A gene encoding a tyrosine tRNA synthetase is located near sacS in Bacillus subtilis.

Within the frame of an attempt to sequence the whole Bacillus subtilis genome, a region of 5.5 kbp of the B. subtilis chromosome near the sacS locus has been sequenced. It contains five complete coding sequences, including the sequence of sacY, three unknown CDS and a sequence coding for a tyrosine tRNA synthetase. That the corresponding CDS encodes a functional synthetase has been demonstrated by complementation of an Escherichia coli mutant possessing a thermosensitive tRNA synthetase. Insertion of a kanamycin resistance cassette in the B. subtilis chromosome at the corresponding locus resulted, however, in no apparent phenotype, demonstrating that this synthetase is dispensable. Finally phylogenetic relationships between known tyrosine and tryptophan tRNA synthetases are discussed.

Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU.

The rates of synthesis of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis are controlled by a signal transduction pathway defined by at least four regulatory genes: degS, degU, degQ (formerly sacQ), and degR (formerly prtR). The DegS-DegU proteins show amino acid similarities with two-component procaryotic modulator-effector pairs such as NtrB-NtrC, CheA-CheY, and EnvZ-OmpR. By analogy with these systems, it is possible that DegS is a protein kinase which could catalyze the transfer of a phosphoryl moiety to DegU, which acts as a positive regulator. DegR and DegQ correspond to polypeptides of 60 and 46 amino acids, respectively, which also activate the synthesis of degradative enzymes. We show that the degS and degU genes are organized in an operon. The putative sigma A promoter of the operon was mapped upstream from degS. Mutations in degS and degU were characterized at the molecular level, and their effects on transformability and cell motility were studied. The expression of degQ was shown to be subject both to catabolite repression and DegS-DegU-mediated control, allowing an increase in the rate of synthesis of degQ under conditions of nitrogen starvation. These results are consistent with the hypothesis that this control system responds to an environmental signal such as limitations of nitrogen, carbon, or phosphate sources.

Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems.

The sacU locus has been cloned by using two independent strategies, and the presence of two open reading frames was deduced from the nucleotide sequence. Open reading frame 1 encodes a 45,000-dalton polypeptide that is similar to the products of the Salmonella typhimurium cheA and Escherichia coli cpxA genes, which act as sensory transducers. Open reading frame 2 encodes a 26,000-dalton polypeptide that is similar to a family of transcriptional activators, including the products of the Bacillus subtilis spoOA and spoOF and the E. coli ompR and dye genes. These similarities suggest that the products of the B. subtilis sacU locus form a sensor-transducer couple, which functions to relay information about specific environmental changes to the transcription apparatus.

Recent aspects of genetic manipulation in Bacillus thuringiensis.

The conjugative plasmid pAM beta 1 was transferred from Streptococcus faecalis to several strains of Bacillus thuringiensis by a filter-mating process. From a transconjugant clone of B. thuringiensis a hybrid plasmid resulting from an in vivo insertion into pAM beta 1 of a 3 Md DNA sequence was isolated. This 3 Md DNA molecule (Th sequence) is related to several host plasmids found in different serotypes of B. thuringiensis. A reciprocal conjugation-like process involving the transfer of pAM beta 1 from B. thuringiensis to S. faecalis was also demonstrated. The comparison of the restriction maps of the crystal genes from plasmid and chromosomal origins of different serotypes, six of which having been cloned in E. coli, revealed the existence of two classes of genes which are very similar in the map corresponding to the N-terminal part of the protein, and which differ essentially in the 3' region. The presence of the transposon-like Th sequence was found in several cases associated with the crystal gene in the same host plasmid, and a model for their structural organization is proposed.

Molecular relationships among plasmids of Bacillus thuringiensis: conserved sequences through 11 crystalliferous strains.

