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B Vosman

Publications and source records attributed to B Vosman.

33 records · Page 2Linked to original sources
PubMed1997

Molecular characterization of GATA/GACA microsatellite repeats in tomato.

Microsatellite repeats like GATA or GACA display a degree of variability that allows their use in cultivar identification. Southern hybridization with oligonucleotide probes complementary to these microsatellites were used for the detection of polymorphisms. To understand the molecular structure of the detected DNA, fragments hybridizing to GATA and GACA probes were cloned and sequenced. In the four clones analyzed, repeats of GATA and GACA were found intertwined. The GATA and GACA arrays were not perfect but were heavily degenerated, in that they contained many tetranucleotides that might have been derived by a single point mutation from GATA or GACA. Some of these derived sequences, like GGTA and GGAT, were present as relatively long stretches that also contained some point mutations. This supports the hypothesis that long stretches of repeats are stabilized by the accumulation of point mutations. Analysis of the flanking sequences of the fragments obtained with the GACA probe showed that one of them was homologous to a Lilium henryi retrotransposon and the other to a sequence upstream of a potato patatin gene. The two fragments obtained using the GATA probe were flanked by DNA that had no homology to any known sequence but they were highly homologous to each other. This DNA was frequently associated with GATA elements and was present in the tomato genome in approximately 4300 copies. The function of this new class of repetitive DNA, here termed U30, is presently unknown.

Base Sequence↗
PubMed1995

Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene.

The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signal sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disulphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.

Acinetobacter calcoaceticus↗
PubMed1995

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase.

Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A. calcoaceticus BD413 is proposed.

Acinetobacter calcoaceticus↗
PubMed1995

GATA- and GACA-repeats are not evenly distributed throughout the tomato genome.

This paper describes the distribution of highly polymorphic GATA- and GACA-containing DNA regions in tomato. To study the distribution of these polymorphic regions, a mapping experiment was done. The segregation of 32 GATA- and GACA-containing loci was analyzed in a F2 population from a cross between Lycopersicon esculentum and L. pennellii. From these loci, 28 could be mapped to 8 of the 12 tomato chromosomes. Both the GATA- and GACA-containing loci seem to cluster in the same chromosomal regions. To our knowledge, this is the first report on mapping of GATA- and GACA-containing loci in plants.

Base Sequence↗
PubMed1994

A gene of Acinetobacter calcoaceticus BD413 encodes a periplasmic peptidyl-prolyl cis-trans isomerase of the cyclophilin sub-class that is not essential for growth.

Downstream of the Acinetobacter calcoaceticus estA gene, encoding a cell-bound esterase, an open reading frame (orf) was identified, which may encode a protein with a mass of 20.4 kDa. This protein shows extensive similarity to both prokaryotic and eukaryotic peptidyl-prolyl cis-trans isomerases (PPIases) of the cyclophilin sub-class, especially to the periplasmic rotamase (RotA) of Escherichia coli. A putative signal sequence suggests that the product of the Acinetobacter gene, we termed rotA, is located outside the cytoplasm. Transcription of the gene is initiated from a promoter, just upstream of the rotA orf. The observation that two A. calcoaceticus rotA deletion mutants display no apparent mutant phenotype, suggests that this PPIase is not essential for growth of the organism. These mutants, to our knowledge, are the first prokaryotic PPIase mutants reported.

Acinetobacter calcoaceticus↗
PubMed1993

Regulation of the expression of the Pseudomonas stutzeri recA gene.

With the aid of recA-lacZ fusion strains, the in vivo regulation of the Pseudomonas stutzeri recA gene has been studied. It is shown that expression of this gene can be induced with a variety of DNA damaging agents, as well as with agents that interfere with DNA replication. For this induction, the presence of an active RecA protein is essential. Sequence analysis of the promoter region of the P. stutzeri recA gene showed that its open reading frame is preceded by an SOS-box, suggesting a regulation of its expression, similar to the regulation of recA expression in Escherichia coli.

