Generation of auxotrophic mutants of Enterococcus faecalis

Abstract

A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.Enterococci, which have been recognized as a cause of many infections, including infectious endocarditis (26), are the secondto third-most-common pathogens found in hospital-acquired infections. Because of an alarming increase in resistance to different antibiotics, therapy of enterococcal infections has become increasingly difficult. 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. This methodology is important for developing genetic approaches for studying enterococcal virulence. We also describe the further characterization of a clone containing pyr biosynthesis genes and the determination of the arrangement of the pyr gene cluster by mapping, sequencing, and complementation. MATERIALS AND METHODSBacterial strains, plasmids, and phage. The bacterial strains used in this work are described in Table 1. 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 c...

The B Form of Dihydroorotate Dehydrogenase from Lactococcus lactis Consists of Two Different Subunits, Encoded by the pyrDb and pyrK Genes, and Contains FMN, FAD, and [FeS] Redox Centers

“…It seems likely that the architecture of the ␦-enzyme is the prototype of the dihydroorotate dehydrogenases from Gram-positive bacteria, since the PyrDB protein shows very high sequence similarity to all known PyrD proteins of Gram-positive bacteria (i.e. Bacillus subtilis, Bacillus caldolyticus, and Enterococcus faecalis (Nielsen et al, 1996)) and since an orf with very high similarity to pyrK is found immediately upstream of the pyrD gene in these bacteria (Andersen et al, 1996;Ghim et al, 1994;Li et al, 1995;Quinn et al, 1991). Moreover, it was shown in the case of B. subtilis that disruption of this orf, now termed pyrDII, imposes a partial pyrimidine requirement on the bacterium and strongly lowers the activity of dihydroorotate dehydrogenase in cell-free extracts (Kahler and Switzer, 1996).…”

“…Clustering of cysteinyl residues in the PyrK homologues of four Gram-positive bacteria. Ll, L. lactis (Andersen et al 1996); Bc, B. caldolyticus (Ghim et al 1994); Bs, B. subtilis (Kahler and Switzer, 1996;Quinn et al, 1991); Ef, E. faecalis (Li et al, 1995). The first residue shown is residue 220 in the PyrK protein of L. lactis.…”

Endocarditis and biofilm-associated pili of Enterococcus faecalis

“…The resulting constructs were transformed into ebp and srtC mutants by electroporation (44). The expression of ebpC and srtC genes in the ebpC strains complemented with ebpC and ebpC plus srtC after nisin induction (28) was determined by RT-PCR.…”

Molecular Typing of Selected Enterococcus faecalis Isolates: Pilot Study Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

“…We chose internal regions of three E. faecalis antigen-encoding genes (ace, efaA, and salA) and one housekeeping gene (pyrC) for sequencing. These four loci were chosen primarily because all these genes were well studied in our laboratory (8,19,31,43), and the sequence diversity of the region coding for the A domain of ace has been recognized in our earlier study (31). The open reading frame (ORF) sizes of the four chosen loci are listed in Table 2.…”