A study on the effects of aminoacids and arginine deiminase (arcA) on growth, metabolism and gene expression in Enterococcus faecalis V583

In this study the roles of amino acids and arginine deiminase (arcA) in Enterococcus faecalis V583‟s growth, metabolism and gene expression were investigated. Literature describes only one pathway of arginine catabolism for Enterococcus faecalis V583 in the ADI-pathway where arginine deiminase catalyzes the deamination of L-arginine, producing L-citrulline and ammonia. A deletion of arcA was constructed in Enterococcus faecalis V583 (ΔarcA), but surprisingly L-arginine was still broken down in a glucose-limited continuous culture, suggesting an alternative arginine deiminase or alternative pathway for arginine catabolism exists in the E. faecalis V583 genome. A significant effect of the arcA deletion was observed on growth and metabolism both in batch culture with no nutritional limitation, and in glucoselimited continuous culture. Growth studies in batch culture using a defined medium with amino acid leave-out compositions showed that amino acid availability played a significant role in both E. faecalis V583 wild type and mutant growth. A significant reduction in growth rate and a more pronounced death phase was observed for the ΔarcA mutant in comparison to wild type. Growth in glucose-limited continuous culture revealed a pH-dependency on the shift between homolactic and mixed acid fermentation. Transcriptional analysis by real-time PCR also showed pH-dependency of pflA transcription, revealing a down-regulation of pflA in pH 6.5 culture compared to pH 7.5 culture. A reduction in free phosphate concentrations in the mutant cultures, together with provisional genomic data of a polyphosphate kinase (ppnK) gene at EF2670 indicates E. faecalis V583 might be able to produce polyphosphates in stressful environments. A significant reduction in biomass for the ΔarcA mutant in comparison to wild type also suggests a less efficient metabolism for the ΔarcA mutant. Complementation of the E. faecalis V583ΔarcA mutant was performed, providing an intact arcA gene in trans, and construction of a double deletion mutant E. faecalis V583ΔglnAΔarcA was also performed. A preliminary growth study in batch culture showed no growth difference between E. faecalis V583ΔarcA and the complemented E. faecalis V583ΔarcA, suggesting that providing an intact arcA gene is not sufficient to restore ADI-pathway function. A significant difference between E. faecalis V583ΔglnA and V583ΔglnAΔarcA was observed, with V583ΔglnAΔarcA growing significantly slower, but reaching a higher OD600 be