A Klier

The two‐component system ArlS–ArlR is a regulator of virulence gene expression in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen that produces many virulence factors in a temporally regulated manner controlled by at least two global virulence regulatory loci (agr and sarA). We identified previously a two‐component system, ArlS–ArlR, that modifies the activity of extracellular serine protease and may be involved in virulence regulation. Here, we show that mutations in either arlR or arlS increase the production of secreted proteins [α‐toxin (Hla), β‐haemolysin, lipase, coagulase, serine protease (Ssp)] and especially protein A (Spa). Furthermore, the pattern of proteins secreted by both mutants was strikingly different from that of the wild‐type strain. Transcriptional fusions showed that expression of hla, ssp and spa was higher in both mutants than in the wild‐type strain, indicating that the arl operon decreases the production of virulence factors by downregulating the transcription of their genes. The arl mutation did not change spa expression in an agrA mutant or in a sarA mutant, suggesting that both the sarA and the agr loci are required for the action of arl on spa. Northern blot analyses indicated that the arl mutation increased the synthesis of both RNA II and RNA III, but decreased sarA transcription. Finally, arl was not autoregulated, but its expression was stimulated by agr and sarA. These results suggest that the Arl system interacts with both agr and sarA regulatory loci to modulate the virulence regulation network.

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT.

Glucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose‐specific phosphoenolpyruvatesugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI ) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsI form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon.

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.

Coding capacity of the transcription products synthesized in vitro by the RNA polymerases from Bacillus thuringiensis

Abstract RNA transcripts synthesized in vitro from B. thuringiensis DNA by the vegetative RNA polymerase and by the form I and the form II of the sporulation enzyme were shown to contain messenger activities in a cell-free system from E. coli . The MW distribution of the resulting polypeptides was determined by SDS-gel electrophoresis. Results based on hybridization-competition experiments suggested that the mRNA coding for the parasporal crystal, a specific sporulation protein, might be transcribed preferentially by the form II of the sporulation enzyme.

Bacillus thuringiensis : risk assessment

Use of biological control agents will dramatically increase in the next decades as farmers move towards environmentally safe agricultural practices, in response to the people request. Bacillus thuringiensis is one of these agents and the derivative products are estimated to rise at least 20% per year. However, there are some species with an increased activity against specific insects and a broader host range. In addition, recombinant DNA technology allows now to reach new derivatives or to introduce the genetic determinants into new hosts, including plants and other microbes. It is obvious that technology combined with the diversity of the Bt species will increase the scope for the application of Bt. The benefits to agriculture and for the environment are considerable, but the possibility of adverse environmental impact for the fauna and/or the flora due to the large scale application of the new Bt derivative products needs to be considered and evaluated. Moreover, little is known about the ecology of Bt and the role of spores in the environment.

CLONING OF B. SUBTILIS GENES IN E. COLI AND EXPRESSION OF SOME CLONED GENES IN B. SUBTILIS

ABSTRACT A collection of 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. Thirty clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. Recombinant plasmids extracted from E. coli were used to transform auxotrophic mutants of B. subtilis. Complementation was observed for several markers such as thr, leuA, hisA, glyB and purB. In B. subtilis rec+ strains in most cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Such loss did not occur when a rec − strain harboring the marker recE4 was used.