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Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus
FEMS Microbiology Letters 222 (2003) 107^113
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Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus Miki Matsuo a , Kenji Kurokawa a; , Satoshi Nishida a , Yan Li a , Haruto Takimura a , Chikara Kaito a , Norio Fukuhara b , Hideki Maki b , Kenji Miura b , Kazuhisa Murakami b , Kazuhisa Sekimizu a a
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b Discovery Research Laboratories, Shionogi and Co., Ltd., 3-1-1, Futaba-Cho, Toyonaka, Osaka 561-0825, Japan Received 15 February 2003; received in revised form 24 March 2003 ; accepted 24 March 2003 First published online 18 April 2003
Abstract The murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the murB gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Staphylococcus aureus; murB; UDP-N-acetylenolpyruvylglucosamine reductase; Temperature-sensitive mutant; Murein; Peptidoglycan
1. Introduction Bacterial peptidoglycan functions as a critical structural barrier against osmotic pressure. Peptidoglycan biosynthesis inhibitors, such as penicillins, cephalosporins, and fosfomycin, have potent bactericidal e¡ects that are important for clinical purposes [1^3]. Peptidoglycan biosynthesis is a continuous process including cytoplasmic and membrane-bound reactions [4^6]. The ¢rst step is MurA-mediated production of UDP-N-acetylenolpyruvylglucosamine (UDPGlcNAcEP) from UDP-N-acetylglucosamine (UDPGlcNAc) and phosphoenolpyruvate. The enolpyruvyl moiety is sequentially reduced by MurB to lactyl ether yielding UDP-N-acetylmuramic acid (UDPMurNAc). The pentapeptide chain is added onto the lactyl ether group of
* Corresponding author. Tel. : +81 (3) 5841-4823; Fax : +81 (3) 5684-2973. E-mail address : [email protected] (K. Kurokawa). Abbreviations : UDPGlcNAcEP, UDP-Nacetylenolpyruvylglucosamine; UDPGlcNAc, UDP-N-acetylglucosamine; UDPMurNAc, UDP-N-acetylmuramic acid
UDPMurNAc by the enzymes MurC, MurD, MurE, and MurF in the cytoplasm [2,4]. The resultant UDPMurNAcpentapeptide is modi¢ed further on the membrane and is included into existing peptidoglycans as a major component. Studies to isolate temperature-sensitive mutants to identify essential genes of Staphylococcus aureus [7,8], a critically important pathogenic Gram-positive bacterium, revealed six independent mutants that were complemented by the murB gene. MurB is a £avoprotein that contains one molecule of £avin adenine dinucleotide (FAD) as a cofactor and is proposed to transfer the protons from NADPH and solvent to FAD and subsequently from FADH2 to UDPGlcNAcEP [9]. Structural analyses of MurB from Escherichia coli and S. aureus revealed overall fold structural similarity composed of three domains [10^12]. It was proposed that domains 1 and 2 are responsible for FAD binding and domain 3 is responsible for binding to the substrates, NADPH and UDPGlcNAcEP. Identi¢cation of the mutation sites of temperature-sensitive mutants is a useful way to determine the functional domain of signi¢cance. The murB gene is essential in E. coli, as demonstrated by the isolation of one temper-
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00260-X
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ature-sensitive murB mutant [13,14]; the mutation site, however, has not been determined. Unsuccessful insertional inactivation of the murB gene in Bacillus subtilis [15] and growth inhibition by antisense RNA expression in S. aureus [16] suggest that it is essential for bacterial growth, however physiologic changes, such as loss of peptidoglycan biosynthesis, were not observed. Functional domain analysis using mutant protein has been limited to one mutant protein of E. coli, MurBS229A [17,18]. In the present study, we isolated six temperature-sensitive murB mutants of S. aureus. The mutation sites of ¢ve of six mutants were located in the proposed substrate binding site, domain 3.
with slight modi¢cations [19]. Cells in overnight culture were diluted in 1 ml of NaCl-depleted LB medium, followed by incubation at 30‡C. When cells reached the exponential growth stage (OD600 = 0.05), 1 WCi of N-acetyl3 31 D-[ H]glucosamine (70^90 Ci mmol ; NEN Life Science Products) was added and the temperature was then shifted to 43‡C for various time periods. An aliquot (200 Wl) was sampled, and trichloroacetic acid was added to a ¢nal concentration of 5%. The resultant suspension was boiled for 15 min, placed on ice for 15 min, followed by ¢ltration through nitrocellulose membrane ¢lters (HA, 0.45 Wm; Millipore). Radioactivity retained on the ¢lters was measured in a liquid scintillation counter (Beckman LS3801). 2.5. MurB enzyme assay for cell lysate
2. Materials and methods 2.1. Bacterial strains and plasmids Temperature-sensitive mutants were obtained by treating RN4220 with ethylmethanesulfate (Sigma, St. Louis, MO, USA) as previously described [7]. Plasmid pND50 is a shuttle vector [7]. pSmurB was constructed by insertion of the DNA fragment, which was ampli¢ed using a polymerase chain reaction (PCR) method performed with chromosomal DNA from RN4220 as a template and primers of 5P-CGGGATCCTGAACTAAAACAACAGGTGCGT-3P and 5P-CCGGAATTCATAATGGCGACAGCGTGTTT-3P, into the SmaI and EcoRI sites in pND50. 2.2. Culture of bacteria S. aureus strains were cultured in LB medium (1% bactotryptone, 0.5% yeast extract, and 1% NaCl). Transformants of S. aureus and E. coli harboring pND50 or its derivatives were cultured in LB medium supplemented with 12.5 Wg ml31 chloramphenicol. Temperature sensitivity of growth and peptidoglycan synthesis of isolated mutants were examined in LB medium without NaCl. Transformants of E. coli containing a plasmid derived from pBluescript II SK(+) for sequencing were cultured in LB medium supplemented with 50 Wg ml31 ampicillin. 2.3. DNA manipulations Transformation of S. aureus cells, extraction of plasmid DNA from S. aureus and E. coli cells, and DNA sequencing methods were as described previously [7]. Sequence data of the wild-type murB gene are registered in the DNA Data Bank of Japan (DDBJ), accession number AB101653.