Amino Acid Sequence↗
PubMed1993

Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases.

Acinetobacter calcoaceticus BD413, when grown in batch culture in nutrient broth, produces both extracellular lipase activity and cell-bound esterase activity during and after the transition between exponential growth and the stationary phase. From a library of A. calcoaceticus DNA in Escherichia coli, plasmids were isolated that enabled E. coli to grow on media with tributyrin as the sole carbon source. Assays with model substrates classified the product of the cloned gene as an esterase. Via deletion analysis, the esterase gene was mapped on a 1.8 kbp chromosomal DNA fragment. This fragment was sequenced and found to contain one open reading frame, termed estA, which encodes a protein of 40.0 kDa. The amino acid sequence of this protein shows homology to a number of lipolytic enzymes, most notably to esterases. Deletion of estA only partially abolished cell-bound esterase activity in A. calcoaceticus, indicating that BD413 forms at least two esterases. Both esterases show the same temporal regulation of expression. beta-Galactosidase activity was measured in strains in which a promoterless lacZ gene was inserted into estA. Induction of lacZ expression in these strains also occurred at the end of exponential growth in batch cultures, indicating that production of the esterase is regulated at the genetic level.

Acinetobacter calcoaceticus↗
PubMed1993

Physiological characterization of natural transformation in Acinetobacter calcoaceticus.

Acinetobacter calcoaceticus BD413 develops competence for natural transformation immediately after the start of the exponential growth-phase and remains competent up to e few hours into the stationary phase, after which competence gradually declines. The transformation frequencies obtained strongly depend on the kind of transforming DNA and the incubation time with DNA. Up to 25% of the cells in a culture can be transformed. DNA uptake in Acinetobacter does not display sequence specificity, is Mg(2+)-, Mn(2+)- or Ca(2+)-dependent and is uncoupler sensitive. The transforming DNA enters the cells in single-stranded form. These properties constitute a unique combination, not previously observed in other bacteria, and make A. caloaceticus ideally suited for detailed studies of the bioenergetics of DNA translocation.

Acinetobacter calcoaceticus↗
PubMed1992

Characterization of transformation-deficient mutants of Acinetobacter calcoaceticus.

Three Acinetobacter calcoaceticus transformation-deficient mutants, obtained by insertional mutagenesis with the nptll gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100-1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal DNA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA-deficient strain.

Acinetobacter calcoaceticus↗
PubMed1991

Molecular cloning and functional characterization of a recA analog from Pseudomonas stutzeri and construction of a P. stutzeri recA mutant.

A recombinant plasmid carrying the Pseudomonas stutzeri recA gene was isolated by complementation of the Escherichia coli recA13 mutation. Subcloning experiments showed that the gene was located on a 1500 bp PvuII-BglII fragment. The cloned gene complements an E. coli recA mutant for resistance to Methylmethanosulphonate (MMS) and UV irradiation. It was also capable of restoring the recombination proficiency in that mutant. The cloned fragment was used to construct a recA deletion mutant of P. stutzeri. This mutant too was shown to be sensitive towards MMS and UV irradiation. The mutant strain was found to be completely deficient in natural transformation with chromosomal DNA, due to the impairment in homologous recombination.

Chromosome Deletion↗
PubMed1988

Cloning and characterization of a Bacillus subtilis transcription unit involved in ATP-dependent DNase synthesis.

By insertional mutagenesis, 36 transformation-deficient, mitomycin C-sensitive Bacillus subtilis mutants were isolated, 16 of which were ATP-dependent DNase (ADD) deficient. PBS1 transduction showed that the mutations were closely linked to the metD marker and weakly linked to the glyB marker. With the aid of one of the mutants, a transcription unit involved in ADD synthesis was cloned. The chromosomal location of the transcription unit was established at the restriction site level by determining the presence or absence of ADD in transformants of wild-type cells obtained with various DNA fragments inserted in pUC derivatives. The transcription unit complemented a mutant in which the add transcription unit had been deleted.

