Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function

Abstract

A defined synthetic medium was used to determine the amino acid requirements of Enterococcus faecalis OG1 and to demonstrate the absence of a requirement for exogenous purines or pyrimidines. Genomic libraries prepared from strain OG1 were transduced into Escherichia coli auxotrophic mutants, and cloned DNAs which complemented pyrC, pyrD, purF, purL, and guaAB mutations were identified. These and other cloned DNAs with known functions were localized on a restriction map of OG1 which was generated with SfiI (5 fragments), AscI (9 fragments), and NotI (15 fragments); the size of the OG1 chromosome was revised from a previous estimate of approximately 2,750 kb to 2,825 kb. The synthetic medium and the restriction map should be useful for studying enterococcal metabolic functions and the relationships between chromosomally encoded genes.

Two Arginine Repressors Regulate Arginine Biosynthesis inLactobacillus plantarum

“…E. faecalis also contains both synteny groups but contains two additional argR copies (argR1 and argR2), which are linked to the ADI arginine catabolic operon (2). Unlike L. plantarum and L. mesenteroides (13), E. faecalis not only synthesizes arginine (25) but also degrades it. L. lactis harbors two genes with ahrC linked to yqxC-recN (Table 3, group 2) and argR linked to the arginine catabolic arc genes (20).…”

Generation of auxotrophic mutants of Enterococcus faecalis

“…The cosmid vector pLAFRx is a derivative of pLAFR which contains oriT of RK2 and a polylinker for cloning (12,17). pKV48 and pKV53 are cosmids from the previously constructed enterococcal genomic libraries from E. faecalis OG1RF (Table 1) which complement E. coli pyrC and purL auxotrophs, respectively (27). The vector used for subcloning was pBluescript II SKϩ phagemid (36).…”

“…M63 salts (25) supplemented with 0.2% glucose, thiamine (100 g/ml), MgSO 4 (1 mM), and 1.5% agar was used as the minimal medium for growth of E. coli. For testing of possible enterococcal auxotrophs, Davis minimal medium with supplements (DMMS) as described by Murray et al (27) was used as the defined synthetic enterococcal broth medium. DMMS agar contains 1.5% Bacto Agar (Difco Laboratories, Detroit, Mich.).…”

“…Thus, there is a need for more basic knowledge of these organisms in order to understand how to control or prevent enterococcal infections through developing either therapeutics or vaccines. In a previous report, we described the physical map of the genome of Enterococcus faecalis OG1RF as well as several cosmid clones that complemented Escherichia coli auxotrophs (27). In this study, the possibility of generating enterococcal pyr and pur auxotrophs by allelic replacement was tested.…”

“…Transducing lysate preparation and transduction were performed by using standard procedures (34). Southern transfer and hybridizations were carried out as described previously (27). PCR was performed by using a PCR Optimizer Kit (Invitrogen, San Diego, Calif.) and a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, Conn.).…”