Crude cell lysates (fraction I) were prepared from 800 ml of S. aureus cell culture of wild-type RN4220 or murB mutants. Cells were washed and lysed by 0.2 mg ml31 lysostaphin in 10 ml bu¡er (25 mM Tris^HCl pH 8.0, 5 mM EGTA, 150 mM KCl) on ice for 45 min, followed by freezing with liquid N2 and thawing. The lysates were clari¢ed by ultracentrifugation (40 000Ug for 20 min). The lysate fractions were incubated for 15 min at room temperature in 100 Wl of reaction bu¡er (50 mM Tris^HCl pH 8.0, 20 mM KCl, 0.5 mM dithiothreitol, 150 WM NADPH, in the presence or absence of 100 WM UDPGlcNAcEP). UDPGlcNAcEP was synthesized from UDPGlcNAc and phosphoenolpyruvate by puri¢ed S. aureus MurA1-mediated enzymatic reaction in vitro, and was puri¢ed by high pressure liquid chromatography as previously described [9]. Oxidation of NADPH was measured by the decrease in absorbance at 340 nm (an extinction coe⁄cient of 6220 M31 cm31 for NADPH) as MurB activity. NADPH oxidation without UDPGlcNAcEP was subtracted as background. Protein concentrations were determined using the Bradford method [20] with bovine serum albumin as a standard. 2.6. Determination of viable cell numbers Overnight culture of the bacterial strain (50 Wl) was diluted to 5 ml in NaCl-depleted LB medium, and cultured at 30‡C in a water bath with vigorous shaking to the exponential growth stage (OD600 = 0.3). The temperature was shifted to 43‡C and incubation was continued for various time periods. Aliquots were appropriately diluted (1038 ^1035 -fold) with LB medium, and samples (100 Wl) were plated onto LB agar plates followed by incubation at 30‡C for 15 h, and the number of colonies was counted. 2.7. Electron microscopy analysis
2.4. Measurement of peptidoglycan synthesis in S. aureus 3
Incorporation of N-acetyl-D-[ H]glucosamine into acidinsoluble fractions was monitored as described previously
Bacteria from an exponential phase (50 ml) culture in LB medium at 30‡C were treated at 43‡C for 2 h. Cells were harvested by centrifugation (1600Ug, 10 min) at
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room temperature, washed with saline, and ¢xed at 4‡C for 16 h with a pre-¢xative solution (1.5 ml) of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate bu¡er (pH 7.3). Samples were post-¢xed with 2% osmium tetroxide in 0.1 M phosphate bu¡er (pH 7.2) at 4‡C for 2 h. The ¢xed samples were washed, dispersed in 3% agar, dehydrated through a graded series of ethanol, substituted with propylene oxide, and embedded with Luveak epoxylated resin. Ultra-thin sections were cut, stained with uranyl acetate and lead citrate for investigating sub-cellular morphology, and examined using a Jeol TEM 1200 EX electron microscope at a magni¢cation of 10 000U.
3. Results 3.1. Complementation of temperature-sensitive phenotype of S. aureus mutants by the murB gene Approximately 400 temperature-sensitive mutants, produced by treating S. aureus RN4220 strain with ethylmethanesulfate, were collected [7,8]. These mutants formed colonies at 30‡C but not at 43‡C on NaCl-depleted LB agar plates. To search for a complementary gene, an EcoRI library of S. aureus genomic DNA, which was a mixture of plasmids carrying EcoRI-digested fragments of S. aureus genomic DNA at the EcoRI site in the shuttle vector pND50, was constructed and electroporated into the mutants. Temperature-resistant transformants were collected, followed by extraction of plasmid DNA and sequencing of the inserted DNA fragments into the vector. A 4.4-kb DNA fragment was found to complement the temperature sensitivity of TS2341. This DNA fragment corresponded to the S. aureus strain N315 genomic DNA nucleotides 791 112^795 517 [21] and carried ¢ve open reading frames including the murB gene, which was determined using the genome information broker (GIB) program in DDBJ. To minimize the region required for com-
Table 1 Complementation of temperature-sensitive phenotypes by the murB gene Strain
2.1/1.8 8.0/8.0 1.4/1.4 3.8/1.8 2.1/1.9 1.8/2.1
plementation, the inserted DNA fragment was digested by HindIII and HincII and the digested fragments were ligated into a pND50 plasmid. The resultant plasmids were used for complementation testing. The results suggested that the murB gene was one of two candidates responsible for the complementation (data not shown). The murB gene was ampli¢ed using PCR methods and ligated into a pND50 plasmid. PCR primers were designed based on the S. aureus N315 genomic sequence. The resultant plasmid, pSmurB, complemented the temperaturesensitive growth of TS2341 (Table 1). Then, other mutants that were complemented by pSmurB in the pool of temperature-sensitive mutants were sought. The temperature-sensitive phenotypes of TS1443, TS2531, TS2557, TS2901, and TS4132 were all complemented by pSmurB (Table 1). 3.2. Determination of mutation sites in the murB gene
Plasmid pSmurB pND50 transformantsU103 (43‡C/30‡C)
TS1443 TS2341 TS2531 TS2557 TS2901 TS4132
Fig. 1. Mutation sites of the temperature-sensitive mutants in the murB gene. Amino acid sequences of S. aureus MurB are represented with secondary structure and domain assignments determined previously [12]. Mutation sites identi¢ed by sequencing are indicated by short vertical lines, and the substituted amino acid residues are indicated with their mutant strain numbers at the bottom.