Bacillus subtilis↗
PubMed1988

Transformation in Bacillus subtilis: involvement of the 17-kilodalton DNA-entry nuclease and the competence-specific 18-kilodalton protein.

A protein complex, consisting of a 17-kilodalton (kDa) nuclease and an 18-kDa protein, is believed to be involved in the binding and entry of donor DNA during transformation of Bacillus subtilis (H. Smith, K. Wiersman, S. Bron, and G. Venema, J. Bacteriol. 156:101-108, 1983). In this paper, the nucleotide sequences of the genes encoding both the nuclease and the 18-kDa protein are presented. The genes are encoded by a 904-base-pair PstI-HindIII fragment. The open reading frames encoding both proteins are partly overlapping. A B. subtilis mutant was constructed by insertion of a Cmr marker into the gene encoding the nuclease. This mutant lacked the competence-specific nuclease activity and the 18-kDa protein but retained 5% residual transformation. The total DNA association of the mutant was higher than that of the wild-type cells, and DNA entry was reduced to 30% of the wild-type level. These results suggest that an alternative pathway exists for the internalization of transforming DNA. A mutant, exclusively deficient for the 18-kDa protein, previously suggested to be involved in the binding of transforming DNA, was constructed by insertion of a kanamycin resistance gene into the coding sequence of the gene. Since the mutant showed wild-type DNA-binding activity, the 18-kDa protein is probably not involved in the binding of donor DNA to competent cells. The transforming activity of the mutant was reduced to 25% of the wild-type level, indicating that the 18-kDa protein has a function in the transformation process. In vitro experiments showed that the 18-kDa protein is capable of inhibiting the activity of the competence-specific nuclease. Its possible role in transformation is discussed.

Amino Acid Sequence↗
PubMed1987

Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis.

With the aim of cloning genes involved in transformation of Bacillus subtilis, a set of transformation-deficient mutants was isolated by means of insertional mutagenesis with plasmid pHV60 (Vosman et al., 1986). Analysis of these mutants showed that those mapping in the aroI region lacked the DNA-entry nuclease activity. Plasmid pHV60 derivatives, containing flanking chromosomal DNA fragments, were isolated from these mutants and were used to screen a library of B. subtilis chromosomal DNA in phage lambda EMBL4. In Escherichia coli lysates, prepared with the phages that hybridized to the pHV60-based probe, a prominent nuclease activity could be detected. The nuclease encoded by the phage DNA had the same Mr as the B. subtilis DNA-entry nuclease and its activity was strongly stimulated by Mn2+, which is also characteristic for the B. subtilis DNA-entry nuclease. From these results it was concluded that the gene specifying the B. subtilis DNA-entry nuclease had been cloned. It was shown that the nuclease activity was specified by a 700-bp EcoRI-PstI fragment.

Bacillus subtilis↗
PubMed1986

Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism.

Using plasmid pHV60, which contains a chloramphenicol resistance (Cmr) gene that is expressed in Bacillus subtilis, a set of transformation-deficient strains of B. subtilis was isolated by insertional mutagenesis. When chromosomal DNA from these mutants was used to transform a transformation-proficient B. subtilis strain, almost all of the Cmr transformants had the mutant phenotype as expected. However, with a frequency of approximately 3 X 10(-4) atypical transformants with the wild-type phenotype were produced. Data concerning amplification of the DNA containing the Cmr marker and duplication of DNA sequences are presented that suggest that these atypical transformants are the result of a Campbell-like integration of the chromosomal DNA containing the integrated plasmid. Transductional mapping showed that in the atypical transformants the vector-containing DNA had a strong tendency to integrate at sites adjacent to the original site of integration, although integration at sites elsewhere on the chromosome was also observed. The production of atypical transformants is explained on the basis of integration of chromosomal DNA by a Campbell-like mechanism. Circularization of vector-containing chromosomal DNA is thought to occur through joining of the extremities of single-stranded DNA molecules by fortuitous base pairing with an independently entered single-stranded DNA molecule.

Bacillus subtilis↗