Three-partner conversion induced by the P-element transposase in Drosophila melanogaster.

Conversion of one P-derived transposon into another has already been shown to occur with a measurable frequency. However, the mechanism responsible for such replacements has remained controversial. We previously proposed a mechanism involving three partners. We assumed that after excision of the P-element inserted at the target site, the double-strand break was repaired using, first, the homologous P sequences on the sister chromatid, and second, a remote template, the donor P-derived transposon. However, two other mechanisms have been proposed. The first involves two partners only, the broken end and the remote template, while the second involves transposition of the donor into the target P-element, followed by a double recombination event. Here we describe the conversion of a defective P-element using as a remote template an enhancer-trap element that is itself unable to transpose because it lacks 21 bp at its 5' end. This result makes it possible to exclude the possibility that this conversion event occurred after transposition. The new allele was molecularly and genetically characterized. The occurrence of a polymorphism at position 33 of the P-element sequence and of an imperfect copy of the template on the 3' side of the converted transposon confirmed that the sister chromatid was absolutely necessary as a partner for repair. Our results show that targeting of a marked P-element is possible, even when this element is unable to transpose. This provides a means of improving recovery of conversion events by eliminating unwanted transpositions catalyzed by the P transposase.

Enhancer-trap targeting at the Broad-Complex locus of Drosophila melanogaster.

Here, we describe the exact replacement of a defective unmarked P element by an enhancer-trap transposon marked by the miniwhite gene and carrying lacZ as a reporter gene. The original defective P element was located in an intron of the Broad-Complex (BRC), a key gene involved in metamorphosis. Replacement events resulted from conversions induced by the P-element transposase from a donor enhancer-trap element located on another chromosome. Six independent conversion events were selected. In all converted chromosomes, the enhancer-trap transposon was in the same orientation as the original P element. From the pattern of X-gal staining observed, lacZ expression likely reflects the regulatory influence of BRC enhancers on the convertant transposon. Reversion to wild type was achieved by excision of the enhancer-trap transposon. The six convertants were analyzed in detail at the nucleotide level. The occurrence of a polymorphism at position 33 of the P-element sequences led us to propose a conversion mechanism involving homologous P sequences for repair. This is in contrast to previously analyzed P-element transposase-induced conversion events and proposed models relying on sequence identity between genomic Drosophila sequences. The lack of any homology requirement other than between P element sequences means that our findings can be easily generalized. Targeting a marked P-element derivative at a precise site without loss or addition of genetic information makes it possible to exploit the hundreds of defective P elements scattered throughout the Drosophila genome by replacing them with engineered P elements, already available.

Developmental regulation of transcription of the Bacillus subtilis ftsAZ operon.

The products of the ftsA and ftsZ genes play a major role in septum formation in Escherichia coli. Their homologues have been found in various bacterial species, such as Bacillus subtilis where they are involved in septation during vegetative growth as well as during sporulation, a developmental process that is initiated by the formation of an asymmetrically positioned septum. Transcription of the B. subtilis ftsAZ operon was studied during exponential growth and sporulation by monitoring beta-galactosidase synthesis in strains harboring fusions of the E. coli lacZ gene with various fragments of the ftsAZ regulatory region. Transcription of the ftsAZ operon was found to be controlled by three promoters which were mapped by primer extension and characterized by their temporal pattern of expression. Two of these promoters, P1 and P3, are dependent on sigma A, the major vegetative sigma factor, and are expressed mainly during growth. The third one, P2, is recognized by sigma H associated RNA polymerase and its activity increases three- to four-fold around the onset of sporulation. The post-exponential enhancement of P2-driven transcription is abolished in a spo0A mutant but partially restored in an abrB spo0A double mutant. After inactivation by oligonucleotide-directed mutagenesis mutated copies of P1 and P2 were introduced into the chromosome upstream from the ftsAZ operon. Transformants could be obtained only when ftsAZ transcription was controlled by a combination of two intact promoters, neither P1, P2 nor P3 being essential for viability. The sporulation efficiency was found to be dependent on the level of transcription of ftsAZ, the absence of P2 still allowing 30% of the normal sporulation rate. Therefore the post-exponential burst of synthesis of the FtsA and FtsZ proteins is not an absolute requirement for the successful completion of the asymmetric septum.

The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains.

Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose-specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane-bound, polypeptide with an apparent molecular weight of 80,000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40-50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl alpha-glucoside. Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl alpha-glucoside transport but retained part of the glucose-transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS. The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non-PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non-phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: nucleotide sequence of ptsX, ptsH and the 5'-end of ptsI and evidence for a ptsHI operon.

The nucleotide sequence of a 1689bp fragment of the Bacillus subtilis locus containing ptsX (a crr-like gene), ptsH (coding for HPr), and the 5'-end of ptsI (coding for Enzyme I) was determined. The deduced amino acid sequences of ptsH and the N-terminal part of ptsI were compared to those of Streptococcus faecalis and Escherichia coli. Transcription fusion demonstrated that ptsHI constitutes an operon. An open reading frame overlapping the main part of ptsH in the opposite sense was shown to be expressed in vivo, using protein fusions with beta-galactosidase. The deduced amino acid sequence of ptsX showed significant homology with that of Salmonella typhimurium glucose-specific Enzyme III. ptsX was preceded by an open reading frame whose amino acid sequence showed strong homology with the C-terminal part of E. coli Enzyme IIGlc.

Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site.

The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins. In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.

5'-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis.

