Specific interaction of the RNA-binding domain of the bacillus subtilis transcriptional antiterminator GlcT with its RNA target, RAT.

Expression of the Bacillus subtilis ptsGHI operon is controlled by transcriptional antitermination mediated by the antiterminator protein GlcT. The antiterminator is inactivated in the absence of glucose, presumably by phosphorylation. A conditional terminator in the ptsG mRNA leader region has been identified. Mutations in this terminator resulted in constitutive expression of the operon. The terminator is overlapped by an inverted repeat (called ribonucleic-antiterminator, RAT) which is thought to form a stem-loop structure upon binding of the antiterminator protein GlcT. The N-terminal 60 amino acid residues of GlcT are able to bind to the RAT and prevent transcriptional termination in vivo. Sequence-specific interaction between the RNA-binding domain and the RAT was demonstrated by surface plasmon resonance analysis. Mutations affecting the RNA-binding domain were isolated and will be discussed with respect to their consequences for dimerization and RNA binding.

The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis.

Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA. An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired. We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport. We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants). Catabolite repression of beta-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant. A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen. An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase. This enzyme is necessary for the assimilation of ammonium. In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.

Regulation of the Bacillus subtilis GlcT antiterminator protein by components of the phosphotransferase system.

Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. The sugar is transported into the cell by a specific permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI operon. Expression of this operon is induced by glucose and requires the action of a positive transcription factor, the GlcT antiterminator protein. Glucose availability is sensed by glucose-specific enzyme II (EIIGlc), the product of ptsG. In the absence of inducer, the glucose permease negatively controls the activity of the antiterminator. The GlcT antiterminator has a modular structure. The isolated N-terminal part contains the RNA-binding protein and acts as a constitutively acting antiterminator. GlcT contains two PTS regulation domains (PRDs) at the C terminus. One (PRD-I) is the target of negative control exerted by EIIGlc. A conserved His residue (His-104 in GlcT) is involved in inactivation of GlcT in the absence of glucose. It was previously proposed that PRD-containing transcriptional antiterminators are phosphorylated and concomitantly inactivated in the absence of the substrate by their corresponding PTS permeases. The results obtained with B. subtilis glucose permease with site-specific mutations suggest, however, that the permease might modulate the phosphorylation reaction without being the phosphate donor.

Characterization of the presumptive phosphorylation sites of the Bacillus subtilis glucose permease by site-directed mutagenesis: implication in glucose transport and catabolite repression.

Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. Glucose is transported and concomitantly phosphorylated by the glucose permease (PtsG) of the phosphoenolpyruvate:sugar phosphotransferase system. The phosphate is transferred from enzyme I via HPr and domains IIA and IIB of the glucose permease to the sugar. In this study mutants affected in the putative phosphorylation sites of glucose permease were constructed and the effect on sugar transport and glucose repression tested. Phosphorylation of both domains IIAGlc and IIBGlc is required for efficient glucose transport and repression of beta-xylosidase and the bglPH operon.

Homozygous antithrombin deficiency: report of two new cases (99 Leu to Phe) associated with arterial and venous thrombosis.

Inherited antithrombin deficiency is associated with an increased risk of thrombosis, primarily venous rather than arterial. Most affected individuals have inherited only a single copy of an abnormal antithrombin (AT) gene. Homozygously affected individuals, although rare, have a severe thrombotic history of early onset and often affecting the arteries. We report two new cases of type II HBS (heparin binding site) deficiency in which the propositi are homozygous for the previously reported mutation 99 Leu to Phe, and who have a severe thrombotic history. These cases are considered alongside existing homozygote and compound heterozygote cases.