Biomedical subjects

G Venema

Publications and source records attributed to G Venema.

At least 19 recordsLinked to original sources

PubMed1992

Increasing the thermostability of the neutral proteinase of Bacillus stearothermophilus by improvement of internal hydrogen-bonding.

In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of Ser-170 could contribute to stability by filling an internal cavity. After the introduction of the mutation, using site-directed-mutagenesis techniques, an increase in stability of 0.7 +/- 0.1 degrees C was obtained.

Alanine↗

PubMed1992

Plasmid maintenance in Bacillus stearothermophilus is strain-dependent.

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

DNA Replication↗

PubMed1992

Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis.

Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot analysis it was found that all monoclonal antibodies recognize all denatured proteinase components. The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule. In a L. lactis proteinase which was inactivated by the substitution Asp30----Asn under native conditions, only one epitope could be detected. This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies.

Amino Acid Sequence↗

PubMed1992

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

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

Amino Acid Sequence↗

PubMed1992

Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases.

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr-ste) a 10 residue beta-hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the beta-hairpin was deleted from Npr-ste and inserted into Npr-sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the beta-hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the interaction between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis.

Amino Acid Sequence↗

PubMed1992

Lysozyme expression in Lactococcus lactis.

Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and lambda were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.

Bacteriophage lambda↗

PubMed1992

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

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

Amino Acid Sequence↗

PubMed1992

Protein export elements from Lactococcus lactis.

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

Amino Acid Sequence↗

PubMed1992

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

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

Bacillus subtilis↗

PubMed1992

Introduction of a stabilizing 10 residue beta-hairpin in Bacillus subtilis neutral protease.

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.

Amino Acid Sequence↗

PubMed1992

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of alpha-helices.

The 247-260 and 289-299 alpha-helices of Bacillus subtilis neutral protease have a lysine in their N-terminal turn. These lysines were replaced by Ser or Asp in order to improve electrostatic interactions with the alpha-helix dipole. After replacing Lys by Ser at positions 249 or 290, the thermostability of the enzyme was increased by 0.3 and 1.0 degrees C, respectively. The Asp249 and Asp290 mutants exhibited a stabilization of 0.6 and 1.2 degrees C, respectively. The results show the feasibility of stabilizing enzymes by introducing favourable residues at the end of alpha-helices.

Amino Acid Sequence↗

PubMed1992

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease.

Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B. thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284----Trp and Met168----Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4 degrees C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core.

Base Sequence↗

PubMed1992

Gene expression in Lactococcus lactis.

Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis. A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis. This feature allowed the expression of a number of L. lactis-derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.

Base Sequence↗

PubMed1992

Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6.

On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp. cremoris 9B4, a third bacteriocin determinant was identified. The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). The fragment was sequenced and analyzed by deletion and mutation analyses. The bacteriocin determinant consisted of two genes which were transcribed as an operon. The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity. The second gene (lciB) contained 91 codons and was responsible for immunity. The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6. Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6. This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene. When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.

Amino Acid Sequence↗

PubMed1992

Characterization of transcription initiation and termination signals of the proteinase genes of Lactococcus lactis Wg2 and enhancement of proteolysis in L. lactis.

The transcription initiation signals of the prtP and prtM genes specifying the proteolytic activity of Lactococcus lactis subsp. cremoris Wg2 were mapped by primer extension. The strength of these promoters was analyzed with promoter-screening vector pGKV410, and they appeared to be weaker than previously isolated promoters of strain Wg2. In addition, a putative transcription terminator downstream of the prtP gene was characterized by using the terminator-screening vector pGKV259. The putative terminator decreased the transcription activity of lactococcal promoter P59 by approximately 70% in both Bacillus subtilis and L. lactis. Deletion of a part of the stem-loop structure of the terminator decreased the negative effect on transcription, indicating that the structure could indeed function as a terminator of transcription. The proteolytic activity of the lactococcal host was enhanced by placing the originally oppositely oriented prt genes in tandem and replacing the relatively weak promoters upstream of the prt genes with the stronger promoter, P32, from the chromosome of L. lactis Wg2.

Bacterial Proteins↗

PubMed1992

Nucleotide sequence of the Synechococcus sp. PCC7942 hemE gene encoding the homologue of mammalian uroporphyrinogen decarboxylase.

We have determined the complete nucleotide sequence of a Synechococcus sp. PCC7942 gene encoding the homologue of mammalian uroporphyrinogen decarboxylase (UROD). The gene, designated hemE, encoded a polypeptide of 354 amino acids with a molecular weight of 39,283. The primary sequences of the polypeptide encoded by hemE and human and rat UROD had 32.5% identical amino acid residues. No invariant cysteine residues were found, despite the fact that UROD isolated from different sources has been shown to be inhibited by sulfhydryl reagents. The knowledge of the primary structure of this cyanobacterial protein may be helpful in better understanding the structural alterations and functional abnormalities of UROD in patients suffering from Porphyria Cutanea Tarda (PCT).

Amino Acid Sequence↗

PubMed1992

The glgB gene from the thermophile Bacillus caldolyticus encodes a thermolabile branching enzyme.

We have cloned the structural gene for the Bacillus caldolyticus glycogen branching enzyme (glgB) in Escherichia coli. The glgB gene consisted of a 1998 bp open reading frame (ORF) encoding a 78,087 Da protein, which was highly similar to the Bacillus stearothermophilus branching enzyme. The 5' end of a second gene that encoded a protein with extensive similarity to E. coli ADP-glucose pyrophosphorylase (ADPGP) partly overlapped the 3' end of the glgB gene. A putative promoter recognized by Bacillus subtilis RNA polymerase containing the sigma factor H (E-sigma H) preceded the genes. These data suggest that in contrast to the situation observed in B. stearothermophilus, the genes involved in glycogen synthesis in B. caldolyticus are clustered on the chromosome, and are presumably coordinately expressed during the early stages of sporulation. An incomplete third gene started upstream of B. caldolyticus glgB. This gene was highly similar to a gene found directly upstream of B. stearothermophilus glgB, which encodes a putative membrane protein with unknown function. The B. caldolyticus glgB gene was expressed in E. coli and B. subtilis. Surprisingly, the branching enzyme appeared to be thermolabile, the temperature of optimal activity being only 39 degrees C.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1991

A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis.

Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression. Mutations were made such that the DNA sequence upstream of the ATG start codon was not changed. Moreover, care was taken that the substitutions, which were all within the first six codons, neither affected the amino acid sequence of the gene product nor introduced codons rarely used in L. lactis. The results suggest that mRNA secondary structure contributes to the efficiency of translation initiation in L. lactis.

Base Sequence↗