Isabelle Martin-Verstraete

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.

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.

CymR, the master regulator of cysteine metabolism in Staphylococcus aureus, controls host sulphur source utilization and plays a role in biofilm formation

We have characterized the master regulator of cysteine metabolism, CymR, in Staphylococcus aureus. CymR repressed the transcription of genes involved in pathways leading to cysteine formation. Eight direct DNA targets were identified using gel‐shift or footprinting experiments. Comparative transcriptome analysis and in vitro studies indicated that CysM, the OAS‐thiol‐lyase, was also implicated in this regulatory system. OAS, the direct precursor of cysteine, prevents CymR‐dependent binding to DNA. This study has allowed us to predict sulphur metabolism functions for previously uncharacterized S. aureus genes. We show that S. aureus is able to grow on homocysteine as the sole sulphur source suggesting efficient MccA and MccB‐dependent conversion of this compound into cysteine. We propose that SA1850 is a new thiosulphate transporter and that TcyP and TcyABC are l‐cystine transporters. CymR directly controls the use of sulphur sources of human origin such as taurine and homocysteine. The cymR mutant also displayed a reduced capacity to form biofilms, indicating that CymR is involved in controlling this process in S. aureus via an ica‐independent mechanism. These data indicate that fine‐tuning of sulphur metabolism plays an important part in the physiology of this major pathogen and its adaptation to environmental conditions and survival in the host.