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G Venema

Publications and source records attributed to G Venema.

At least 91 records · Page 5Linked to original sources
PubMed1996

Effects of lysine-to-glycine mutations in the ATP-binding consensus sequences in the AddA and AddB subunits on the Bacillus subtilis AddAB enzyme activities.

The N-terminal regions of both subunits AddA and AddB of the Bacillus subtilis AddAB enzyme contain amino acid sequences, designated motif I, which are commonly found in ATP-binding enzymes. The functional significance of the motif I regions was studied by replacing the highly conserved lysine residues of the regions in both subunits by glycines and by examination of the resulting mutant enzymes with respect to their enzymatic properties. This study shows that the mutation in subunit AddB hardly affected the ATPase, helicase, and exonuclease activities of the AddAB enzyme. However, the mutation in subunit AddA drastically reduced these activities, as well as the kcat for ATP hydrolysis. The apparent Km for ATP in ATP hydrolysis did not significantly deviate from that of the wild-type enzyme. These results suggest that the lysine residue in motif I of subunit AddA of the AddAB enzyme is not essential for the binding of the nucleotide but has a role in ATP hydrolysis, which is required for the exonuclease and helicase activities of the enzyme.

Adenosine Triphosphatases↗
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

Lactococcal bacteriocins: mode of action and immunity.

Bacteriocins are antimicrobial peptides produced by bacteria. Some of those synthesized by Lactococcus lactis have been characterized in great detail recently. The lactococcal bacteriocins are hydrophobic cationic peptides, which form pores in the cytoplasmic membrane of sensitive cells.

Amino Acid Sequence↗
PubMed1995

Structural determinants of the stability of thermolysin-like proteinases.

Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Site-directed mutagenesis studies of the thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to thermolysin, is due to only some of the 44 naturally occurring amino-acid differences between them. In fact TLP-ste becomes more resistant than thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localized to a solvent-exposed region in the N-terminal domain of TLP-ste.

Amino Acid Sequence↗
PubMed1995

Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing.

A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55.5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods of immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.

Amino Acid Sequence↗
PubMed1995

Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme.

The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

ATP-Binding Cassette Transporters↗
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↗
PubMed1995

A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis.

The valine-activation domain-encoding portion of the srfA locus (srfA-d4) is not only involved in the non-ribosomal synthesis of surfactin, but is also required for the regulation of competence development. In this study we show that impairment of the adenylation activity of the valine-activating domain did not affect competence development. Deletion analysis and complementation studies delineated the competence-required portion of srfA-d4 to a 168 bp fragment, which contains a small open reading frame (ORF), designated comS, encoding a polypeptide of 46 amino acids, embedded within, but translated in, a frame different from that of srfA-d4. Introduction of an amber mutation in the comS-coding frame prevented competence development, demonstrating the involvement of comS in this prokaryotic specialization process.

Amino Acid Sequence↗
PubMed1995

Expression of the ATP-dependent deoxyribonuclease of Bacillus subtilis is under competence-mediated control.

Transcription of the ATP-dependent deoxynuclease operon (addAB), as monitored by means of an addAB-lacZ transcriptional fusion, has a low, constitutive level and is initiated from a sigma A type promoter. Transcription of addAB is independent of DNA-damaging agents known to induce the SOS response in Bacillus subtilis. However, addAB transcription increased significantly during competence development. This competence-specific induction was dependent on the gene products of srfA, degU and comK, but not on that of recA. Deletion analysis of the addAB promoter region demonstrated that the competence-specific transcription induction requires DNA sequences located upstream of the addAB promoter that associated with ComK, the competence transcription factor. The latter finding indicates that a direct regulatory link exists between the establishment of the competent state and the synthesis of AddAB, required for recombination of internalized donor DNA.

Amino Acid Sequence↗