The Essential Staphylococcus Aureus Gene Fmhb Is Involved In
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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9351–9356, August 1999 Microbiology The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation (cell wall biosynthesis͞murein͞glycyl-tRNA͞methicillin resistance͞drug target) S. ROHRER†‡,K.EHLERT‡§,M.TSCHIERSKE†,H.LABISCHINSKI§, AND B. BERGER-BA¨ CHI†¶ †Institute of Medical Microbiology, University of Zu¨rich, Gloriastr. 32, Postfach, CH-8028 Zu¨rich, Switzerland; and §Bayer AG, PH-Research Antiinfectives I, D-42096 Wuppertal, Germany Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved June 2, 1999 (received for review April 19, 1999) ABSTRACT The factor catalyzing the first step in the glycine side-chain formation reduces methicillin resistance synthesis of the characteristic pentaglycine interpeptide in without affecting PBP2Ј synthesis, resulting in -lactam hy- Staphylococcus aureus peptidoglycan was found to be encoded persusceptibility (6–8). Thus, the interpeptide has several by the essential gene fmhB. We have analyzed murein com- functions related to bacterial growth, cell wall stability, patho- position and structure synthesized when fmhB expression is genicity, and antibiotic resistance. reduced. The endogenous fmhB promoter was substituted with Synthesis of the pentaglycine chain occurs at the membrane- the xylose regulon from Staphylococcus xylosus, which allowed bound lipid II precursor GlcNAc-(-1,4)-N-acetylmuramic glucose-controlled repression of fmhB transcription. Repres- acid(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala)-pyrophosphoryl- sion of fmhB reduced growth and triggered a drastic accu- undecaprenol by sequential addition of glycine to the -amino mulation of uncrosslinked, unmodified muropeptide mono- group of lysine, using glycyl-tRNA as donor, in a ribosome- mer precursors at the expense of the oligomeric fraction, independent fashion (9). Chain formation depends on proteins leading to a substantial decrease in overall peptidoglycan of which two, FemA and FemB, have been identified previ- crosslinking. The composition of the predominant muropep- ously. FemA is involved in the incorporation of glycyl residues tide was confirmed by MS to be N-acetylglucosamine-(-1,4)- 2 and 3 (6, 7), and FemB in that of glycyl residues 4 and 5 (10). N-acetylmuramic acid(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala), Although FemA and FemB have a relatively high amino acid proving that FmhB is involved in the attachment of the first sequence identity (40%) and similarity (64%) they are specific glycine to the pentaglycine interpeptide. This interpeptide and cannot substitute for each other (11). A third factor, plays an important role in crosslinking and stability of the S. FemX, has been postulated to be responsible for the incorpo- aureus cell wall, acts as an anchor for cell wall-associated ration of the first glycine (12). Because femAB null mutants are proteins, determinants of pathogenicity, and is essential for barely viable and depend on compensatory mutations for the expression of methicillin resistance. Any shortening of the survival (8), femX is predicted to be a lethal target. Interest- pentaglycine side chain reduces or even abolishes methicillin ingly, S. aureus contains one glycyl-tRNA gene for protein resistance, as occurred with fmhB repression. Because of its biosynthesis and three nonproteinogenic glycyl-tRNA genes key role FmhB is a potential target for novel antibacterial that are involved in cell wall biosynthesis (13, 14). It is tempting agents that could control the threat of emerging multiresis- to speculate that each Fem factor may preferentially recognize tant S. aureus. one of the three glycyl-tRNA species. Other FemAB-like factors have been identified in staphy- The staphylococcal cell wall plays an important role in infec- lococci, such as Lif in Staphylococcus simulans biovar staph- tion and pathogenicity. Because of the uniqueness of the ylolyticus (15) and Epr in Staphylococcus capitis (16), which peptidoglycan structure and assembly it is one of the preferred protect these strains from their own glycyl-glycine endopepti- targets of antibiotics. The Staphylococcus aureus peptidoglycan dase (17). Genome analysis of a nonpublic database produced consists of linear sugar chains of alternating units of N- three novel femAB-like sequences, fmhA, fmhB, and fmhC acetylglucosamine (GlcNAc) and N-acetylmuramic acid sub- (synonym eprh; ref. 18) in S. aureus (19). Whereas inactivation stituted with the pentapeptide L-Ala-D-iGln-L-Lys-D-Ala-D- of fmhA and fmhC apparently has no effect on growth and Ala. Characteristic for S. aureus is the pentaglycine side chain physiology, fmhB was shown to be essential and was postulated that connects L-Lys of the stem peptide to the D-Ala in position to be femX (19). To investigate its function in peptidoglycan 4 of a neighboring subunit, whereby the D-Ala in position 5 is biosynthesis we placed fmhB under the control of the xylose split off by transpeptidation. The flexible pentaglycine