Biomedical subjects

G Venema

Publications and source records attributed to G Venema.

At least 37 records · Page 2Linked to original sources

PubMed1999

Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.

The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.

Bacillus subtilis↗

PubMed1999

Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin a maturation.

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for externalization. The secretion apparatus for the lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD. LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems. This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA. By making translational fusions of LcnC to the reporter proteins beta-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm. As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.

ATP-Binding Cassette Transporters↗

PubMed1999

The role of lipoprotein processing by signal peptidase II in the Gram-positive eubacterium bacillus subtilis. Signal peptidase II is required for the efficient secretion of alpha-amylase, a non-lipoprotein.

Computer-assisted analyses indicate that Bacillus subtilis contains approximately 300 genes for exported proteins with an amino-terminal signal peptide. About 114 of these are lipoproteins, which are retained in the cytoplasmic membrane. We have investigated the importance of lipoprotein processing by signal peptidase II (SPase II) for cellular homeostasis, using cells lacking SPase II. The results show that lipoprotein processing is important for cell viability at low and high temperatures, suggesting that lipoproteins are essential for growth under these conditions. Although certain lipoproteins are required for the development of genetic competence, sporulation, and germination, these developmental processes were not affected in the absence of SPase II. Cells lacking SPase II accumulated lipid-modified precursor and mature-like forms of PrsA, a folding catalyst for secreted proteins. These forms of PrsA seem to have a reduced activity, as the secretion of alpha-amylase was strongly impaired. Unexpectedly, type I signal peptidases, which process secretory preproteins, were not involved in alternative amino-terminal processing of pre-PrsA in the absence of SPase II. In conclusion, processing of lipoproteins by SPase II in B. subtilis is not strictly required for lipoprotein function, which is surprising as lipoproteins and type II SPases seem to be conserved in all eubacteria.

Amino Acid Sequence↗

PubMed1999

Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis.

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha-amylase (AmyL), Escherichia coli TEM beta-lactamase (Bla), human pancreatic alpha-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.

Amino Acid Sequence↗

PubMed1999

The plasmid-encoded signal peptidase SipP can functionally replace the major signal peptidases SipS and SipT of Bacillus subtilis.

The gram-positive eubacterium Bacillus subtilis is the organism with the largest number of paralogous type I signal peptidases (SPases) known. These are specified both by chromosomal and plasmid-borne genes. The chromosomally encoded SPases SipS and SipT have a major function in precursor processing, and cells depleted of SipS and SipT stop growing and die. In this study, we show that the SPase SipP, specified by the B. subtilis plasmid pTA1015, can functionally replace SipS and SipT, unlike the three chromosomally encoded SPases with a minor function in protein secretion (i.e., SipU, SipV, and SipW). Unexpectedly, SipP is not specifically required for the processing and secretion of Orf1p, which is specified by a gene that is cotranscribed with sipP. These two genes form a conserved structural module of rolling-circle plasmids from B. subtilis. As previously shown for the chromosomal sipS and sipT genes, the transcription of plasmid-borne copies of sipP is temporally controlled, reaching maximal levels during the post-exponential growth phase when the cells secrete proteins at high levels. However, increased transcription of sipP starts at the end of exponential growth, about 2 h earlier than that of sipS and sipT. These data suggest that SipP fulfills a general role in the secretory precursor processing of pTA1015-containing cells.

Bacillus subtilis↗

PubMed1999

Characterization of a novel stable biocatalyst obtained by protein engineering.

Protein engineering is a powerful tool for the improvement of the properties of biocatalysts. Previously we have applied protein engineering technologies to obtain an extremely stable variant of the thermolysin-like protease from Bacillus stearothermophilus [Van den Burg, Vriend, Veltman, Venema and Eijsink (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2056-2060]. This variant is much more resistant to denaturing conditions (temperature and denaturing agents) than the wild-type enzyme. An extensive enzymic characterization was undertaken to explore the suitability of the variant as a biocatalyst at high temperatures. By comparing a range of variants with increasing thermal stabilities we show that the additivity of the mutations is accompanied by an increase in activity at elevated temperatures in accordance with the Arrhenius law. The results suggest that the constructed protease variants could be suitable alternatives to proteases that are currently used industrially. Our studies demonstrate how protein engineering can be exploited to obtain high-performance biocatalysts.

Catalysis↗

PubMed1998

Early steps in the unfolding of thermolysin-like proteases.

Several series of site-directed mutations in thermolysin-like proteases are presented that show remarkable nonadditivity in their effect on thermal stability. A simple model is proposed that relates this nonadditivity to the occurrence of independent partial unfolding processes that occur in parallel at elevated temperatures. To prove this model, a thermolysin-like protease was designed in which two mutations located approximately 35 A apart in the structure individually exert small stabilizing effects of 2.3 and 4. 1 degreesC, respectively, but when combined stabilize the protease by 14.6 degreesC. This overadditivity, which follows directly from the model, confirms that unfolding of this engineered protease starts in parallel at two different regions of the protein.

