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J Delcour

Publications and source records attributed to J Delcour.

At least 37 records · Page 2Linked to original sources
PubMed1994

NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization.

A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD(+)-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched beta-carbon.

Alcohol Oxidoreductases↗
PubMed1994

The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 3'-terminal loop of 18 S rRNA is essential in yeast.

Biogenesis of cytoplasmic ribosomes universally involves methylation of ribosomal RNA. Little genetic evidence is available about the functional role(s) of this conserved posttranscriptional modification. The only known methylase gene involved in rRNA maturation is ksgA in Escherichia coli, which directs dimethylation of two adjacent adenosines (m6(2)A1518m6(2)A1519) in the loop of a conserved hairpin near the 3'-end of 16 S rRNA. This tandem methylation is the only rRNA modification common to pro and eukaryotes. Disruption of ksgA confers resistance to the aminoglycoside antibiotic kasugamycin without significantly impairing viability. Here we report the cloning of the DIM1 gene encoding the homolog 18 S rRNA dimethylase in Saccharomyces cerevisiae. The yeast enzyme is evolutionary related to the ksgA protein. It carries a distinctive lysine-rich-N-terminal extension with a potential protein kinase C phosphorylation site. Like ksgA, DIM1 belongs to the erm family of prokaryotic 23 S rRNA dimethylases responsible for erythromycin resistance. Surprisingly, disruption of DIM1 turns out to be lethal in yeast.

Adenosine↗
PubMed1994

IS231 and other Bacillus thuringiensis transposable elements: a review.

Bacillus thuringiensis is an entomopathogenic bacterium whose toxicity is due to the presence in the sporangia of delta-endotoxin crystals active against agricultural pests and vectors of human and animal diseases. Most of the genes coding for these toxin proteins are plasmid-borne and are generally structurally associated with insertion sequences (IS231, IS232, IS240, ISBT1 and ISBT2) and transposons (Tn4430 and Tn5401). Several of these mobile elements have been shown to be active and are believed to participate in the crystal gene mobility, thereby contributing to the variation of bacterial toxicity. Structural analysis of the iso-IS231 elements indicates that they are related to IS1151 from Clostridium perfringens and distantly related to IS4 and IS186 from Escherichia coli. Like the other IS4 family members, they contain a conserved transposase-integrase motif found in other IS families and retroviruses. Moreover, functional data gathered from IS231A in Escherichia coli indicate a non-replicative mode of transposition, with a marked preference for specific targets. Similar results were also obtained in Bacillus subtilis and B. thuringiensis, and a working model for DNA-protein interactions at the target site is proposed.

Amino Acid Sequence↗
PubMed1994

IS231A insertion specificity: consensus sequence and DNA bending at the target site.

In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430. Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli. Sequence analysis reveals that IS231A insertion sites match the 5'-GGG(N)5CCC-3' consensus. However, this consensus is not the only determinant of IS231A insertion specificity. Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random. We demonstrate that this preference is determined by the flanking regions of the site. These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly 'S'-shaped structure centered on the target consensus. DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions. We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA.

Bacillus thuringiensis↗
PubMed1994

Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus alpha-amylase gene.

An amylolytic Lactobacillus plantarum silage strain with the starch-degrading ability displayed by Lactobacillus amylovorus was developed. An active fragment of the gene coding for alpha-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp80 at the cbh locus. The alpha-amylase gene fragment was also introduced into L. plantarum Lp80 on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp80 derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp80 derivative containing the L. amylovorus alpha-amylase gene fragment integrated into the host chromosome secreted alpha-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell density than either the parent strain or L. amylovorus in MRS medium containing a mixture of starch and glucose as the fermentable carbohydrate source. This recombinant alpha-amylolytic L. plantarum strain would therefore seem to have considerable potential as a silage inoculant for crops such as alfalfa, in which water-soluble carbohydrate levels are frequently low but starch is present as an alternative carbohydrate source.

Amino Acid Sequence↗
PubMed1994

Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression.

