The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation - PubMed (original) (raw)

The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation

S Rohrer et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The factor catalyzing the first step in the synthesis of the characteristic pentaglycine interpeptide in Staphylococcus aureus peptidoglycan was found to be encoded by the essential gene fmhB. We have analyzed murein composition and structure synthesized when fmhB expression is reduced. The endogenous fmhB promoter was substituted with the xylose regulon from Staphylococcus xylosus, which allowed glucose-controlled repression of fmhB transcription. Repression of fmhB reduced growth and triggered a drastic accumulation of uncrosslinked, unmodified muropeptide monomer precursors at the expense of the oligomeric fraction, leading to a substantial decrease in overall peptidoglycan crosslinking. The composition of the predominant muropeptide was confirmed by MS to be N-acetylglucosamine-(beta-1,4)-N-acetylmuramic acid(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala), proving that FmhB is involved in the attachment of the first glycine to the pentaglycine interpeptide. This interpeptide plays an important role in crosslinking and stability of the S. aureus cell wall, acts as an anchor for cell wall-associated proteins, determinants of pathogenicity, and is essential for the expression of methicillin resistance. Any shortening of the pentaglycine side chain reduces or even abolishes methicillin resistance, as occurred with fmhB repression. Because of its key role FmhB is a potential target for novel antibacterial agents that could control the threat of emerging multiresistant S. aureus.

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Figures

Figure 1

Figure 1

Integration of pSR1 into the S. aureus chromosome. Black: fmhB gene; hatched: xylose regulon (xylR and the operator region of xylA); white: fmhB promoter region; bold line: pOX7 plasmid DNA. Relevant restriction sites are indicated as follows: H, _Hin_dIII; B, _Bam_HI; K, _Kpn_I; E, _Eco_RI. Primers used for cloning and PCR controls are indicated by arrows and numbered as in Materials and Methods. The region spanned by the internal DNA probe used for Southern and Northern blots is indicated by a line above fmhB. Not to scale.

Figure 2

Figure 2

Northern blots of fmhB mRNA. Strain SR18 was grown in LB at 42°C to exponential phase in presence of glucose (lane 1), unsupplemented LB (lane 2), or xylose (lane 3). RNA was probed with the fmhB probe shown in Fig. 1.

Figure 3

Figure 3

Muropeptide profiles. Isolated peptidoglycan was digested with muramidase and muropeptides were subjected to reversed-phase HPLC. Strain BB270 grown at 42°C (a) with xylose, (b) with glucose. Strain SR18 grown (c) with xylose, (d) with glucose. The muropeptide fractions are indicated as follows: monomers, corresponding to uncrosslinked subunits (retention time 10–45 min); dimers, corresponding to two crosslinked muropeptides (45–85 min); trimers, corresponding to three crosslinked muropeptides (85–105 min); and oligomers, corresponding to three or more crosslinked muropeptides. More highly crosslinked muropeptides in the oligomeric fraction are indicated by numbers.

Figure 4

Figure 4

Detailed view of the monomeric and dimeric muropeptide pattern of SR18 grown with fmhB induction (a) and repression (b). Peaks were identified by their retention time upon comparison with standard samples. Peak M1 was analyzed by MS. The peak nomenclature is shown in Table 3.

Figure 5

Figure 5

Pentaglycine side-chain formation and peptidoglycan crosslinking. (Upper) Transpeptidase reaction crosslinking the fifth glycine residue of the acceptor muropeptide to the

d

-Ala of the donor stem peptide with release of the terminal

d

-Ala. Direct linkage to the ɛ-amino group of lysine does not occur in S. aureus. (Lower) Synthesis of the pentaglycine side chain at the membrane-bound lipid II intermediate.

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