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G Venema

Publications and source records attributed to G Venema.

At least 19 recordsLinked to original sources
PubMed2006

Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations.

A putative protective protein from Plasmodium falciparum merozoites, MSA2, was expressed in two different ways on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. The first display format exploits an LPXTG-type anchoring motif of the lactococcal proteinase PrtP to covalently anchor MSA2 to the genetically modified producer cells. In a second display format, MSA2 was fused to the peptidoglycan-binding domain (Protein Anchor) of the lactococcal cell wall hydrolase AcmA and was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells, termed Gram-positive enhancer matrix (GEM) particles. The L. lactis recombinants carrying covalently bound MSA2 were used to immunise rabbits through nasal and oral routes. The highest levels of IgG antibodies reacting with near-native MSA2 on merozoites was elicited by oral administration. Intestinal antibodies to MSA2 were produced only after oral immunisation. MSA2-specific T(h)-cell activation could be demonstrated. Based on these results, the immunogenicity in oral immunisations of MSA2, bound non-covalently to non-genetically modified L. lactis GEM particles, was compared with MSA2 that was bound covalently to genetically modified L. lactis. These two forms elicited similar titres of serum antibodies. The results illustrate the potential of using non-genetically modified L. lactis as a safe vaccine delivery vehicle to elicit systemic antibodies, thereby avoiding the dissemination of recombinant DNA into the environment.

Administration, Intranasal↗
PubMed2004

Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis.

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type alpha-subunits and with C-terminal deletion mutants of the alpha-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

Bacillus subtilis↗
PubMed2001

Proteins of the lactococcin A secretion system: lcnD encodes two in-frame proteins.

Polyclonal antibodies were raised against LcnC and LcnD proteins of the Lactococcus lactis bacteriocin lactococcin A secretory system to examine their cellular location and interaction. Two major reacting bands were detected by Western immunoblot with the anti-LcnD antibody: one of 52 kDa (LcnD) and another of 45 kDa, called here LcnD*. LcnD* was still detectable after removing the AUG start codon for LcnD. Chemical cross-linking analyses of membrane fractions of L. lactis cells expressing the LcnC/D secretion machinery were performed. Our results indicate that LcnD is present in the secretion machinery complex as a dimer and is able to interact with LcnD* and LcnC.

ATP-Binding Cassette Transporters↗
PubMed2001

The Bacillus subtilis competence transcription factor, ComK, overrides LexA-imposed transcriptional inhibition without physically displacing LexA.

During the development of competence in Bacillus subtilis the recA gene is activated by the competence transcription factor, ComK, which is presumably required to alleviate the transcriptional repression of recA by LexA. To investigate the mechanism by which ComK activates recA transcription we examined the binding of ComK and LexA to the recA promoter in vitro. Using hydroxyl radical protection analyses to establish the location of ComK dimer-binding sites within the recA promoter, we identified four AT-boxes in a configuration unique for ComK-regulated promoters. Gel mobility shift experiments showed that all four ComK dimer-binding sites were occupied at ComK concentrations in the physiological range. In addition, occupation of all ComK-binding sites did not prevent LexA from binding to the recA promoter, despite the fact that the ComK and LexA recognition motifs partially overlap. Although ComK did not replace LexA from the recA promoter, in vitro transcription analyses indicated that the presence of ComK is sufficient to alleviate LexA repression of recA.

Bacillus subtilis↗
PubMed2001

Distinction between major and minor Bacillus signal peptidases based on phylogenetic and structural criteria.

The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability. As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential. This allows a distinction between SPases that are of major and minor importance for cell viability. Notably, the functional difference between major and minor SPases is not reflected clearly in sequence alignments. Here, we have successfully used molecular phylogeny to predict major and minor SPases. The results were verified with SPases from various bacilli. As predicted, the latter enzymes behaved as major or minor SPases when expressed in B. subtilis. Strikingly, molecular modeling indicated that the active site geometry is not a critical parameter for the classification of major and minor Bacillus SPases. Even though the substrate binding site of the minor SPase SipV is smaller than that of other known SPases, SipV could be converted into a major SPase without changing this site. Instead, replacement of amino-terminal residues of SipV with corresponding residues of the major SPase SipS was sufficient for conversion of SipV into a major SPase. This suggests that differences between major and minor SPases are based on activities other than substrate cleavage site selection.

Amino Acid Sequence↗
PubMed2001

Regulation of bacteriocin production in Lactobacillus plantarum depends on a conserved promoter arrangement with consensus binding sequence.

Bacteriocin production in Lactobacillus plantarum C11 is regulated by a three-component signal transduction system comprising a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two homologous response regulators (RRs; PlnC and PlnD). Both RRs are DNA-binding proteins that bind to promoter-proximal elements in the pln regulon. The binding site for the two regulators consists of two 9-bp direct repeats, that conform to the consensus sequence 5'-TACGTTAAT-3', and the repeats are separated by an intervening 12-bp AT-rich spacer region. In the present work, the plhA promoter was used as a model to evaluate the significance of the binding sequence and conserved promoter arrangement. Point substitutions in the consensus sequence, particularly those in invariant positions, either abolished or significantly reduced binding of PlnC and PlnD. Both regulators bind as homodimers to DNA fragments containing a complete set of regulatory elements, while removal of either repeat, or alterations in the length of the spacer region, significantly weakened binding of both protein dimers. DNase I footprinting demonstrated that PlnC and PlnD both bind to, and protect, the direct repeats. By fusing the plnA promoter region to the beta-glucuronidase (GUS) gene, it was shown that promoter activity is dependent on an intact set of accurately organized repeats. The in vitro and in vivo results presented here confirm the involvement of the repeats as regulatory elements in the regulation of bacteriocin production.

