Gene Structure, Organization, Expression, and Potential Regulatory Mechanisms of Arginine Catabolism inEnterococcus faecalis

Abstract

Although Enteroccus faecalis is the paradigm for biochemical studies on the arginine deiminase (ADI) pathway of fermentative arginine catabolism, little genetic information exists on this pathway in this organism. We fill this important gap by characterizing, in an 8,228-bp region cloned from a gt11 genomic library of E. faecalis, a five-gene cluster forming a transcriptional unit (revealed by Northern blots and primer extension in E. faecalis) that corresponds to the ADI operon. Four additional genes in the opposite DNA strand and one in the same DNA strand are also identified. Studies on the protein products, including heterologous expression and/or sequence comparisons, allow us to ascertain or propose functions for all but 1 of the 10 genes. The ADI operon genes, arcABCRD, encode, respectively, ADI, ornithine transcarbamylase, carbamate kinase, a putative Crp/Fnr-type regulator (ArcR), and a putative ornithine-arginine antiporter (ArcD). Arginine induces the expression of arcABCRD, most likely by means of two homologous ArgR/AhrC-type regulators encoded by two genes, argR1 and argR2, that precede arcABCRD in each DNA strand and that are transcribed monocistronically, their transcription being influenced differentially by glucose and arginine. Potential ArgR1/ArgR2 (double and single) binding sequences are found in the promoter regions of arcA and of argR1/argR2 themselves. In addition, putative binding sequences for ArcR and for CcpA are found, respectively, in the argR1/ argR2 and arcA promoter regions. Of the three other genes identified, two form a transcriptional unit and encode a putative metal-sensitive transcriptional regulator (ArsR) and a cysteine protease.A number of microorganisms generate ATP fermentatively from arginine by a route (1) (Fig. 1) in which arginine is deiminated by arginine deiminase (ADI; EC 3.5.3.6), the resulting citrulline is phosphorolyzed by ornithine transcarbamylase (OTC; EC 2.1.3.3), giving ornithine and carbamoyl phosphate, and finally the carbamoyl phosphate is used to phosphorylate ADP in a reaction catalyzed by carbamate kinase (CK; EC 2.7.2.2), yielding ATP. This route, called the ADI pathway, operates in many eubacteria (1,10,12,64) and in some archaea (53) and unicellular eukaryotes (59, 71), but it has not been found in multicellular organisms.Possibly no microbial species has been more important for the biochemical characterization of the ADI pathway than Enterococcus faecalis (formerly Streptococcus faecalis). The ADI pathway was originally demonstrated and characterized in this microorganism (22,25,62), and its enzymes were purified (40, 41, 50) and shown to be coordinately induced by arginine (61). Recently the first three-dimensional structure of a CK was determined (38) on the enzyme isolated from what was believed to be Enterococcus faecium (6) but that we presently show to be E. faecalis (called here E. faecalis SD10).Despite the abundant information derived from E. faecalis on the biochemistry of the ADI pathway, there was no other genetic information about...

ArgR and AhrC Are Both Required for Regulation of Arginine Metabolism in Lactococcus lactis

“…The presence of two arginine regulator homologues in Enterococcus faecalis has recently been described (2). Only a single Asp residue, as is the case for ArgR Ll , is present in the putative arginine-binding region of both E. faecalis homologues, leading to the suggestion that these regulators may bind metabolites other than arginine.…”

Regulation and Physiologic Significance of the Agmatine Deiminase System of Streptococcus mutans UA159

“…In S. mutans and E. faecalis, the AgDS closely resembles the arginine deiminase system (ADS), a pathway that generates ammonia, CO 2 , and ATP from arginine (4,6,13,23,33,37,39). The ADS is considered a primary mechanism of acid tolerance used by some oral bacteria to survive the frequent cycles of acidification encountered in den-tal plaque, but it is not present in the genome of S. mutans (9).…”

Interaction between ArgR and AhrC Controls Regulation of Arginine Metabolism in Lactococcus lactis

“…Despite differences in the organization of genes involved in arginine metabolism, experimental evidence indicates that the mechanism of arginine-dependent regulation of these genes is highly conserved among a range of different organisms, including Gram-negative, Gram-positive and extremophilic bacteria (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Regulation is exerted by binding of single transcriptional regulators of the ArgR family to so-called ARG operator sites preceding the relevant target genes, generally leading to repression of arginine biosynthetic genes and activation of catabolic genes, in the presence of arginine.…”

“…A few recent investigations have proven that these ArgR homologues are not merely orthologous but fulfill distinct functions in these organisms. A study in Enterococcus faecalis revealed the presence, upstream of the arginine catabolic arcABCRD operon, of two genes named argR1 and argR2 (10). Although the function of the E. faecalis ArgR-type regulators was not investigated, it was proven that the divergently transcribed argR1 and argR2 genes were differentially expressed in response to arginine and glucose, possibly via putative ARG boxes preceding the genes (10).…”

“…A study in Enterococcus faecalis revealed the presence, upstream of the arginine catabolic arcABCRD operon, of two genes named argR1 and argR2 (10). Although the function of the E. faecalis ArgR-type regulators was not investigated, it was proven that the divergently transcribed argR1 and argR2 genes were differentially expressed in response to arginine and glucose, possibly via putative ARG boxes preceding the genes (10). In our laboratory, a random knock-out screening led to the identification of the argR and ahrC genes in L. lactis, the gene products of which were responsible for repression of the arginine biosynthetic gltSargE operon (12).…”