In general, mutation of the glycosyl-transferase bgsA and bgsB

yielded similar phenotypes, suggesting that the phenotypic changes observed for both mutants are mainly the result of the depletion of DGlcDAG or altered LTA Thiazovivin structure. On the other hand, MGlcDAG seems to play a minor role in bacterial physiology and virulence. Conclusions We have shown that the bgsB gene is responsible for the glycosylation of DAG to form MGlcDAG, the first step in glycolipid synthesis in E. faecalis. bgsB deletion led to reduced biofilm formation and attachment to colonic cells, and to impaired virulence in vivo. Methods Bacterial Pinometostat mw strains, plasmids, and growth conditions The bacterial strains and plasmids used in this study are shown in Table 1. Enterococci MLN2238 were grown at 37°C without agitation in tryptic soy broth (TSB; Merck), M17 broth (Difco Laboratories), or TSB plus 1% glucose (TSBG) as indicated. In addition, tryptic soy agar or M17 agar plates were used. Escherichia coli DH5α and TOP10 (Invitrogen) were cultivated aerobically in LB-broth. Kanamycin was added for enterococci (1 mg/ml) and for E. coli (50 μg/ml); tetracycline was used at 12.5 μg/ml for E. coli and at 10 μg/ml for enterococci.

Table 1 E. faecalis strains and plasmids used in this study. strain or plasmid characterization reference strains     E. faecalis 12030 Clinical isolate, strong biofilm producer [33] E. faecalis ATCC 29212 Reference strain   E. faecalis 12030ΔbgsA (EF2891) bsgA mutant [5] E. faecalis 12030ΔbgsB bgsB deletion mutant This study E. faecalis 12030ΔbgsB_rec. Reconstituted mutant This study Escherichia coli DH5α Gram-negative cloning host   Escherichia coli TOP10 Gram-negative cloning host Invitrogen plasmids     pCASPER Gram-positive, temperature-sensitive mutagenesis vector [34] pCRII-TOPO Gram-negative cloning vector Invitrogen pCASPER/ΔbgsB   This

study pMAD/bsgB   This study pMAD oripE194ts, EmR, AmpR, bgaB [35] Construction of a nonpolar deletion mutant Terminal deoxynucleotidyl transferase of bgsB Molecular techniques used in this study have been described previously [5]. In brief, the bgsB mutant was constructed in E. faecalis 12030 by homologous recombination. The deletion of a portion of the gene bgsB (790 bp) (EF_2890 in the E. faecalis V583 genome, GenBank accession no. AAO82579.1) was created as described elsewhere [5]. Primers 1 and 2 (Table 2) were used to amplify a 581-bp fragment downstream, and primers 3 and 4 were used to amplify a 563-bp fragment upstream of the target gene. Primers 2 and 3 contain a 21-bp complementary sequence (underlined in Table 2). Overlap extension PCR was performed to generate a PCR product lacking a fragment of 790 bp in the center of bgsB (Figure 1). The resulting construct was cloned into the Gram-positive shuttle vector pCASPER containing a temperature-sensitive replicon; the resulting plasmid, pCASPER-ΔbgsB, was transformed into E. faecalis 12030 by electroporation.

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