Screening for the plasmid content of 11 strains belonging to nine different serotypes of B. thuringiensis was carried out by electron microscopic examination and electrophoresis in agarose gels. All the strains contained at lest two covalently closed, circular (CCC) DNA species. In one strain (berliner 1715), 17 extrachromosomal elements could be distinguished with regard to their size, ranging from 3.9 to 180 Mdal. Southern hybridisation experiments showed that most of these plasmids fell into two categories (inferior to 15 Mdal and superior to 15 Mdal) which have no homology between them. Within these two size groups there is partial conservation of DNa sequences through various serotypes. Further relationships among the plasmids were investigated by a two dimensional version of the Southern's blotting technique. Possible homology between plasmids and the chromosomal DNA was studied. It was shown that the smaller plasmids from the berliner 1715 and kurstaki HD1 strains contained no sequence related to chromosomal DNA, whereas among the larger plasmids a few showed homologous sequences.

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA).poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid. Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such at thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec- recipient strain of B. subtilis was used.

The phosphoenolpyruvate : methyl-alpha-d-glucoside phosphotransferase system in Bacillus subtilis Marburg : kinetic studies of enzyme ii and evidence for a phosphoryl enzyme ii intermediate.

The Enzyme II complex catalyzing the phosphoryl transfer from P-HPr to sugar in the inducible methyl-alpha-D-glucoside : phosphotransferase system in Bacillus subtilis acts according to a ping-pong mechanism, implying a phosphorylated Enzyme II intermediate. This result is supported by the demonstration of a specific transphosphorylation between [14C] alphaMG and glucose-6-phosphate in the presence of an induced Enzyme II preparation.

Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168.

A set of nine reference strains bringing convenient markers in the genetic background of Bacillus subtilis Marburg 168 has been prepared to allow rapid mapping of new markers.

Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168.

A beta-D-fructofuranosidase -- called levanase -- capable of the hydrolysis of sucrose, inulin and levans has been identified in Bacillus subtilis Marburg. This enzyme can not be detected in strain 168. However, sacL mutations -- mapped on the chromosome of strain 168 between the pheA and aroD reference markers -- lead to constitutive levanase synthesis. This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose.

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis. Identity of the sacUh, amyB and pap mutations.

The sacUh, amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location. Strains bearing these mutations overproduce several exocellular enzymes: alpha amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella. These mutations are tightly linked to the sacU- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers. It is possible that the sacUh, amyB and pap mutations on one hand and the sacU- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions. Furthermore an amy- mutation, leading to the lack of alpha-amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.

The phosphoenolpyruvate : methyl-alpha-D-glucoside phosphotransferase system in Bacillus subtilis Marburg 168 : purification and identification of the phosphocarrier protein (HPr).

The phosphocarrier protein (HPr) of the phosphoenol pyruvate : alpha-methyl-D-glucoside-phosphotransferase system (PTS) has been purified from Bacillus subtilis Marburg 168. The molecular weight is about 8300. HPr contains 1 histidine residue. Phophoenzyme I appears to be an intermediate in the initial phosphoryl transfer from phosphoenolpyruvate (PEP) to HPr. Phospho-HPr is isolated and characterized as a component of the complete system.

Revision of the linkage map of Bacillus subtilis 168: indications for circularity of the chromosome.

A revision of the linkage map of the Bacillus subtilis 168 chromosome has been undertaken with the use of the generalized transducing phage PBS1. The mapping of four new markers (narB1, mtlB1, aroI906, and tre-12) has allowed a determination of the relative orientation of the purB-dal segment and its linkage with the lin markers. The chromosomal segment comprised between the sacQ36 and gtaA12 markers has been linked with the narA1, ctrA1, and sacA321 markers. The recA1 marker has been mapped relative to the thyA and citB17 markers. Indications of linkage have been found between the tre-12 and catA markers and the aroG932 and sacQ36 markers. According to these results, a circular genetic map of the chromosome of B. subtilis 168 is presented. Taken together, the transduction data and the order of marker replication determined by Harford in the accompanying paper support strongly the hypothesis of a symmetrical and fully bidirectional mode of replication for the B. subtilis 168 chromosome.

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.

Identification of the structural gene of the PEP-phosphotransferase enzyme I in Bacillus subtilis Marburg.

A thermosensitive mutation pt8I1 in the gene coding for the Enzyme I of the PEP-phosphotransferase system pathway has been isolated. The mutant enzyme was shown to be sensitive to high temperature, but this effect is dependent on the ionic strength. The ptsI1 mutation was shown to belong to the previously described ctr locus. Following Lin (1970) it is proposed to retain the symbole ptsI for this locus.