0.0/1.8 0.0/5.0 0.0/1.4 0.0/2.0 0.0/2.0 0.0/1.9
Plasmids were transformed into six temperature-sensitive mutants. The colony numbers at 30‡C and 43‡C on LB agar plates containing chloramphenicol were counted. Data represent mean numbers of colonies obtained from three di¡erent experiments when 22 Wl of competent cells and 1 Wg of DNA were used.
The results of the complementation test suggested that mutations in the murB gene were responsible for the temperature-sensitive phenotypes. Therefore, we determined whether mutations occur in the chromosomal murB gene of mutant strains. Sequencing analysis indicated that each mutant had only one nucleotide transition mutation, resulting in a substitution of an amino acid within the coding region of the murB gene. As shown in Fig. 1, TS2341, TS1443, TS2531, TS2557, TS2901, and TS4132 had amino acid substitutions of Gly251Asp, Leu277Phe, Ser243Phe, Glu275Lys, Gly78Asp, and Glu275Lys, respectively, via
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Fig. 2. Decrease in peptidoglycan biosynthesis in the temperature-sensitive murB mutants. Exponentially grown cell cultures (OD600 = 0.05) at 30‡C were added to N-acetyl-D-[3 H]glucosamine and then shifted to 30‡C (A) or 43‡C (B) for the indicated periods. Incorporation of N-acetyl-D-[3 H]glucosamine into the acid-insoluble fraction was measured and shown in the vertical axis.
transition mutations from G:C to A:T (G752A, C829T, C728T, G823A, G233A, and G823A). TS2557 and TS4132 had the same transition mutation. The amino acid sequence of the MurB protein of strain RN4220 was identical to that of strain ISP3 (GenBank accession number AF300988) and was di¡erent from that of strain N315 in that Asp93 in RN4220 was replaced with Glu. 3.3. Temperature sensitivity of peptidoglycan synthesis in the mutants Genetic studies in E. coli revealed that the murB gene is essential for biosynthesis of cell wall peptidoglycans [13,14]. Therefore, we examined whether peptidoglycan synthesis decreases at the restrictive temperature in these mutant strains. Peptidoglycan biosynthesis was monitored by incorporation of N-acetyl-D-[3 H]glucosamine into cell wall peptidoglycans. Incorporation of N-acetyl-D-[3 H]glucosamine decreased in these six mutants but continued in wild-type RN4220 at 43‡C, whereas it continued at 30‡C
in the six mutants much the same as RN4220 (Fig. 2; data not shown for TS4132). This result suggests temperaturesensitive biosynthesis of peptidoglycans in these mutant strains. Most E. coli temperature-sensitive mutants for peptidoglycan biosynthesis can be grown by the addition of 20% sucrose to the medium at a restrictive temperature [13,22]. Consistent with the literature, the growth of ¢ve mutants (TS1443, TS2341, TS2531, TS2557, and TS4132) at 43‡C was restored by adding 20% sucrose to the LB agar plate, but their growth was rather slow (data not shown). 3.4. Thinner cell walls in the mutants Electron microscopy was used to examine whether cell walls in these mutant strains become thinner at the restrictive temperature. Cells were grown exponentially at 30‡C and temperature was shifted to 43‡C for 2 h. Cell walls were stained homogeneously but more lightly than the cytoplasmic region and were three to ¢ve times thicker
Table 2 Decrease in UDPGlcNAcEP-dependent NADPH oxidation activity in the murB temperature-sensitive mutants Strain
murB allele
Speci¢c activity (nmol min31 mg protein31 )
%
RN4220 TS2341 TS2531 TS2557 TS2901
Wild-type G251D S243F E275K G78D
3.8 T 0.8 0.54 0.27 1.1 0.54
100 14 7 29 14
Soluble protein extracts (fraction I) were prepared from each strain and assayed for UDPGlcNAcEP-dependent NADPH oxidation activity as described in Section 2. Activity is represented as amount of NADPH oxidation under our experimental conditions. The background NADPH oxidation in the absence of UDPGlcNAcEP was 5.9, 4.6, 5.4, 5.6, and 5.9 nmol min31 mg protein31 in RN4220, TS2341, TS2531, TS2557, and TS2901, respectively.
