Raymond Dedonder

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis

SummaryThe 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: α-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 α-amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.

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.

Identification of the structural gene of levansucrase in Bacillus subtilis Marburg

SummaryTwo mutations affecting either the temperature sensitivity or the catalytic properties of the levansucrase of Bacillus subtilis Marburg were characterized. The relevant altered levansucrases were purified and their properties compared with those of the unmodified levansucrase. The results of this analysis and the genetic mapping of these mutations indicates that the structural gene of levansucrase is located in the sacB locus on the Bacillus subtilis chromosome between the cysB3 and hisA1 markers.

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome

SummaryA 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 as 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-α-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.

Identification of the structural gene for sucrase in Bacillus subtilis marburg

Summary Mutants of Bacillus subtilis unable to grow on 0.1 p. cent sucrose were shown on the basis of enzymatic characterization and genetic mapping to be affected in either of two adjacent loci sacA and sacP. The sacP locus is defined by mutations impairing the activity of a phosphorylating sucrose transport system and the sacA locus by sucrase defective mutations. Proteins showing a crossreaction with antibodies directed against purified sucrase have been detected in crude extracts of two sacA mutants. According to these results it is proposed that sacA is the structural gene of sucrase and that the sacA and sacP loci are part of an operon.

The phosphoenolpyruvate : methyl-α-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.