Biomedical subjects

D van Sinderen

Publications and source records attributed to D van Sinderen.

33 records · Page 2Linked to original sources

PubMed1996

Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage r1t.

The temperate lactococcal bacteriophage r1t was isolated from its lysogenic host and its genome was subjected to nucleotide sequence analysis. The linear r1t genome is composed of 33,350 bp and was shown to possess 3' staggered cohesive ends. Fifty open reading frames (ORFs) were identified which are, probably, organized in a life-cycle-specific manner. Nucleotide sequence comparisons, N-terminal amino acid sequencing and functional analyses enabled the assignment of possible functions to a number of DNA sequences and ORFs. In this way, ORFs specifying regulatory proteins, proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and a dUTPase were putatively identified. One ORF seems to be contained within a self-splicing group I intron. In addition, the bacteriophage att site required for site-specific integration into the host chromosome was determined.

Amino Acid Sequence↗

PubMed1996

Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis.

It has been hypothesized that the dinR gene product of Bacillus subtilis acts as a repressor of the SOS regulon by binding to DNA sequences located upstream of SOS genes, including dinR and recA. Following activation as a result of DNA damage, RecA is believed to catalyse DinR-autocleavage, thus derepressing the SOS regulon. The present results support this hypothesis: a dinR insertion mutation caused a high, constitutive expression of both dinR and recA, which could not be further elevated by SOS-induction. In addition, gel-retardation assays demonstrated a direct interaction between the dinR gene product and the recA and dinR promoter regions. Epistatic interactions and gel-retardation assays demonstrated that the previously reported competence-specific expression of recA directly depended upon the gene product of comK, the competence transcription factor. These data demonstrate the existence of a direct regulatory link between the competence signal-transduction pathway and the SOS reguion.

Bacillus subtilis↗

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↗

PubMed1995

Differential expression of two closely related deoxyribonuclease genes, nucA and nucB, in Bacillus subtilis.

Despite the lack of involvement of the competence-specific, membrane-associated deoxyribonuclease (DNase) in competence development, the expression of the gene encoding this protein, nucA, was shown to be dependent on the competence signal transduction pathway, and in particular on ComK, the competence transcription factor, which was shown to bind to the DNA region upstream of nucA. The expression of nucB, specifying an extracellular DNase, which was cloned on the basis of its homology to nucA, was shown to be sporulation-specific and dependent on the gene products of spo0A and spoIIG, the latter constituting an operon responsible for the synthesis of the mother-cell-specific sigma factor sigma E. The observed differential expression of nucA and nucB demarcates the appearance of DNase activities which are either associated with the cytoplasmic membrane or secreted into the medium during different post-exponential growth-phase processes.

Amino Acid Sequence↗

PubMed1995

comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis.

comK is a positive autoregulatory gene occupying a central position in the competence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA-binding and uptake apparatus. In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK). Using portions of the region upstream of comC we show that the ComK-binding sequences are essential for the expression of competence. Moreover, we demonstrate that the presence of ComK stimulates the expression of comF-lacZ and comG-lacZ translational fusions in vivo in Escherichia coli. These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF).

Amino Acid Sequence↗

PubMed1994

Molecular cloning and sequence of comK, a gene required for genetic competence in Bacillus subtilis.

The transformation-deficient strain E26, isolated as a pHV60 insertion mutant, was used to isolate comK, a novel transcription unit required for genetic competence in Bacillus subtilis. Mutational analysis and sequence determination showed that comK contained one open reading frame (ORF), which could encode a protein of 192 amino acid residues with a predicted molecular weight of 22,500. An integrated copy of comK not only complemented the competence deficiency of a comK deletion mutant, but also that of strains E26 and FB93. Expression of comK occurred exclusively in glucose-based minimal medium during the transition to stationary growth phase. Furthermore, the expression of late competence genes appeared to be dependent on the gene product of comK, the expression of which in turn depended on the presence of a functional comL (or srfA) transcription unit. These epistatic interactions indicate that comK is a competence locus occupying an intermediate position in the competence signal transduction network. Primer extension analysis showed that comK has one major transcription start site, preceded by a sequence resembling the consensus promoter used by the sigma A form of RNA polymerase.

Amino Acid Sequence↗

PubMed1994

comK acts as an autoregulatory control switch in the signal transduction route to competence in Bacillus subtilis.