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3.5. Decrease in UDPGlcNAcEP reductase activity To con¢rm that temperature-sensitive phenotypes of isolated mutants were caused by mutations in the murB gene, the enzyme activity of the murB gene product, UDPGlcNAcEP reductase, was measured and found to be lower in the crude extracts prepared from the mutants than in those from the wild-type strain. The UDPGlcNAcEP reductase activity was determined by measuring NADPH oxidation, which depends on UDPGlcNAcEP. The results indicated that speci¢c activity of the extract from each mutant (TS2341, TS2531, TS2557, and TS2901) was much lower than that of wild-type RN4220 (Table 2). 3.6. Loss of cell viability after temperature increase To examine whether inactivation of MurB protein causes cell death, the number of viable cells of the mutants at the restrictive temperature was determined. The number of viable cells of the mutants (TS2341, TS2531, TS2557, and TS2901) decreased at the restrictive temperature in a time-dependent manner, whereas that of the wild-type
Fig. 3. Transmission electron micrograph of the murB mutant. Exponentially growing cells at 30‡C in LB medium were shifted to 43‡C for 2 h, harvested, ¢xed, and examined using an electron microscope as described in Section 2. Wild-type RN4220 cells (A), murB mutant TS2901 harboring pND50 (B), and TS2901 harboring pSmurB (C). Scale bars represent 250 nm.
than the cell membrane, which was a deeply stained thin layer surrounding the cytoplasmic region on the inside of the cell wall structure (Fig. 3). The cell wall thickness of strain TS2901 was thinner than that of wild-type RN4220 and TS2901 harboring pSmurB (Fig. 3). Similarly, a thinner cell wall at high temperature and its complementation by pSmurB was also observed in mutant strains TS2341, TS2531, and TS2557 (data not shown).
Fig. 4. Decrease in viable cell numbers of the murB mutants at the restrictive temperature. Cultures grown exponentially (OD600 = 0.3) at 30‡C in LB medium were shifted to 43‡C for the indicated time. The viable cell numbers were examined by plating of diluted aliquots on LB agar plate followed by incubation for 15 h at 30‡C. Relative percentage values of the viable cell numbers are presented. The 100% value was approximately 5.0U108 cells ml31 for each mutant.
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continued to increase (Fig. 4). Consistent with the observation of a bactericidal e¡ect of antibiotic drugs that targeted peptidoglycan biosynthesis, MurB protein is suggested to be a valuable target of antibiotics.
seems to be a promising target for development of novel antibiotics.
Acknowledgements 4. Discussion In this study, six temperature-sensitive mutants of S. aureus were isolated whose temperature sensitivity was complemented by the murB gene. These mutant strains had decreased peptidoglycan biosynthesis at the restrictive temperature, consistent with the observations that MurB protein has UDPGlcNAcEP reductase activity, which is involved in peptidoglycan biosynthesis [9,13,14]. In addition, cellular extracts prepared from the mutants had a lower speci¢c UDPGlcNAcEP reductase activity than did wild-type. Moreover, each mutant had a single amino acid substitution in the chromosomal murB gene. These results suggested that the mutations in the murB gene were responsible for the temperature sensitivity of these mutants, that is, the murB gene is essential for S. aureus cell growth. Mutation site determination of temperature-sensitive genes contributes to reveal regions that are important for their enzymatic activities. Although MurB protein structures are characterized as having three domains [10,12], the importance of these domains for its enzymatic activity has not been fully examined. One of six mutation sites of MurB protein in this study was located in the L-4 of domain 1, which is a proposed FAD binding site composed of domains 1 and 2 (Fig. 1). The other ¢ve mutation sites were located in K-5, K-6, or in the loop between L-17 and L-18 of domain 3, which was proposed to be a substrate binding site (Fig. 1). Because ¢ve of six mutation sites were concentrated in domain 3, domain 3 seems to be critical for MurB activity. These amino acid residues might not be important for direct binding to either substrates or FAD as judged by the location of these amino acid residues determined by X-ray crystal analysis. These amino acid substitutions might cause conformational instability in each domain at the high temperature and result in a substrate recognition defect and then loss of UDPGlcNAcEP reductase activity. Searching for compounds that bind to domain 3 might lead to antibiotics that inhibit MurB activity. S. aureus is a Gram-positive pathogen that can cause opportunistic infections in humans. The failure of therapy against methicillin-resistant S. aureus is an important clinical problem; therefore, the development of a new drug with novel action is necessary. Enzymes that are essential for cell growth are desirable targets for antibiotics that inhibit bacterial proliferation. Because the temperaturesensitive murB mutants had a dramatic decrease in cell viability after the temperature increase, that is, inactivation of MurB protein led to cell death, MurB protein
We thank M. Ikeda and M. Notoya for performing electron microscopy experiments. We thank Misses Yoriko Okada, Noriko Aota, Makiko Miyatani, Hiromi Komaki and Kozue Saito for their technical assistance.
