Although MGlcDAG and DGlcDAG are the major glycolipids in many Gram-positive bacteria, distinct differences are found between species in biosynthetic pathways. In staphylococci and Bacillus, a single processive glucosyltransferase YpfP adds two glucose residues to DAG to synthesize DGlcDAG [12, 16, 17]. Depending on the bacterial species and strain background, the deletion of this enzyme may result in an increased LTA content and turnover , or loss of LTA from the cell membrane, associated with a reduced rate of autolysis and impaired biofilm formation . In listeria, streptococci, and enterococci, genome analysis revealed two putative glycosyltransferases involved in the biosynthetic pathway of glycolipids [7, 14, 15, 18]. Homologues of a (1→2) glucosyltransferase have been investigated in listeria (LafA), group B streptococci (IagA), and E. faecalis (BgsA) [5, 15, 18]. In group B streptococci, deletion of iagA results in the absence of capsule expression, reduced retention of LTA on the bacterial cell surface, and increased release of LTA into the culture medium . Inactivation of lafA in L. monocytogenes strongly depletes LTA from both the cell wall and the culture medium . In contrast to these findings, deletion of bgsA in E. faecalis results in an increased concentration of LTA in the bacterial cell envelope, most likely related to the longer glycerol-phosphate polymer. The different makeup of glycolipids and LTA in this mutant strongly impaired biofilm-formation and affected virulence in vivo .
In the current study, we constructed a deletion mutant by targeted mutagenesis of the putative glycosyltransferase bgsB located immediately downstream of bgsA. After inactivation of bgsB in E. faecalis 12030, no glycolipids or glycolipid-derivatives were recovered from the cell envelope of the 12030ΔbgsB mutant, indicating that BgsB is a 1,2-diacylglycerol 3-glucosyltransferase. BgsA cannot take the place of BgsB, which suggests that BgsA has higher substrate specificity than YpfP in S. aureus and B. subtilis [13, 17]. The putative function assigned to BgsA and BgsB by this work is in agreement with data obtained for their homologues LafA and LafB in L. monocytogenes . Although the lipid anchor of LTA from 12030ΔbgsB was not characterized chemically, indirect evidence suggests that DAG instead of DGlcDAG anchors LTA to the cell membrane in this mutant. LTA extracted from 12030ΔbgsB migrated more slowly than wild-type LTA in SDS PAGE, a feature that has been described for homologous LTA molecules substituted with DAG instead of DGlcDAG in S. aureus and L. monocytogenes [13, 15]. In staphylococci and listeria it has been also demonstrated that, in the absence of glycolipids, the enzyme that transfers glycerolphosphate residues to the glycolipid anchor (LtaS) can utilize DAG as glycerolphosphate acceptor for the synthesis of the LTA backbone [13, 15]. Deletion mutants of the glucosyltransferases bgsB and bgsA enabled us to study the individual roles of the two major glycolipids MGlcDAG and DGlcDAG in the physiology and virulence of E. faecalis. To our surprise, the complete loss of glycolipids from the cell membrane in 12030ΔbgsB had only minor effects on bacterial morphology, cell growth, and autolysis.
In contrast, MGlcDAG and DGlcDAG are critical for cell membrane elasticity and fluidity and important for the function of membrane-bound proteins in Acholeplasma laidlawii [6, 7, 14]. It is possible, however, that up-regulation of other cell membrane amphiphiles may compensate for the lack of glycolipids in the bgsB mutant . In fact, the concentration of LTA was increased in 12030ΔbgsB and possibly compensates for the loss of phosphoglycolipid derivatives of MGlcDAG and DGlcDAG in the 12030ΔbgsB mutant . A characteristic feature of both mutants is the increased chain length of the glycerol-phosphate polymer. However, the mechanism underlying this alteration in LTA structure remains unclear and deserves further attention.
The most notable feature of 12030ΔbgsB is its impairment in biofilm formation and adherence to colonic cells. As observed previously in the bgsA mutant, initial attachment to polystyrene was not impaired in 12030ΔbgsB, but the accumulation of bacteria in the growing biofilm was impaired. This is in contrast to other biofilm-defective mutants in E. faecalis, in which attachment to the foreign surface is the feature primarily affected and underlines the importance of cell envelope amphiphiles in the retention of bacteria within the biofilm architecture [20, 21]. Several mechanisms may explain the biofilm phenotype of the mutants. As in the bgsA mutant, impaired biofilm formation in 12030ΔbgsB was associated with reduced hydrophobicity, a well-known determinant of biofilm formation in bacteria [22, 23]. Also, increased LTA concentration in the cell envelope of the bgsB-mutant may impair biofilm formation by increasing the net negative charge of the cell envelope. The impact of the higher negative charge of the LTA molecule on biofilm formation has been demonstrated by mutants in the D-alanine-D-alanyl-carrier protein ligase DltA [24, 25]. Finally, the increased amount of LTA released into the biofilm matrix (as observed with 12030ΔbgsB and 12030ΔbgsA) may act as a biosurfactant, promoting detachment of bacterial cells from the biofilm and thereby impeding its growth . In contrast to our results the inactivation of the glycosyltransferase YpfP in S. aureus leads to depletion of LTA from the cell surface and to a reduced ability to form biofilm .
Aside from its effects on biofilm formation, the increased density of negative charges of the LTA molecule of the mutant may also explain the slight increase in sensitivity of 12030ΔbgsB to the antimicrobial peptides colistin and polymyxin B. If this difference explains the significantly impaired virulence in our mouse bacteremia model, however, is unclear. On balance, we observed a 2-log reduction in the number of CFU recovered for both mutants, suggesting that glycolipids, either as a cell membrane component or as an anchor of LTA, play a critical role in the cell envelope of enterococci during infection. 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 structure. On the other hand, MGlcDAG seems to play a minor role in bacterial physiology and virulence.