The comK gene is a regulatory transcription unit which is essential for the development of genetic competence in Bacillus subtilis. The transcription of comK is under strict nutritional and growth phase-dependent control and has been shown to depend on the gene products of comA and srfA. In this report, we show that expression of comK is dependent on its own gene product as well as on the gene products of all other tested regulatory genes known to be involved in competence development (abrB, comA, comP, degU, sin, spo0A, spo0H, spo0K, and srfA). A mecA mutation is able to suppress the competence deficiency of mutations in any of these regulatory loci except for mutations in spo0A and, as we show here, in comK. Furthermore, we show that the presence of comK on a multiple copy plasmid leads to derepression of comK expression, causing an almost constitutive expression of competence in minimal medium as well as permitting competence development in complex medium. We infer from these results that the signals which trigger competence development, after having been received and processed by the various components of the competence signal transduction pathway, all converge at the level of comK expression. As soon as derepression of comK expression occurs, the positive autoregulation rapidly results in accumulation of the comK gene product, which subsequently induces competence.

Bacillus subtilis↗

PubMed1993

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis.

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilobases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.

Amino Acid Isomerases↗

PubMed1993

Characterization of the srfA locus of Bacillus subtilis: only the valine-activating domain of srfA is involved in the establishment of genetic competence.

srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5' portion of the srfA operon affect all three of these processes, whereas mutations in the 3' portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid-dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colinearity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5' region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.

Amino Acid Sequence↗

PubMed1990

An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis.

The recently described beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis [Haima et al., Gene 86 (1990) 63-69]was optimized in several ways. First, the efficiency of translation of the lac Z delta M15 gene was improved. Second, the plasmid-borne lacZ delta M15 gene was segregationally stabilized by integration into the B. subtilis chromosome. Third, a new lacZ alpha complementing cloning vector was constructed, containing more unique target sites. It was shown that large heterologous DNA fragments (up to at least 29 kb) could be cloned with lacZ alpha-complementing vectors carrying the replication functions of the cryptic B. subtilis plasmid pTA1060, and that these inserts were structurally stably maintained for at least 100 generations of growth.

Bacillus subtilis↗

PubMed1990

Development of a beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis.

A versatile beta-galactosidase alpha-complementation system for Bacillus subtilis was developed, which can be used for molecular cloning in this Gram+ organism. The cloning system, which is based on the highly efficient host-vector system 6GM-pHP13, offers several advantages over previously described systems: (1) convenient direct selection of recombinants; (2) the cloning of large heterologous DNA fragments with high efficiency; and (3) the availability of six unique target sites: SphI, NdeI, NheI, BamHI, SmaI and EcoRI.

Bacillus subtilis↗

PubMed1990

Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis.

Using the transformation-deficient mutant M465, which was previously isolated by means of insertional mutagenesis with plasmid pHV60, a transcription unit comL required for genetic competence of Bacillus subtilis was identified. A chromosomal DNA fragment flanking the inserted pHV60 was isolated and used to screen two different libraries of B. subtilis DNA in phage lambda EMBL4 and lambda EMBL12, respectively. With the aid of six recombinant phages that hybridize with this chromosomal fragment a restriction map of about 23 kb of B. subtilis chromosomal DNA was constructed. Using small adjoining pieces of this chromosomal DNA in Campbell integrations, the size of the transcription unit involved in competence development could be delimited to about 15 kb. By insertion of a promoterless lacZ gene into comL, the transcriptional regulation of comL was analysed and epistatic interactions among various other com genes were determined. The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn is dependent on the gene products of comA and comB.

Bacillus subtilis↗

PubMed1987

Cell Wall-Associated Proteases of Streptococcus cremoris Wg2.

Two components of the proteolytic system, proteins A and B (J. Hugenholtz, F. Exterkate, and W. N. Konings, Appl. Environ. Microbiol. 48:1105-1110, 1984), have been studied in Streptococcus cremoris Wg2 by immunological methods. The components could not be separated by standard chromatography techniques because both proteins had almost identical molecular weights (about 140,000) and isoelectric points (pH 4.5). Specific antibodies were raised against proteins A and B by excision of the different immunoprecipitates from crossed immunoelectrophoresis gels. With these antibodies, protein A or B was removed from solutions containing both proteins. The purified proteins A and B possessed proteolytic activity and were inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Each of these proteins accounted for approximately 50% of the total proteolytic activity isolated from S. cremoris Wg2. The specific antibodies against the proteases were also used for immuno-gold labeling studies. The proteases were clearly seen to be located at the outside of the cell wall. The proteases had the same location when the genetic information coding for the proteases was cloned in Streptococcus lactis and Bacillus subtilis.

Journal Article↗

PubMedDate not supplied

From DNA sequence to application: possibilities and complications.

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF's) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Bacteriophages↗