GabR, a member of a novel protein family, regulates the utilization of γ‐aminobutyrate in Bacillus subtilis

Abstract

Summary The Bacillus subtilis ycnG (gabT) and ycnH (gabD) genes were shown to encode γ‐aminobutyrate (GABA) aminotransferase and succinic semi‐aldehyde dehydrogenase, respectively, and to form a GABA‐inducible operon. Null mutations in gabT, gabD or the divergently transcribed ycnF (gabR) gene blocked the utilization of GABA as sole nitrogen source. GabR proved to be a transcriptional activator of the gabTD operon and a negative autoregulator. The target of GabR action was localized to an 87 bp region that includes both gabR and gabT promoters. GabR is a member of a novel but widespread family of chimeric bacterial proteins that have apparent DNA‐binding and aminotransferase domains. Mutations in conserved residues of the putative aminotransferase domain abolished GabR function as a transcriptional activator, but did not affect its activity as a negative autoregulator.

Positive transcriptional control of the pyridoxal phosphate biosynthesis genes pdxST by the MocR-type regulator PdxR of Corynebacterium glutamicum ATCC 13032

“…This result is in marked contrast with observations made with the regulatory protein GabR from Bacillus subtilis, a MocRtype transcription regulator that positively controls the utilization of c-aminobutyric acid and represses its own expression (Belitsky & Sonenshein, 2002). The aminotransferase domain of GabR is probably essential for the proper functioning of this protein, as modification of the conserved lysine residue to alanine by site-directed mutagenesis led to a mutant form of the regulator that was unable to activate the expression of the divergently orientated gabTD operon, but retained the full ability to bind to the target DNA and to repress the gabR promoter (Belitsky & Sonenshein, 2002). The atypical amino acid sequence of the PLP attachment site in the actinobacterial PdxR-like proteins implies moreover that the covalent binding of PLP to the E-b/O domain is dispensable for the regulatory activity of PdxR, suggesting that the E-b/O domain has a different and hitherto unknown function in these proteins.…”

“…Replacement of this essential lysine residue by serine, threonine or histidine may result in the loss of an enzymic function, which is consistent with a previous functional analysis in C. glutamicum, as aminotransferase activity was not detectable in enzyme assays with the purified PdxR protein (Marienhagen et al, 2005). This result is in marked contrast with observations made with the regulatory protein GabR from Bacillus subtilis, a MocRtype transcription regulator that positively controls the utilization of c-aminobutyric acid and represses its own expression (Belitsky & Sonenshein, 2002). The aminotransferase domain of GabR is probably essential for the proper functioning of this protein, as modification of the conserved lysine residue to alanine by site-directed mutagenesis led to a mutant form of the regulator that was unable to activate the expression of the divergently orientated gabTD operon, but retained the full ability to bind to the target DNA and to repress the gabR promoter (Belitsky & Sonenshein, 2002).…”

“…Another interesting aspect of MocR-type transcription regulators relates to their environmental sensing mechanism. The GabR protein from B. subtilis binds to its target DNA independently of PLP and c-aminobutyric acid, but requires both metabolites for the activation of the gabTD operon (Belitsky & Sonenshein, 2002). Disruption of the pdxR gene in C. glutamicum resulted in decreased expression of the pdxST genes, which was also observed in the wild-type strain when supplementing the growth medium with either pyridoxal or PLP.…”

Physical and Enzymological Interaction of Bacillus subtilis Proteins Required for De Novo Pyridoxal 5′-Phosphate Biosynthesis

“…Virtually no growth was observed in glucose-glutamate or glucose-1 mM NH 4 Cl medium, but growth at almost the wild-type rate was observed in the presence of 60 to 120 mM NH 4 Cl (Fig. 2); intermediate growth was observed in glucose-0.2% (37.4 mM) NH 4 Cl medium. Growth of strain BB2256 was restored to the wild-type rate by addition of PL.…”

“…Methods for plasmid isolation, agarose and polyacrylamide gel electrophoresis, use of restriction and DNA modification enzymes, DNA ligation, PCR, and electroporation of E. coli JM107 cells were as described by Sambrook et al (35). Growth of B. subtilis cells, transformation by plasmid DNA, and procedures for gene replacement were as described previously (4,5).…”