References [1] Rolinson, G.N. (1998) Forty years of beta-lactam research. J. Antimicrob. Chemother. 41, 589^603. [2] Zoeiby, A.E., Sanschagrin, F. and Levesque, R.C. (2003) Structure and function of the Mur enzymes : development of novel inhibitors. Mol. Microbiol. 47, 1^12. [3] Reynolds, P.E. (1989) Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur. J. Clin. Microbiol. Infect. Dis. 8, 943^950. [4] van Heijenoort, J. (1996) Murein synthesis. In: Escherichia coli and Salmonella : Cellular and Molecular Biology, Vol. 1 (Neidhardt, F.C., Curtiss III, R., Ingraham, J.L., Lin, E.C.C., Low, K.B., Magasanik, B., Rezniko¡, W.S., Riley, M., Schaechter, M. and Umbarger, H.E., Eds.), pp. 1025^1034. American Society for Microbiology, Washington, DC. [5] Holtje, J.V. (1998) Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 62, 181^203. [6] Navarre, W.W. and Schneewind, O. (1999) Surface proteins of Grampositive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63, 174^229. [7] Inoue, R., Kaito, C., Tanabe, M., Kamura, K., Akimitsu, N. and Sekimizu, K. (2001) Genetic identi¢cation of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus. Mol. Genet. Genomics 266, 564^571. [8] Kaito, C., Kurokawa, K., Hossain, M.S., Akimitsu, N. and Sekimizu, K. (2002) Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene. FEMS Microbiol. Lett. 210, 157^164. [9] Benson, T.E., Marquardt, J.L., Marquardt, A.C., Etzkorn, F.A. and Walsh, C.T. (1993) Overexpression, puri¢cation, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase. Biochemistry 32, 2024^2030. [10] Benson, T.E., Filman, D.J., Walsh, C.T. and Hogle, J.M. (1995) An enzyme-substrate complex involved in bacterial cell wall biosynthesis. Nat. Struct. Biol. 2, 644^653. [11] Constantine, K.L., Mueller, L., Goldfarb, V., Wittekind, M., Metzler, W.J., Yanchunas Jr., J., Robertson, J.G., Malley, M.F., Friedrichs, M.S. and Farmer II, B.T. (1997) Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes. J. Mol. Biol. 267, 1223^1246. [12] Benson, T.E., Harris, M.S., Choi, G.H., Cialdella, J.I., Herberg, J.T., Martin Jr., J.P. and Baldwin, E.T. (2001) A structural variation for MurB: X-ray crystal structure of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Biochemistry 40, 2340^ 2350. [13] Miyakawa, T., Matsuzawa, H., Matsuhashi, M. and Sugino, Y. (1972) Cell wall peptidoglycan mutants of Escherichia coli K-12: existence of two clusters of genes, mra and mrb, for cell wall peptidoglycan biosynthesis. J. Bacteriol. 112, 950^958.
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Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus Miki Matsuo a , Kenji Kurokawa a; , Satoshi Nishida a , Yan Li a , Haruto Takimura a , Chikara Kaito a , Norio Fukuhara b , Hideki Maki b , Kenji Miura b , Kazuhisa Murakami b , Kazuhisa Sekimizu a a
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b Discovery Research Laboratories, Shionogi and Co., Ltd., 3-1-1, Futaba-Cho, Toyonaka, Osaka 561-0825, Japan Received 15 February 2003; received in revised form 24 March 2003 ; accepted 24 March 2003 First published online 18 April 2003
Abstract The murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the murB gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Staphylococcus aureus; murB; UDP-N-acetylenolpyruvylglucosamine reductase; Temperature-sensitive mutant; Murein; Peptidoglycan
1. Introduction Bacterial peptidoglycan functions as a critical structural barrier against osmotic pressure. Peptidoglycan biosynthesis inhibitors, such as penicillins, cephalosporins, and fosfomycin, have potent bactericidal e¡ects that are important for clinical purposes [1^3]. Peptidoglycan biosynthesis is a continuous process including cytoplasmic and membrane-bound reactions [4^6]. The ¢rst step is MurA-mediated production of UDP-N-acetylenolpyruvylglucosamine (UDPGlcNAcEP) from UDP-N-acetylglucosamine (UDPGlcNAc) and phosphoenolpyruvate. The enolpyruvyl moiety is sequentially reduced by MurB to lactyl ether yielding UDP-N-acetylmuramic acid (UDPMurNAc). The pentapeptide chain is added onto the lactyl ether group of
* Corresponding author. Tel. : +81 (3) 5841-4823; Fax : +81 (3) 5684-2973. E-mail address : [email protected] (K. Kurokawa). Abbreviations : UDPGlcNAcEP, UDP-Nacetylenolpyruvylglucosamine; UDPGlcNAc, UDP-N-acetylglucosamine; UDPMurNAc, UDP-N-acetylmuramic acid
UDPMurNAc by the enzymes MurC, MurD, MurE, and MurF in the cytoplasm [2,4]. The resultant UDPMurNAcpentapeptide is modi¢ed further on the membrane and is included into existing peptidoglycans as a major component. Studies to isolate temperature-sensitive mutants to identify essential genes of Staphylococcus aureus [7,8], a critically important pathogenic Gram-positive bacterium, revealed six independent mutants that were complemented by the murB gene. MurB is a £avoprotein that contains one molecule of £avin adenine dinucleotide (FAD) as a cofactor and is proposed to transfer the protons from NADPH and solvent to FAD and subsequently from FADH2 to UDPGlcNAcEP [9]. Structural analyses of MurB from Escherichia coli and S. aureus revealed overall fold structural similarity composed of three domains [10^12]. It was proposed that domains 1 and 2 are responsible for FAD binding and domain 3 is responsible for binding to the substrates, NADPH and UDPGlcNAcEP. Identi¢cation of the mutation sites of temperature-sensitive mutants is a useful way to determine the functional domain of signi¢cance. The murB gene is essential in E. coli, as demonstrated by the isolation of one temper-
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00260-X
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ature-sensitive murB mutant [13,14]; the mutation site, however, has not been determined. Unsuccessful insertional inactivation of the murB gene in Bacillus subtilis [15] and growth inhibition by antisense RNA expression in S. aureus [16] suggest that it is essential for bacterial growth, however physiologic changes, such as loss of peptidoglycan biosynthesis, were not observed. Functional domain analysis using mutant protein has been limited to one mutant protein of E. coli, MurBS229A [17,18]. In the present study, we isolated six temperature-sensitive murB mutants of S. aureus. The mutation sites of ¢ve of six mutants were located in the proposed substrate binding site, domain 3.