The regulation of the levansucrase gene sacB was studied in Bacillus subtilis strains. Fusions were constructed in which genes of cytoplasmic proteins such as lacZ were placed immediately downstream from sacR, the regulatory region located upstream from sacB. These fusions were introduced in mutants affected in sacB regulation. In all cases the marker gene was affected in the same way as sacB by the genetic context. This result is of particular interest for the sacU pleiotropic mutations, which affect sacB expression and other cellular functions such as the synthesis of several exocellular enzymes. We also showed that strains harboring sacU+ or sacU-hyperproducing alleles contained different amounts of sacB mRNA, which was proportional to their levansucrase secretion. We concluded that the sacU gene does not affect sacB expression at the level of secretion but acts on a target within sacR. We discuss the possibility that sacU acts on a part of sacR, a homologous copy of which was found upstream from the gene of another sacU-dependent secreted enzyme of B. subtilis, beta-glucanase.

The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites.

We present the sequence of a 2 kb fragment of the Bacillus subtilis Marburg genome containing sacB, the structural gene of levansucrase, a secreted enzyme inducible by sucrose. The peptide sequence deduced for the secreted enzyme is very similar to that directly determined by Delfour (1981) for levansucrase of the non-Marburg strain BS5. The peptide sequence is preceded by a 29 amino acid signal peptide. Codon usage in sacB is rather different from that in the sequenced genes of other secreted enzymes in B. subtilis, especially alpha-amylase. Genetic evidence has shown that the sacB promotor is rather far from the beginning of sacB (200 bp or more). The 200 bp region preceding sacB shows some of the features of an attenuator. A preliminary discussion of the putative workings and roles of this attenuator-like structure is proposed. sacRc mutations, which allow constitutive expression of levansucrase, have been located within the 450 bp upstream of sacB. It is shown that sacRc and sacR+ alleles control in cis the expression of the adjacent sacB gene.

Levansucrase of Bacillus subtilis. Characterization of a stabilized fructosyl-enzyme complex and identification of an aspartly residue as the binding site of the fructosyl group.

A covalently linked fructosyl-enzyme complex was isolated from a reaction mixture of enzyme and sucrose submitted to the quenching effect of a large decrease of the pH. The fructosyl-enzyme bond was shown to be stable under acidic and neutral conditions in the presence of high concentration of urea and of sodium dodecyl sulfate. This intermediate did not transfer at a measurable rate its fructosyl group to the usual fructosyl acceptors of the enzyme reaction under the usual conditions of enzyme activity. However stability measurements of the fructosyl-enzyme bond indicated a marked lability at pH values above 8.5. The apparent rate constant of the hydrolytic reaction of this bond evaluated under the standard state of molar concentration of hydroxide ion was of the same order of magnitude as the apparent rate constant of the hydrolytic reaction of the transient fructosyl-enzyme postulated from the kinetic analysis of levansucrase. Furthermore, nucleophilic agents like imidazole enhanced the hydrolytic reaction of the fructosyl-enzyme bond. Identification of the fructosyl binding site on the enzyme was accomplished by proteolytic hydrolysis of the trapped complex. Peptic digestion followed by pronase digestion released a fructosyl-aspartate compound that we have isolated in a high state of purity. The lability of the fructosyl-aspartate bond under mild alkaline conditions suggested that the fructosyl was linked through an ester bond involving the beta-carboxyl of the aspartate residue. Treatment of the trapped complex with cyanogen bromide released only one fructosylated peptide. The apparent molecular weight of this peptide was estimated to be lower than 10000.

Levansucrase of Bacillus subtilis: kinetic and thermodynamic aspects of transfructosylation processes.

Simple kinetic considerations derived from the ping-pong mechanism previously proposed for levansucrase of Bacillus subtillis allowed us to predict an easily operated method to approach the kinetic studies of exchange and hydrolytic activities of this enzyme. The experimental kinetic pattern obtained from the study of both activities is in close agreement with those predicted by theoretical approach. The combination of kinetic results enabled us to determine with a good accuracy the values of the apparent rate constant of the step of fructosylation of the enzyme from the sucrose-enzyme Michaelis complex and the apparent rate constants of the steps of defructosylation of the fructosyl enzyme to water or to glucose. The standard free energy reaction coordinate diagram for the transfructosylation process from sucrose to water was constructed. We found that the high energy of the glycosidic linkage of sucrose is preserved in the fructosyl-enzyme intermediate. The temperature dependence studies of the rate constants of fructosylation and defructosylation of the enzyme show that the entropy of activation for the two steps of defructosylation of the fructosyl enzyme are nearly the same. However the enthalpy of activation for the transfructosylation step to water is greater than that to glucose. We attempted to explain this discrepancy. Furthermore comparison of mechanism and efficiency of enzymatic and acid catalysis of sucrose hydrolysis was developed.

Levansucrase of Bacillus subtilis : reexamination of some physical and chemical properties.

To give some support to researchs presently in progress in this institute, on the sequence elucidation and the X-Ray pattern of the levansucrase of B. subtilis, some physical and chemical properties of this enzyme were carefully reexaminated. The results explicit and on some points rectify previous reports from this laboratory. The molecular weight was measured by three different methods: sedimentation equilibrium, SDS-gel electrophoresis, gel filtration. They give an average value of 54000 g. From this molecular weight and the value of the Stokes' radius, an estimate of the frictional ratio f/fo was calculated. These results provide some knowledge about the size and the shape of the molecule. They are consistent with the electronic microscopy observations obtained elsewhere. The amino acid composition was determined from the acid hydrolysate. The nature of the sulfur containing aminoacid was established by analysis of (35-S)-labelled levansucrase : neither cysteine nor cystine were found in the molecule. The methionine residues appear essentially under unoxidized form. One terminal residue was characterized by the dansylation method using (14-C)-labelled dansyl chloride. An explanation of the affinity of the levansucrase on hydroxyapatite was attempted.