Enterococcus faecalis makes ATP from agmatine in three steps catalyzed by agmatine deiminase (AgDI), putrescine transcarbamylase (PTC), and carbamate kinase (CK). An antiporter exchanges putrescine for agmatine. We have cloned the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for agmatine catabolism, overexpressed them in Escherichia coli, purified the products, characterized them functionally as PTC and AgDI, and crystallized and X-ray diffracted them. The 1.65-Å-resolution structure of AgDI forming a covalent adduct with an agmatine-derived amidine reactional intermediate is described. We provide definitive identification of the gene cluster for agmatine catabolism and confirm that ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to be trimeric) evolved from ornithine transcarbamylase. N-(Phosphonoacetyl)-putrescine was prepared and shown to strongly (K i ‫؍‬ 10 nM) and selectively inhibit PTC and to improve PTC crystallization. We find that E. faecalis AgDI, which is committed to ATP generation, closely resembles the AgDIs involved in making polyamines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably supplied the genes for PTC and CK but not those for the agmatine/putrescine antiporter, and thus the AgDI and ADI pathways are not related by a single "en bloc" duplication event. The AgDI crystal structure reveals a tetramer with a five-blade propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of sequence identity, and explains substrate affinity, selectivity, and Cys357-mediated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks access to the active center.In addition to the fermentation of carbohydrates, Enterococcus faecalis (formerly Streptococcus faecalis) is able to use arginine and its decarboxylated derivative agmatine as energy sources for growth (8,10,45,48,49). Arginine and agmatine are metabolized via the arginine deiminase (ADI) and agmatine deiminase (AgDI) pathways, respectively. The two metabolic routes are very similar and include the sequential action of three enzymes (48, 49) and one antiporter (11), which are analogous in the two pathways. Arginine and agmatine are deiminated by ADI (EC 3.5.3.6) and AgDI (EC 3.5.3.12), respectively, yielding citrulline and carbamoyl putrescine, which are phosphorolyzed by ornithine transcarbamylase (OTC) (EC 2.1.3.3) and putrescine transcarbamylase (PTC) (EC 2.1.3.6). This generates carbamoyl phosphate for use in ADP phosphorylation by pathway-specific carbamate kinase (CK) (EC 2.7.2.2) isozymes, producing one ATP molecule (48, 49). The resulting ornithine and putrescine are exchanged with external arginine or agmatine by an arginine/ ornithine antiporter in one pathway and by an agmatine/putrescine antiporter in the other pathway (11).Possibly no microbial species has been more important for the biochemical characterization of the ADI and AgDI pathways tha...

Aims:  To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ‐PCR procedure suitable for the routine detection and quantification of this pathogen in food. Methods and Results:  Specificity of nuc targeted primers (Pri1–Pri2 and the newly designed RTQ‐PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 103 CFU g−1. The two RTQ‐PCR systems, incorporating SYBR‐Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93·3%). Conclusions:  PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. Significance and Impact of the Study:  The SYBR‐Green I real‐time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.

Aims: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. Methods and Results: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g)1 were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30°C and a 24-h enrichment step at 37°C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method.Conclusions: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. Significance and Impact of the Study: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.