with slight modi¢cations [19]. Cells in overnight culture were diluted in 1 ml of NaCl-depleted LB medium, followed by incubation at 30‡C. When cells reached the exponential growth stage (OD600 = 0.05), 1 WCi of N-acetyl3 31 D-[ H]glucosamine (70^90 Ci mmol ; NEN Life Science Products) was added and the temperature was then shifted to 43‡C for various time periods. An aliquot (200 Wl) was sampled, and trichloroacetic acid was added to a ¢nal concentration of 5%. The resultant suspension was boiled for 15 min, placed on ice for 15 min, followed by ¢ltration through nitrocellulose membrane ¢lters (HA, 0.45 Wm; Millipore). Radioactivity retained on the ¢lters was measured in a liquid scintillation counter (Beckman LS3801). 2.5. MurB enzyme assay for cell lysate
2. Materials and methods 2.1. Bacterial strains and plasmids Temperature-sensitive mutants were obtained by treating RN4220 with ethylmethanesulfate (Sigma, St. Louis, MO, USA) as previously described [7]. Plasmid pND50 is a shuttle vector [7]. pSmurB was constructed by insertion of the DNA fragment, which was ampli¢ed using a polymerase chain reaction (PCR) method performed with chromosomal DNA from RN4220 as a template and primers of 5P-CGGGATCCTGAACTAAAACAACAGGTGCGT-3P and 5P-CCGGAATTCATAATGGCGACAGCGTGTTT-3P, into the SmaI and EcoRI sites in pND50. 2.2. Culture of bacteria S. aureus strains were cultured in LB medium (1% bactotryptone, 0.5% yeast extract, and 1% NaCl). Transformants of S. aureus and E. coli harboring pND50 or its derivatives were cultured in LB medium supplemented with 12.5 Wg ml31 chloramphenicol. Temperature sensitivity of growth and peptidoglycan synthesis of isolated mutants were examined in LB medium without NaCl. Transformants of E. coli containing a plasmid derived from pBluescript II SK(+) for sequencing were cultured in LB medium supplemented with 50 Wg ml31 ampicillin. 2.3. DNA manipulations Transformation of S. aureus cells, extraction of plasmid DNA from S. aureus and E. coli cells, and DNA sequencing methods were as described previously [7]. Sequence data of the wild-type murB gene are registered in the DNA Data Bank of Japan (DDBJ), accession number AB101653.
Crude cell lysates (fraction I) were prepared from 800 ml of S. aureus cell culture of wild-type RN4220 or murB mutants. Cells were washed and lysed by 0.2 mg ml31 lysostaphin in 10 ml bu¡er (25 mM Tris^HCl pH 8.0, 5 mM EGTA, 150 mM KCl) on ice for 45 min, followed by freezing with liquid N2 and thawing. The lysates were clari¢ed by ultracentrifugation (40 000Ug for 20 min). The lysate fractions were incubated for 15 min at room temperature in 100 Wl of reaction bu¡er (50 mM Tris^HCl pH 8.0, 20 mM KCl, 0.5 mM dithiothreitol, 150 WM NADPH, in the presence or absence of 100 WM UDPGlcNAcEP). UDPGlcNAcEP was synthesized from UDPGlcNAc and phosphoenolpyruvate by puri¢ed S. aureus MurA1-mediated enzymatic reaction in vitro, and was puri¢ed by high pressure liquid chromatography as previously described [9]. Oxidation of NADPH was measured by the decrease in absorbance at 340 nm (an extinction coe⁄cient of 6220 M31 cm31 for NADPH) as MurB activity. NADPH oxidation without UDPGlcNAcEP was subtracted as background. Protein concentrations were determined using the Bradford method [20] with bovine serum albumin as a standard. 2.6. Determination of viable cell numbers Overnight culture of the bacterial strain (50 Wl) was diluted to 5 ml in NaCl-depleted LB medium, and cultured at 30‡C in a water bath with vigorous shaking to the exponential growth stage (OD600 = 0.3). The temperature was shifted to 43‡C and incubation was continued for various time periods. Aliquots were appropriately diluted (1038 ^1035 -fold) with LB medium, and samples (100 Wl) were plated onto LB agar plates followed by incubation at 30‡C for 15 h, and the number of colonies was counted. 2.7. Electron microscopy analysis
2.4. Measurement of peptidoglycan synthesis in S. aureus 3
Incorporation of N-acetyl-D-[ H]glucosamine into acidinsoluble fractions was monitored as described previously
Bacteria from an exponential phase (50 ml) culture in LB medium at 30‡C were treated at 43‡C for 2 h. Cells were harvested by centrifugation (1600Ug, 10 min) at
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room temperature, washed with saline, and ¢xed at 4‡C for 16 h with a pre-¢xative solution (1.5 ml) of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate bu¡er (pH 7.3). Samples were post-¢xed with 2% osmium tetroxide in 0.1 M phosphate bu¡er (pH 7.2) at 4‡C for 2 h. The ¢xed samples were washed, dispersed in 3% agar, dehydrated through a graded series of ethanol, substituted with propylene oxide, and embedded with Luveak epoxylated resin. Ultra-thin sections were cut, stained with uranyl acetate and lead citrate for investigating sub-cellular morphology, and examined using a Jeol TEM 1200 EX electron microscope at a magni¢cation of 10 000U.
