Maryvonne Arnaud

New Vector for Efficient Allelic Replacement in Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria

ABSTRACT A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.

PRD--a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria.

Several operon‐specific transcriptional regulators, including antiterminators and activators, contain a duplicated conserved domain, the PTS regulation domain (PRD). These duplicated domains modify the activity of the transcriptional regulators both positively and negatively. PRD‐containing regulators are very common in Gram‐positive bacteria. In contrast, antiterminators controlling β‐glucoside utilization are the only functionally characterized members of this family from Gram‐negative bacteria. PRD‐containing regulators are controlled by PTS‐dependent phosphorylation with different consequences: (i) In the absence of inducer, the phosphorylated EIIB component of the sugar permease donates its phosphate to a PRD, thereby inactivating the regulator. In the presence of the substrate, the regulator is dephosphorylated, and the phosphate is transferred to the sugar, resulting in induction of the operon. (ii) In Gram‐positive bacteria, a novel mechanism of carbon catabolite repression mediated by PRD‐containing regulators has been demonstrated. In the absence of PTS substrates, the HPr protein is phosphorylated by enzyme I at His‐15. This form of HPr can, in turn, phosphorylate PRD‐containing regulators and stimulate their activity. In the presence of rapidly metabolizable carbon sources, ATP‐dependent phosphorylation of HPr at Ser‐46 by HPr kinase inhibits phosphorylation by enzyme I, and PRD‐containing regulators cannot, therefore, be stimulated and are inactive. All regulators of this family contain two copies of PRD, which are functionally specialized in either induction or catabolite repression.

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety‐two open reading frames were predicted to encode putative proteins among which only forty‐two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.

Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis

Bacillus subtilis can utilize several sugars as single sources of carbon and energy. Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B. subtilis. In this study, an analysis of the complete set of PTS proteins encoded within the B. subtilis genome is presented. Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants. Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified. Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B. subtilis; by contrast only five Enzyme IIA(Glc)-like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the EIIA(Glc)-like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families. In addition, 13 PTS-associated proteins are encoded within the B. subtilis genome. These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction. The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.

In Vitro Reconstitution of Transcriptional Antitermination by the SacT and SacY Proteins of Bacillus subtilis

Expression of the sacPA and sacB genes of Bacillus subtilis is positively modulated by transcriptional regulatory proteins encoded by the sacT and sacY genes, respectively. Previous genetic studies led to the suggestion that SacT and SacY function as nascent mRNA binding proteins preventing early termination of transcription at terminators located in the leader regions of the corresponding genes. Here we report the overproduction, purification to near homogeneity, and characterization of the two antiterminators, SacT and SacY. Using mRNA band migration retardation assays and a reconstituted transcriptional antitermination system, the mRNA binding functions and antitermination activities of purified SacT and SacY are demonstrated under in vitro conditions. The results establish for the first time that members of the BglG family of antiterminators function in antitermination in the absence of other proteins in vitro. Purified SacT is shown to be phosphorylated by phosphoenolpyruvate in a phosphotransferase-catalyzed reaction dependent on Enzyme I and HPr. Unexpectedly, the purified SacT is shown to be functional in mRNA binding and in transcriptional antitermination independently of its phosphorylation state.

Expression of a New Operon from Bacillus subtilis, ykzB-ykoL, under the Control of the TnrA and PhoP-PhoR Global Regulators

The ykzB and ykoL genes encode two peptides, of 51 and 60 amino acids, the functions of which are unknown. The ykzB and tnrA genes are contiguous and transcribed divergently. Expression of ykzB and ykoL is induced by glutamate and is under the control of the TnrA global regulator of nitrogen utilization. TnrA regulated its own synthesis in glutamate minimal medium. Two DNA sequences (TnrAB1 and TnrAB2) homologous to the TnrA binding site are present in the region between tnrA and ykzB. Deletion mapping indicated that the TnrAB2 binding site was involved in activation of the ykzB promoter. In addition, transcription of tnrA depends on the presence of the TnrAB1 binding site. The ykzB and ykoL genes are probably in the same transcriptional unit. A single promoter involved in transcription in the presence of glutamate was mapped by primer extension. ykoL expression was induced by phosphate limitation and depended on the PhoP-PhoR two-component regulatory system. Its promoter was mapped to the region between ykoL and ykzB. Four boxes similar to the PhoP binding site are present upstream from the ykoL promoter. These boxes are probably recognized by PhoP approximately P during the activation of transcription in phosphate limitation conditions.

Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis

Expression of the cloned levansucrase gene (sacB) was demonstrated in E. coli minicells by assay of the enzyme in crude extracts, SDS-polyacrylamide gel electrophoresis and immunoblotting. The existence of a precursor form of the enzyme of MW 53000 was also demonstrated and confirmed by the DNA sequence corresponding to the NH2 terminal region of the protein.

Some properties of a sporulating Bacillus subtilis mutant containing heavy DNA.

SUMMARY: A mutant (Cbl-1) was isolated from Bacillus subtilis 168 wild type (wt) sporulating cultures grown on nutrient agar. This mutant differs from the parental strain as follows. (1) In nutrient broth the mutant excretes a substance which kills the wt cells. Therefore when grown in a mixed culture (Cbl-1 plus wt), this property leads to a take-over pattern by the Cbl-1 mutant, and the spores obtained from such a culture are entirely of the mutant type. (2) The DNA of the Cbl-1 strain has a buoyant density in CsCl of 1·714g./cm.3 while that of the normal B. subtilis 168 is 1·703 g./cm.3 although both strains have a G + C content of 42 moles %. An explanation for the higher buoyant density of the Cbl-1 DNA has not yet been found. In addition to the base ratio data, transformation and transduction experiments indicate that Cbl-1 is a mutant of B. subtilis 168.