Dataset Information

Isolation and molecular analysis of the gene cluster for the arginine deiminase system from Streptococcus gordonii DL1.

ABSTRACT: 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 sigma(70) promoter (ParcA [TTGTGT-N(19)-TAGAAT]) 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.

SUBMITTER: Dong Y

PROVIDER: S-EPMC129940 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Isolation and molecular analysis of the gene cluster for the arginine deiminase system from Streptococcus gordonii DL1.

Dong Yiqian Y Chen Yi-Ywan M YY Snyder Jennifer A JA Burne R A RA

Applied and environmental microbiology 20021101 11

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 ...[more]

PMID: 12406748

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Project description:Penicillin tolerance is an incompletely understood phenomenon that allows bacteria to resist drug-induced killing. Tolerance was studied with independent Streptococcus gordonii mutants generated by cyclic exposure to 500 times the MIC of penicillin. Parent cultures lost 4 to 5 log(10) CFU/ml of viable counts/24 h. In contrast, each of four independent mutant cultures lost < or =2 log(10) CFU/ml/24 h. The mutants had unchanged penicillin-binding proteins but contained increased amounts of two proteins with respective masses of ca. 50 and 45 kDa. One mutant (Tol1) was further characterized. The two proteins showing increased levels were homologous to the arginine deiminase and ornithine carbamoyl transferase of other gram-positive bacteria and were encoded by an operon that was >80% similar to the arginine-deiminase (arc) operon of these organisms. Partial nucleotide sequencing and insertion inactivation of the S. gordonii arc locus indicated that tolerance was not a direct consequence of arc alteration. On the other hand, genetic transformation of tolerance by Tol1 DNA always conferred arc deregulation. In nontolerant recipients, arc was repressed during exponential growth and up-regulated during postexponential growth. In tolerant transformants, arc was constitutively expressed. Tol1 DNA transformed tolerance at the same rate as transformation of a point mutation (10(-2) to 10(-3)). The tolerance mutation mapped on a specific chromosomal fragment but was physically distant from arc. Importantly, arc deregulation was observed in most (6 of 10) of additional independent penicillin-tolerant mutants. Thus, although not exclusive, the association between arc deregulation and tolerance was not fortuitous. Since penicillin selection mimicked the antibiotic pressure operating in the clinical environment, arc deregulation might be an important correlate of naturally occurring tolerance and help in understanding the mechanism(s) underlying this clinically problematic phenotype.

Project description:Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions.

Project description:UnlabelledStreptococcus pneumoniae is auxotrophic for arginine, and molecular analysis of the pneumococcal genome showed that the gene encoding an arginine-ornithine antiporter (ArcD) is organized in a cluster together with the arcABC genes encoding the arginine deiminase system (ADS) of pneumococci. The ADS consists of the arginine deiminase (AD), the catabolic ornithine carbamoyltransferase (cOCT), and the carbamate kinase (CK). Pneumococcal genomes contain three ArgR-type regulators (ArgR1, ArgR2, and AhrC) that are supposed to be involved in the regulation of arginine metabolism. Here, we identified ArgR2 of TIGR4 as the regulator of the ADS and ArcD. ArgR2 binds to promoter sequences of the arc operon, and the deficiency of ArgR2 in TIGR4 abrogates expression of the ADS, including the arginine-ornithine antiporter ArcD. Intranasal infection of mice and real-time bioimaging revealed that deletion of the arcABCDT genes attenuates TIGR4. However, the acute-pneumonia model and coinfection experiments indicated that the arginine-ornithine antiporter ArcD is essential to maintain fitness, while the deficiency of ADS enzymes has a minor impact on pneumococcal fitness under in vivo conditions. Strikingly, argR2 mutant TIGR4 outcompeted the wild type in the respiratory tract, suggesting an increase in fitness and further regulatory functions of ArgR2. In contrast to TIGR4, other pneumococci, such as D39, lacking expression of ArgR2, constitutively express the ADS with a truncated nonfunctional AD. On the basis of these results, we propose that the arginine-ornithine antiporter is essential to maintain pneumococcal fitness and that the genes of the ADS cluster are positively regulated in a strain-specific manner by ArgR2.ImportancePneumococci are the major etiologic agents of community-acquired pneumonia, causing more than 1.5 million deaths annually worldwide. These versatile pathogens are highly adapted to the nutrients provided by the host niches encountered. Physiological fitness is of major importance for colonization of the nasopharyngeal cavity and dissemination during invasive infections. This work identifies the regulator ArgR2 as the activator of the S. pneumoniae TIGR4 ADS and the arginine-ornithine transporter ArcD, which is needed for uptake of the essential amino acid arginine. Although ArgR2 activates ArcD expression and uptake of arginine is required to maintain pneumococcal fitness, the deficiency of ArgR2 increases TIGR4 virulence under in vivo conditions, suggesting that other factors regulated by ArgR2 counterbalance the reduced uptake of arginine by ArcD. Thus, this work illustrates that the physiological homeostasis of pneumococci is complex and that ArgR2 plays a key role in maintaining bacterial fitness.

Dataset Information

Isolation and molecular analysis of the gene cluster for the arginine deiminase system from Streptococcus gordonii DL1.

Publications

Isolation and molecular analysis of the gene cluster for the arginine deiminase system from Streptococcus gordonii DL1.

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