3. Results 3.1. Complementation of temperature-sensitive phenotype of S. aureus mutants by the murB gene Approximately 400 temperature-sensitive mutants, produced by treating S. aureus RN4220 strain with ethylmethanesulfate, were collected [7,8]. These mutants formed colonies at 30‡C but not at 43‡C on NaCl-depleted LB agar plates. To search for a complementary gene, an EcoRI library of S. aureus genomic DNA, which was a mixture of plasmids carrying EcoRI-digested fragments of S. aureus genomic DNA at the EcoRI site in the shuttle vector pND50, was constructed and electroporated into the mutants. Temperature-resistant transformants were collected, followed by extraction of plasmid DNA and sequencing of the inserted DNA fragments into the vector. A 4.4-kb DNA fragment was found to complement the temperature sensitivity of TS2341. This DNA fragment corresponded to the S. aureus strain N315 genomic DNA nucleotides 791 112^795 517 [21] and carried ¢ve open reading frames including the murB gene, which was determined using the genome information broker (GIB) program in DDBJ. To minimize the region required for com-
Table 1 Complementation of temperature-sensitive phenotypes by the murB gene Strain
2.1/1.8 8.0/8.0 1.4/1.4 3.8/1.8 2.1/1.9 1.8/2.1
plementation, the inserted DNA fragment was digested by HindIII and HincII and the digested fragments were ligated into a pND50 plasmid. The resultant plasmids were used for complementation testing. The results suggested that the murB gene was one of two candidates responsible for the complementation (data not shown). The murB gene was ampli¢ed using PCR methods and ligated into a pND50 plasmid. PCR primers were designed based on the S. aureus N315 genomic sequence. The resultant plasmid, pSmurB, complemented the temperaturesensitive growth of TS2341 (Table 1). Then, other mutants that were complemented by pSmurB in the pool of temperature-sensitive mutants were sought. The temperature-sensitive phenotypes of TS1443, TS2531, TS2557, TS2901, and TS4132 were all complemented by pSmurB (Table 1). 3.2. Determination of mutation sites in the murB gene
Plasmid pSmurB pND50 transformantsU103 (43‡C/30‡C)
TS1443 TS2341 TS2531 TS2557 TS2901 TS4132
Fig. 1. Mutation sites of the temperature-sensitive mutants in the murB gene. Amino acid sequences of S. aureus MurB are represented with secondary structure and domain assignments determined previously [12]. Mutation sites identi¢ed by sequencing are indicated by short vertical lines, and the substituted amino acid residues are indicated with their mutant strain numbers at the bottom.
0.0/1.8 0.0/5.0 0.0/1.4 0.0/2.0 0.0/2.0 0.0/1.9
Plasmids were transformed into six temperature-sensitive mutants. The colony numbers at 30‡C and 43‡C on LB agar plates containing chloramphenicol were counted. Data represent mean numbers of colonies obtained from three di¡erent experiments when 22 Wl of competent cells and 1 Wg of DNA were used.
The results of the complementation test suggested that mutations in the murB gene were responsible for the temperature-sensitive phenotypes. Therefore, we determined whether mutations occur in the chromosomal murB gene of mutant strains. Sequencing analysis indicated that each mutant had only one nucleotide transition mutation, resulting in a substitution of an amino acid within the coding region of the murB gene. As shown in Fig. 1, TS2341, TS1443, TS2531, TS2557, TS2901, and TS4132 had amino acid substitutions of Gly251Asp, Leu277Phe, Ser243Phe, Glu275Lys, Gly78Asp, and Glu275Lys, respectively, via
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Fig. 2. Decrease in peptidoglycan biosynthesis in the temperature-sensitive murB mutants. Exponentially grown cell cultures (OD600 = 0.05) at 30‡C were added to N-acetyl-D-[3 H]glucosamine and then shifted to 30‡C (A) or 43‡C (B) for the indicated periods. Incorporation of N-acetyl-D-[3 H]glucosamine into the acid-insoluble fraction was measured and shown in the vertical axis.
transition mutations from G:C to A:T (G752A, C829T, C728T, G823A, G233A, and G823A). TS2557 and TS4132 had the same transition mutation. The amino acid sequence of the MurB protein of strain RN4220 was identical to that of strain ISP3 (GenBank accession number AF300988) and was di¡erent from that of strain N315 in that Asp93 in RN4220 was replaced with Glu. 3.3. Temperature sensitivity of peptidoglycan synthesis in the mutants Genetic studies in E. coli revealed that the murB gene is essential for biosynthesis of cell wall peptidoglycans [13,14]. Therefore, we examined whether peptidoglycan synthesis decreases at the restrictive temperature in these mutant strains. Peptidoglycan biosynthesis was monitored by incorporation of N-acetyl-D-[3 H]glucosamine into cell wall peptidoglycans. Incorporation of N-acetyl-D-[3 H]glucosamine decreased in these six mutants but continued in wild-type RN4220 at 43‡C, whereas it continued at 30‡C
in the six mutants much the same as RN4220 (Fig. 2; data not shown for TS4132). This result suggests temperaturesensitive biosynthesis of peptidoglycans in these mutant strains. Most E. coli temperature-sensitive mutants for peptidoglycan biosynthesis can be grown by the addition of 20% sucrose to the medium at a restrictive temperature [13,22]. Consistent with the literature, the growth of ¢ve mutants (TS1443, TS2341, TS2531, TS2557, and TS4132) at 43‡C was restored by adding 20% sucrose to the LB agar plate, but their growth was rather slow (data not shown). 3.4. Thinner cell walls in the mutants Electron microscopy was used to examine whether cell walls in these mutant strains become thinner at the restrictive temperature. Cells were grown exponentially at 30‡C and temperature was shifted to 43‡C for 2 h. Cell walls were stained homogeneously but more lightly than the cytoplasmic region and were three to ¢ve times thicker
Table 2 Decrease in UDPGlcNAcEP-dependent NADPH oxidation activity in the murB temperature-sensitive mutants Strain
murB allele
Speci¢c activity (nmol min31 mg protein31 )
%
RN4220 TS2341 TS2531 TS2557 TS2901
Wild-type G251D S243F E275K G78D
3.8 T 0.8 0.54 0.27 1.1 0.54
100 14 7 29 14
Soluble protein extracts (fraction I) were prepared from each strain and assayed for UDPGlcNAcEP-dependent NADPH oxidation activity as described in Section 2. Activity is represented as amount of NADPH oxidation under our experimental conditions. The background NADPH oxidation in the absence of UDPGlcNAcEP was 5.9, 4.6, 5.4, 5.6, and 5.9 nmol min31 mg protein31 in RN4220, TS2341, TS2531, TS2557, and TS2901, respectively.