Bacillus↗

PubMed1998

Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli.

Bacillus subtilis is one of the best known Gram-positive bacteria at both the genetic and physiological level. The entire sequence of its chromosome is known and efficient tools for the genetic modification of this bacterium are available. Moreover, B. subtilis and related Bacillus species are widely used in biotechnology, in particular for the production of secreted enzymes. Although bacilli can secrete large amounts of several native enzymes, the use of these bacteria for the production of heterologous enzymes has frequently resulted in low yields. Here we describe the identification of several components of the Bacillus protein secretion machinery. These components can now be engineered for optimal protein secretion. Special emphasis is given on type I signal peptidases, which remove signal peptides from secretory precursor proteins. Five genes specifying such enzymes (sip, for signal peptidase) are present on the B. subtilis chromosome. Although none of the sip genes is essential by itself, a specific combination of mutations in these genes is lethal. The expression pattern of some of the sip genes coincides with that of many secretory proteins, which seems to reflect an adaptation to high demands on the secretion machinery. Although the various B. subtilis type I signal peptidases have at least partially overlapping substrate specificities, clear differences in substrate preferences are also evident. These observations have implications for the engineering of the processing apparatus for improved secretion of native and heterologous proteins by Bacillus.

Bacillus↗

PubMed1998

SecDF of Bacillus subtilis, a molecular Siamese twin required for the efficient secretion of proteins.

In the present studies, we show that the SecD and SecF equivalents of the Gram-positive bacterium Bacillus subtilis are jointly present in one polypeptide, denoted SecDF, that is required to maintain a high capacity for protein secretion. Unlike the SecD subunit of the pre-protein translocase of Escherichia coli, SecDF of B. subtilis was not required for the release of a mature secretory protein from the membrane, indicating that SecDF is involved in earlier translocation steps. Strains lacking intact SecDF showed a cold-sensitive phenotype, which was exacerbated by high level production of secretory proteins, indicating that protein translocation in B. subtilis is intrinsically cold-sensitive. Comparison with SecD and SecF proteins from other organisms revealed the presence of 10 conserved regions in SecDF, some of which appear to be important for SecDF function. Interestingly, the SecDF protein of B. subtilis has 12 putative transmembrane domains. Thus, SecDF does not only show sequence similarity but also structural similarity to secondary solute transporters. Our data suggest that SecDF of B. subtilis represents a novel type of the SecD and SecF proteins, which seems to be present in at least two other organisms.

Amino Acid Sequence↗

PubMed1998

Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases.

Approximately 47% of the genes of the Gram-positive bacterium Bacillus subtilis belong to paralogous gene families. The present studies were aimed at the functional analysis of the sip gene family of B. subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, and sipW. All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins. Interestingly, strains lacking as many as four of these SPases could be obtained. As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins. Thus, SipS and SipT are of major importance for protein secretion. This conclusion is underscored by the observation that only the transcription of the sipS and sipT genes is temporally controlled via the DegS-DegU regulatory system, in concert with the transcription of most genes for secretory preproteins. Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.

Amino Acid Sequence↗

PubMed1998

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I.

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.

Bacillus subtilis↗

PubMed1998

The competence transcription factor of Bacillus subtilis recognizes short A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix.

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comG promoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of approximately 65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addAB promoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN5TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK-dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK. The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.

Bacillus subtilis↗

PubMed1998

Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.

Alanine↗

PubMed1998

A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases.

Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability. The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation. Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.

Amino Acid Sequence↗

PubMed1998

Engineering an enzyme to resist boiling.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Amino Acid Substitution↗

PubMed1998

Construction of a food-grade multiple-copy integration system for Lactococcus lactis.

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Blotting, Southern↗

PubMed1998

Identification of a sodium chloride-regulated promoter in Lactococcus lactis by single-copy chromosomal fusion with a reporter gene.

An integration vector, pORI13, was developed to screen in Lactococcus lactis for expression signals induced by changes in the environment and to assay transcriptional activity of genes in single copy. The plasmid carries a promoterless Escherichia coli lacZ gene preceded by a start codon, a lactococcal ribosome binding site, and a multiple cloning site. Chromosomal Sau3AI fragments of L. lactis MG1363 DNA were cloned in pORI13 using a RepA+ E. coli as host. The resulting bank of plasmids was used for Campbell-type integration into the chromosome of L. lactis MG1363. The relatively large size of the chromosomal fragments used increases the chance of retaining complete genes in the targeted region. Screening of integrants in the presence of 0.3 M NaCl resulted in the isolation of a clone (NS3) in which expression of lacZ was dependent on the concentration of chloride ions.

Amino Acid Sequence↗

PubMed1998

A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation.

Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB. GadC is homologous to putative glutamate-gamma-aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane-spanning domains. GadB shows similarity to glutamate decarboxylases. A L. lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L. lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate-dependent acid resistance mechanism of L. lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride-dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii. gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3kb gadCB mRNA that is found in wild-type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.

Amino Acid Sequence↗