In Streptococcus gordonii DL1, inactivation of the ccpA gene and a gene encoding an Fnr-like protein (Flp) demonstrated that CcpA was essential for carbohydrate catabolite repression and that Flp was required for optimal expression and anaerobic induction of the arginine deiminase system.

The arginine deiminase (AD) system (ADS) is one of two major ammonia-generating pathways in the oral cavity that play important roles in oral biofilm pH homeostasis and oral biofilm ecology. To initiate a study of the Streptococcus gordonii ADS, the ADS gene cluster was isolated from subgenomic DNA libraries of S. gordonii DL1 by using an arcB-specific probe. Nucleotide sequence analysis revealed six open reading frames (ORFs) that were arranged contiguously; the first five ORFs were transcribed in the same direction, as an apparent operon, and the sixth was transcribed in the opposite direction. The ORFs were found to share significant homologies and to correspond closely in molecular mass to previously characterized arc genes; thus, they were designated arcA (AD), arcB (ornithine carbamyltransferase), arcC (carbamate kinase), arcD (arginine-ornithine antiporter), arcT (dipeptidase), and arcR (regulator). A putative 70 promoter (ParcA [TTGTGT-N 19 -T AGAAT]) was mapped 5 to arcA by primer extension, and the expression of ParcA was shown to be inducible by arginine and repressible by glucose, in agreement with AD specific activities measured in the wild-type strain. To investigate the function of ArcR in the differential expression of the arc operon, arcR was insertionally inactivated by a KM resistance marker flanked by T4 transcription/translation termination signals, and the expression of ParcA was monitored by primer extension in the wild-type and ArcR-deficient strains. Lower levels of arcA expression, as well as lower levels of AD activity, were consistently observed in the ArcR-deficient strain compared to wild-type cells, regardless of the growth conditions. Thus, ArcR is a transcriptional activator that is required for induction and optimal expression of the S. gordonii ADS gene cluster.The arginine deiminase (AD) system (ADS) is a three-enzyme pathway that catalyzes the conversion of arginine to ornithine, ammonia, and CO 2 with the concomitant production of ATP (16). Arginine is first hydrolyzed by AD, encoded by arcA, to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamylphosphate via a catabolic ornithine carbamyltransferase (cOTC) encoded by arcB. Finally, carbamate kinase (CK), encoded by arcC, transfers phosphate from carbamylphosphate to ADP to produce ATP, CO 2 , and ammonia. In some cases, an arginine-ornithine antiporter (ArcD), which catalyzes the uptake of arginine and concomitant export of ornithine, and a putative transaminase or peptidase (ArcT) have been found to be part of arc gene clusters (37).Arginine metabolism via the ADS is widely distributed in both bacteria and archaebacteria, and the primary structures of the enzymes involved in the pathway have been reasonably conserved throughout evolution. In contrast, the physiologic role and genetic regulation of expression of the ADS vary among microorganisms. For instance, both Bacillus licheniformis (32) and Pseudomonas aeruginosa (26) utilize the ADS exclusively under anaerobic conditions and the pr...

A 1,026-bp open reading frame sharing significant similarity with queA, which encodes a predicted Sadenosylmethionine:tRNA ribosyltransferase-isomerase responsible for queosine modification of tRNAs, was found immediately 5 of the gene for the transcriptional activator (ArcR) of the arginine deiminase system (ADS) operon of Streptococcus gordonii. The role of QueA in bacterial physiology is enigmatic, but loss of QueA has been shown to compromise stationary-phase survival or virulence in certain enteric bacteria. Interestingly, S. gordonii appears to be unique among ADS-positive bacteria in the linkage of queA with the ADS genes. A putative 70 promoter (p queA ; TTGCCA-N 21 -TATAAT) was mapped 5 of queA by primer extension, and queA and arcR were shown to be cotranscribed. The expression from p queA was found to be constitutive under all conditions tested, but the expression of p arcA , which drives the expression of the arc structural genes, was enhanced in stationary phase and could be induced by low pH and arginine. QueA and CcpA acted repressively on arc transcription, but neither QueA-deficient strains nor CcpA-deficient strains showed significant differences in arginine deiminase enzyme activities compared with the wild-type strain. The growth rate of a QueA-deficient strain did not differ significantly from that of the parental strain, but the QueA-deficient strain did not compete well with the wild-type during serial passage. In addition to the finding that ADS expression can be regulated separately by growth phase and pH, a significant linkage between the ADS, translational efficiency modulated by QueA, and post-exponential-phase survival of S. gordonii was found.

The arginine deiminase system (ADS) is responsible for the production of ornithine, CO 2 , ammonia, and ATP from arginine. The ADS of the oral bacterium Streptococcus gordonii plays major roles in physiologic homeostasis, acid tolerance, and oral biofilm ecology. To further our understanding of the transcriptional regulation of the ADS (arc) operon, the binding of the ArcR transcriptional activator, which governs expression of the ADS in response to arginine, was investigated by DNase I protection and gel mobility shift assays. An ArcR binding sequence was found that was 27 bp in length and had little sequence similarity to binding sites of other arginine metabolism regulators. The presence of arginine at physiologically relevant concentrations enhanced the binding of ArcR to its target. Using cat fusions, various deletion and substitution mutations within the putative ArcR footprint were shown to cause dramatic reductions in expression from the arcA promoter in vivo, confirming that the 27-bp sequence is required for optimal expression and induction of the ADS by arginine. Mutation of two putative catabolite response elements (CREs) within the arc promoter region showed that both CREs contribute to catabolite repression. A thorough understanding of the regulation of the ADS in S. gordonii and related organisms is needed to develop ways to exploit arginine catabolism for the control of oral diseases. Identification of the ArcR and CcpA binding sites lays the foundation for a more complete understanding of the complex interactions of multiple regulatory proteins with elements in the arc promoter region.