Bacteriocins↗
PubMed2001

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.

Amino Acid Substitution↗
PubMed2000

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.

Amino Acid Sequence↗
PubMed2000

Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids.

The members of the M4 peptidase family are involved in processes as diverse as pathogenicity and industrial applications. For the first time a number of M4 family members, also known as thermolysin-like proteases, has been characterized with an identical substrate set and a uniform set of assay conditions. Characterization with peptide substrates as well as high performance liquid chromatography analysis of beta-casein digests shows that the M4 family is a homogeneous family in terms of catalysis, even though there is a significant degree of amino acid sequence variation. The results of this study show that differences in substrate specificity within the M4 family do not correlate with overall sequence differences but depend on a small number of identifiable amino acids. Indeed, molecular modeling followed by site-directed mutagenesis of one of the substrate binding pocket residues of the thermolysin-like proteases of Bacillus stearothermophilus converted the catalytic characteristics of this variant into that of thermolysin.

Amino Acids↗
PubMed2000

Conserved serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis.

Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalent, but structurally quite different, forming two distinct SPase families that share only few conserved residues. P-type SPases were, so far, exclusively identified in eubacteria and organelles, whereas ER-type SPases were found in the three kingdoms of life. Strikingly, the presence of ER-type SPases appears to be limited to sporulating Gram-positive eubacteria. The present studies were aimed at the identification of potential active site residues of the ER-type SPase SipW of Bacillus subtilis, which is required for processing of the spore-associated protein TasA. Conserved serine, histidine, and aspartic acid residues are critical for SipW activity, suggesting that the ER-type SPases employ a Ser-His-Asp catalytic triad or, alternatively, a Ser-His catalytic dyad. In contrast, the P-type SPases employ a Ser-Lys catalytic dyad (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). Notably, catalytic activity of SipW was not only essential for pre-TasA processing, but also for the incorporation of mature TasA into spores.

Amino Acid Sequence↗
PubMed2000

The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis.

The response regulator DegU is involved in various late-growth developmental processes in Bacillus subtilis, including the production of degradative enzymes and the development of genetic competence. DegU is essential for the expression of the competence transcription factor, encoded by comK. ComK is required for the transcription of genes encoding the DNA uptake and integration machinery, as well as for the transcription of its own gene. We have purified DegU to study its role in the expression of comK, and we demonstrate here that DegU binds specifically to the comK promoter. The binding of the response regulator DegU to a promoter target had not been reported previously. DNase I protection analyses show that the DegU binding site overlaps with the ComK binding site, and gel retardation experiments indicate that DegU strongly stimulates the binding of ComK to the comK promoter. We propose that DegU functions at the initiation of competence development, when ComK concentrations are insufficient to support comK transcription, by facilitating ComK binding to the comK promoter. DegU therefore acts as a priming protein that primes the autostimulatory transcription of comK. Such priming activity adds a function to the class of response regulator proteins.

Bacillus subtilis↗
PubMed2000

Expression of the neutral protease gene from a thermophilic Bacillus sp. BT1 strain in Bacillus subtilis and its natural host: identification of a functional promoter.

The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5' to the gene. In contrast, in heterologous B. subtilis this thermophilic npr promoter does not function, and expression of the npr gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5' to ORF X that is required for efficient expression of the npr gene in Bacillus subtilis as verified by primer extension, reverse transcription-PCR, and 5' rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic Bacillus sp. BT1 strain are different from those used in B. subtilis.

Bacillus↗
PubMed2000

A truncated soluble Bacillus signal peptidase produced in Escherichia coli is subject to self-cleavage at its active site.

Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of Escherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

Amino Acid Sequence↗
PubMed1999

The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase.

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a beta-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

Adenosine Monophosphate↗
PubMed1999

The potential active site of the lipoprotein-specific (type II) signal peptidase of Bacillus subtilis.

Type II signal peptidases (SPase II) remove signal peptides from lipid-modified preproteins of eubacteria. As the catalytic mechanism employed by type II SPases was not known, the present studies were aimed at the identification of their potential active site residues. Comparison of the deduced amino acid sequences of 19 known type II SPases revealed the presence of five conserved domains. The importance of the 15 best conserved residues in these domains was investigated using the type II SPase of Bacillus subtilis, which, unlike SPase II of Escherichia coli, is not essential for viability. The results showed that only six residues are important for SPase II activity. These are Asp-14, Asn-99, Asp-102, Asn-126, Ala-128, and Asp-129. Only Asp-14 was required for stability of SPase II, indicating that the other five residues are required for catalysis, the active site geometry, or the specific recognition of lipid-modified preproteins. As Asp-102 and Asp-129 are the only residues invoked in the known catalytic mechanisms of proteases, we hypothesize that these two residues are directly involved in SPase II-mediated catalysis. This implies that type II SPases belong to a novel family of aspartic proteases.

Amino Acid Sequence↗
PubMed1999

Functional analysis of paralogous thiol-disulfide oxidoreductases in Bacillus subtilis.

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.

Alkaline Phosphatase↗
PubMed1999

Signal peptide peptidase- and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins.

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.

Adenosine Triphosphatases↗