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3.5. Decrease in UDPGlcNAcEP reductase activity To con¢rm that temperature-sensitive phenotypes of isolated mutants were caused by mutations in the murB gene, the enzyme activity of the murB gene product, UDPGlcNAcEP reductase, was measured and found to be lower in the crude extracts prepared from the mutants than in those from the wild-type strain. The UDPGlcNAcEP reductase activity was determined by measuring NADPH oxidation, which depends on UDPGlcNAcEP. The results indicated that speci¢c activity of the extract from each mutant (TS2341, TS2531, TS2557, and TS2901) was much lower than that of wild-type RN4220 (Table 2). 3.6. Loss of cell viability after temperature increase To examine whether inactivation of MurB protein causes cell death, the number of viable cells of the mutants at the restrictive temperature was determined. The number of viable cells of the mutants (TS2341, TS2531, TS2557, and TS2901) decreased at the restrictive temperature in a time-dependent manner, whereas that of the wild-type
Fig. 3. Transmission electron micrograph of the murB mutant. Exponentially growing cells at 30‡C in LB medium were shifted to 43‡C for 2 h, harvested, ¢xed, and examined using an electron microscope as described in Section 2. Wild-type RN4220 cells (A), murB mutant TS2901 harboring pND50 (B), and TS2901 harboring pSmurB (C). Scale bars represent 250 nm.
than the cell membrane, which was a deeply stained thin layer surrounding the cytoplasmic region on the inside of the cell wall structure (Fig. 3). The cell wall thickness of strain TS2901 was thinner than that of wild-type RN4220 and TS2901 harboring pSmurB (Fig. 3). Similarly, a thinner cell wall at high temperature and its complementation by pSmurB was also observed in mutant strains TS2341, TS2531, and TS2557 (data not shown).
Fig. 4. Decrease in viable cell numbers of the murB mutants at the restrictive temperature. Cultures grown exponentially (OD600 = 0.3) at 30‡C in LB medium were shifted to 43‡C for the indicated time. The viable cell numbers were examined by plating of diluted aliquots on LB agar plate followed by incubation for 15 h at 30‡C. Relative percentage values of the viable cell numbers are presented. The 100% value was approximately 5.0U108 cells ml31 for each mutant.
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continued to increase (Fig. 4). Consistent with the observation of a bactericidal e¡ect of antibiotic drugs that targeted peptidoglycan biosynthesis, MurB protein is suggested to be a valuable target of antibiotics.
seems to be a promising target for development of novel antibiotics.
Acknowledgements 4. Discussion In this study, six temperature-sensitive mutants of S. aureus were isolated whose temperature sensitivity was complemented by the murB gene. These mutant strains had decreased peptidoglycan biosynthesis at the restrictive temperature, consistent with the observations that MurB protein has UDPGlcNAcEP reductase activity, which is involved in peptidoglycan biosynthesis [9,13,14]. In addition, cellular extracts prepared from the mutants had a lower speci¢c UDPGlcNAcEP reductase activity than did wild-type. Moreover, each mutant had a single amino acid substitution in the chromosomal murB gene. These results suggested that the mutations in the murB gene were responsible for the temperature sensitivity of these mutants, that is, the murB gene is essential for S. aureus cell growth. Mutation site determination of temperature-sensitive genes contributes to reveal regions that are important for their enzymatic activities. Although MurB protein structures are characterized as having three domains [10,12], the importance of these domains for its enzymatic activity has not been fully examined. One of six mutation sites of MurB protein in this study was located in the L-4 of domain 1, which is a proposed FAD binding site composed of domains 1 and 2 (Fig. 1). The other ¢ve mutation sites were located in K-5, K-6, or in the loop between L-17 and L-18 of domain 3, which was proposed to be a substrate binding site (Fig. 1). Because ¢ve of six mutation sites were concentrated in domain 3, domain 3 seems to be critical for MurB activity. These amino acid residues might not be important for direct binding to either substrates or FAD as judged by the location of these amino acid residues determined by X-ray crystal analysis. These amino acid substitutions might cause conformational instability in each domain at the high temperature and result in a substrate recognition defect and then loss of UDPGlcNAcEP reductase activity. Searching for compounds that bind to domain 3 might lead to antibiotics that inhibit MurB activity. S. aureus is a Gram-positive pathogen that can cause opportunistic infections in humans. The failure of therapy against methicillin-resistant S. aureus is an important clinical problem; therefore, the development of a new drug with novel action is necessary. Enzymes that are essential for cell growth are desirable targets for antibiotics that inhibit bacterial proliferation. Because the temperaturesensitive murB mutants had a dramatic decrease in cell viability after the temperature increase, that is, inactivation of MurB protein led to cell death, MurB protein
We thank M. Ikeda and M. Notoya for performing electron microscopy experiments. We thank Misses Yoriko Okada, Noriko Aota, Makiko Miyatani, Hiromi Komaki and Kozue Saito for their technical assistance.
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