the rolling-circle-type plasmid pWV01 or the theta-type plasmid pAMbeta1. All pAMbeta1 derivatives were stably maintained. pWV01 derivatives, however, showed size-dependent segregational instability, in particular when large DNA fragments were inserted. All recombinant pWV01 derivatives generated high-molecular-weight plasmid multimers (HMW) in amounts that were positively correlated with plasmid size and inversely correlated with the copy numbers of the monomeric plasmid forms. Formation of HMW or reductions in copy numbers were not observed with pAMbeta1 derivatives. The results indicate that HMW formation and/or reduction in plasmid copy numbers is an important factor in the maintenance of pWV01 derivatives. It is concluded that theta-type plasmids are superior to rolling-circle-type plasmids for cloning in lactococci.

Journal Article↗

PubMed1993

Theta replication of the lactococcal plasmid pWVO2.

pWVO2 is a 3.8 kb narrow-host-range plasmid from Lactococcus lactis ssp. cremoris Wg2, which does not replicate in Bacillus subtilis or Escherichia coli. Single-stranded pWVO2 DNA was not observed in lactococcal cells, indicating that this plasmid does not replicate via a rolling-circle mechanism. The sequence of pWVO2 neither showed the structural organization typical for rolling-circle plasmids, nor were sequence similarities with known rolling-circle plasmids present. By 2-D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism. This is the first proof for the existence of theta-replicating plasmids in lactococci. The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons. It contains one open reading frame encoding the replication protein, which is preceded by a 22 bp sequence tandemly repeated three and a half times. Further upstream is another 10 bp direct repeat present in an A/T-rich sequence. This structural organization resembles that of several iteron-containing theta-type plasmids from E. coli. Derivatives of pWVO2 were stably maintained in L. lactis and are good candidates for the development of stable food-grade cloning vectors for this organism.

Amino Acid Sequence↗