inter- regulon of Staphylococcus xylosus (20) and could demonstrate peptide allows a peptidoglycan crosslinking degree of up to a clear correlation between fmhB repression and accumulation 90%, thus contributing substantially to cell wall stability (1). In of unsubstituted peptidoglycan monomer precursors, proving addition, this pentaglycine chain acts as a recipient for staph- the assumption that FmhB has the activity of the hypothetical ylococcal surface proteins that are covalently anchored to it by FemX. a transpeptidase-like reaction (2). Surface proteins play an important role in adhesion and pathogenicity by interacting MATERIALS AND METHODS with host matrix proteins (3, 4). Staphylococci that have acquired the low-affinity penicillin-binding protein PBP2Ј Strains, Plasmids, and Culture Conditions. All S. aureus express intrinsic resistance to virtually all -lactam antibiotics strains used in this study are derivatives of strain NCTC8325. S. (5). Genetic studies have revealed that inhibition of penta- This paper was submitted directly (Track II) to the Proceedings office. The publication costs of this article were defrayed in part by page charge Abbreviations: PBP, penicillin-binding protein; MIC, minimal inhib- itory concentration. payment. This article must therefore be hereby marked ‘‘advertisement’’ in ‡S.R. and K.E. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. E-mail: bberger@ PNAS is available online at www.pnas.org. immv.unizh.ch. 9351 Downloaded by guest on September 28, 2021 9352 Microbiology: Rohrer et al. Proc. Natl. Acad. Sci. USA 96 (1999) aureus RN4220 is a phage-less, restriction negative strain trans- ribosome binding site and the 5Ј region of fmhB (GenBank formable by electroporation (21). BB255 (22) was used as tem- accession no. AF106849), amplified with the sense primer 1 plate for gene amplifications, and its methicillin-resistant deriv- (5Ј-GCGGATCCATTGTTAAATAGAAGGAGATATC-3Ј) ative, strain BB270 (22) was used for fmhB promoter replace- and reverse primer 2 (5Ј-GCGGTACCCCCAGTGATTT- ment. Plasmid pCX15 containing the xylose regulatory system of TCATTAATTC-3Ј) from S. aureus BB255, was cloned into the Staphylocccus xylosus was obtained from K. P. Wieland, Univer- BamHI and KpnI sites downstream of the xylA promoter region. sityofTu¨bingen, Tu¨bingen, Germany (20). The Escherichia The resulting fusion of the xylose regulon with the 5Ј fragment of coli-S. aureus shuttle vector pOX7, temperature sensitive for fmhB was sequenced to ensure fidelity of the PCR. The fusion replication in S. aureus, conferring ampicillin resistance to E. coli product was excised by HindIII and EcoRI and subcloned into and erythromycin resistance to S. aureus, was obtained from K. shuttle vector pOX7. The resulting plasmid pSR1 (Fig. 1) was Dyke, University of Oxford (23). Growth medium was LB (Difco) electroporated into RN4220 from where it was transduced into supplemented with 0.5% glucose or 0.5% xylose where indicated. BB270. To promote integration of the plasmid pSR1 into the Transductions in S. aureus were made with generalized transduc- chromosome of BB270, transductants grown at 30°C to stationary ͞ ing phage 80␣ (24). Transformants and transductants were se- phase in LB broth containing 20 g ml of erythromycin were ͞ lected in the presence of 20 g͞ml of erythromycin at 30°C. diluted 1:100 into fresh LB containing 2.5 g ml of erythromycin Strains with chromosomally integrated recombinant tempera- plus xylose, incubated at 40°C for 8 h, then rediluted 1:100 into ͞ ture-sensitive plasmid were propagated in presence of 10 g͞ml freshLBwith2.5mgml of erythromycin plus xylose and of erythromycin at 42°C to maintain plasmid integration. incubated overnight at 40°C. Appropriate dilutions were spread ͞ Susceptibility Tests. Minimal inhibitory concentration (MIC) on LB-agar plates containing 2.5 g ml of erythromycin plus of the glycyl-glycine endopeptidase lysostaphin (Ambi, Trow- xylose and incubated at 42°C. Colonies were checked for plasmid 4 integration by PCR using sense primer 3 (5Ј-AAATGAACAAT- bridge, U.K.) was determined by microbroth dilution using 10 Ј Ј cells in 100 l of LB per well as described earlier (7). The MIC GTGCTATATTACC-3 ) and reverse primer 4 (5 -GCCAG- CAAACATTAATAGTGC-3Ј), as well as sense primer 5 (5Ј- of methicillin was measured by Etest (AB Biodisk, Solna, Swe- Ј den) (25). When determining MICs in the presence of glucose, GCGAATTCTTACACAAGCTCTGAATCGAC-3 ) and reverse primer 6 (5Ј-GCCTGCAGATTTAGCTATTTCGGC- the inoculum was prepared from overnight cultures grown on Ј plates containing glucose. Growth was read after 24-h incubation ATGAAG-3 ) as indicated in Fig. 1. In the resulting strain SR18 at either 35°C or 42°C. (mec, fmhB::pSR1) chromosomal integration was verified by DNA Manipulations. Routine DNA manipulations were done Southern blot using a probe covering fmhB from the start codon according to protocols of Ausubel et al. (26) and Maniatis et al. to the HindIII site (Fig. 1). (27). Sequencing was performed with an ABI 310 automated Isolation of Peptidoglycan, Preparation, and Fractionation of Muropeptides.