The genuine alpha-amylase gene from Bacillus licheniformis (amyL) is not expressed in Lactobacillus plantarum, but replacement of the amyL promoter by a strong L. plantarum promoter leads to efficient expression of the gene and secretion of more than 90% of the alpha-amylase into the culture supernatant. A series of L. plantarum genetic cassettes (transcription and translation with or without secretion) were cloned by translation fusion of random DNA fragments to the silent amyL coding frame in the pGIP212 probe vector (P. Hols, A. Baulard, D. Garmyn, B. Delplace, S. Hogan, and J. Delcour, Gene 118:21-30, 1992). Five different cassettes were sequenced and found to harbor genetic signals similar to those of other gram-positive bacteria. The functions of the cloned cassettes and the cassettes isolated previously from Enterococcus faecalis were compared in E. faecalis and L. plantarum, respectively. All signals were well recognized in L. plantarum, but cassettes isolated from L. plantarum led to a low level of amylase production in E. faecalis, suggesting that the L. plantarum signals are more species specific. Six transcriptional or translational fusions were constructed to express the Bacillus subtilis levanase gene (sacC) in L. plantarum. All of these constructions were capable of inducing levanase production and secretion in the culture supernatant, and, furthermore, L. plantarum strains harboring the most efficient fusions could grow in MRS medium containing inulin as the major carbon source. Finally, a two-step chromosomal integration procedure was used to achieve efficient stabilization of an amylase construction without any residual resistance marker or vector sequence.

Amino Acid Sequence↗
PubMed1994

Lactobacillus plantarum ldhL gene: overexpression and deletion.

Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged.

Amino Acid Sequence↗
PubMed1993

Sequence of a gene coding for a cytoplasmic alcohol dehydrogenase from Kluyveromyces marxianus ATCC 12424.

Using a Saccharomyces cerevisiae ADH1 probe, a gene coding for a cytoplasmic alcohol dehydrogenase from Kluyveromyces marxianus ATCC 12424 (formerly K. fragilis) has been cloned. This gene is able to restore alcoholic fermentation in an ADH-null strain of S. cerevisiae and its encoded protein shows strong similarity with other yeast alcohol dehydrogenases (from S. cerevisiae, Schizosaccharomyces pombe and its close relative K. lactis). The product of the gene expressed in S. cerevisiae co-migrates on native gel with a K. marxianus ADH isozyme which is more active in cells growing on non-fermentable carbon sources than on glucose. This behaviour is in contrast with that of the two cytoplasmic ADH isozymes of K. lactis which are both more active in glucose-growing than in ethanol-growing cells.

Alcohol Dehydrogenase↗
PubMed1993

IS231V and W from Bacillus thuringiensis subsp. israelensis, two distant members of the IS231 family of insertion sequences.

IS231 constitutes a family of related insertion sequences (IS) from Bacillus thuringiensis. Two new IS231-related elements, IS231V and IS231W, have been isolated from the 72-MDa plasmid of B. thuringiensis subsp. israelensis. These closely related 1964-bp IS are delimited by 22-bp imperfect inverted repeats strongly similar to those of the other iso-IS231. Although the other known IS231 harbor a single long open reading frame (ORF), IS231V and W display two slightly overlapping ORF on the same DNA strand. They show about 50% identity with the transposase of the other iso-IS231. A frameshifting model is proposed for the synthesis of a fusion product which would constitute their active transposase.

Amino Acid Sequence↗
PubMed1993

The IS4 family of insertion sequences: evidence for a conserved transposase motif.

The eight IS231 variants characterized so far (IS231 A-F, V and W) display similar transposases with an overall 40% identity. Comparison with all the prokaryotic transposable elements sequenced so far revealed that the IS231 transposases share two conserved regions with those of 35 other insertion sequences of wide origins. These insertion sequences, defining the IS4 family, have a common bipartite organization of their ends and are divided into two similarity groups. Interestingly, the transposase domains conserved within this family display similarities with the well known integrase domain shared by transposases of the IS3 and IS15 families, and integrases of retroelements. This domain is also found in IS30-related elements and Tn7 TnsB protein. Amino acid residues conserved throughout all these prokaryotic and eukaryotic mobile genetic elements define a major transposase/integrase motif, likely to play an important role in the transposition process.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of genetic expression and secretion signals from Enterococcus faecalis through the use of broad-host-range alpha-amylase probe vectors.

We have constructed two broad-host-range Gram+/Gram- probe vectors designed for the cloning of bacterial genetic expression and secretion signals. These vectors make use of a silent reporter gene encoding the mature alpha-amylase from Bacillus licheniformis whose reactivation can easily be monitored on iodine-stained starch plates. Shotgun cloning of Enterococcus faecalis DNA fragments allowed recovery of several cassettes directing transcription, translation of the reporter gene and secretion of alpha-amylase. Sequence analysis revealed, in each case, the presence of a putative promoter, ribosome-binding site and signal peptide similar to those described in other Gram+ bacteria.

Amino Acid Sequence↗
PubMed1992

A strategy to construct vector-free amylolytic strains through nondisruptive homologous recombination: application to Enterococcus faecalis.

In order to direct chromosomal integration of the alpha-amylase-encoding gene from Bacillus licheniformis (amyL) under the control of expression and secretion signals from Enterococcus faecalis, the chromosomal fragment (named AB) from the pGIP3124 plasmid [Hols et al., Gene 118 (1992) 21-30] was chosen and split into two fragments (A and B). A translation fusion between the A fragment and 'amyL, deleted of its expression and secretion signals, was made and this fusion was flanked with the AB fragment at its right end. The A::'amyL:AB integration module was cloned into a thermosensitive pE194 replicon (chloramphenicol resistant; CmR) and electro-transformed into E. faecalis OG1X. After an overnight culture in selective liquid medium, the offspring from the amylolytic transformants obtained was shown to yield CmR colonies with two distinct halo sizes on iodine-stained starch plates. Southern analysis clearly showed that the smaller halos corresponded to descendants in which the plasmid had integrated into the chromosome through homologous recombination. One such Amy+ integrant in the AB site was further cultured under nonselective conditions at 42 degrees C for about 20 generations, and the offspring was screened for Amy+/CmS clones. Such revertants were indeed found, and Southern analysis clearly showed that the vector matrix had been excised through homologous recombination between the redundant A sites, leaving the integrated amy gene intact.

Amino Acid Sequence↗
PubMed1992

IS231D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family.

IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231F). Like the previously described IS231A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478-amino-acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso-IS231 terminal inverted repeats are strongly conserved 20 bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231F is bordered by a 12 bp direct repeat. No direct repeats were found flanking IS231D or IS231E.

Amino Acid Sequence↗
PubMed1991

Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli.

A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive cloned showed LDH activity and production of D(-)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var. marxianus ATCC 12424.

Cell wall inulinase (EC 3.2.1.7) was purified from Kluyveromyces marxianus var. marxianus (formerly K. fragilis) and its N-terminal 33-amino acid sequence was established. PCR amplification of cDNA with 2 sets of degenerate primers yielded a genomic probe which was then used to screen a genomic library established in the YEp351 yeast shuttle vector. One of the selected recombinant plasmids allowed an invertase-negative Saccharomyces cerevisiae mutant to grow on inulin. It was shown to contain an inulinase gene (INU 1) encoding a 555-amino acid precursor protein with a typical N-terminal signal peptide. The sequence of inulinase displays a high similarity (67%) to S. cerevisiae invertase, suggesting a common evolutionary origin for yeast beta-fructosidases with different substrate preferences.

Amino Acid Sequence↗
PubMed1991

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.

Bacillus thuringiensis↗
PubMed1988

Cloning and partial characterization of three small cryptic plasmids from Bacillus thuringiensis.

The strain H1.1 of Bacillus thuringiensis var. thuringiensis harbors three small cryptic plasmids: pGI1, pGI2, and pGI3 (8.2, 9.2, and 10.6 kb, respectively). Two of these plasmids (i.e., pGI2 and pGI3) were successfully cloned in their entirety into the vector pBR322, whereas only overlapping DNA fragments covering pGI1 were obtained in Escherichia coli. A curing-hybridization technique was used to obtain isolates of B. thuringiensis missing one or another small cryptic plasmid. These derivatives were examined for any change in a phenotypic trait, but no specific function could be assigned to one of these plasmids. Hybridization and restriction mapping data revealed that the transposon Tn4430 accounts for 45% of the pGI2 plasmid DNA.

Bacillus thuringiensis↗