1 Department of Biological Sciences, Illinois State University, Normal, IL 61790, USA
2 Laboratory of Biochemistry, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence
Jackob Moskovitz
MoskoviJ{at}NHLBI.NIH.GOV
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oxidation of methionine usually leads to a diastereomeric mixture of methionine sulfoxide (MetO): R-MetO and S-MetO. Recently, two distinct activities of the enzyme methionine sulfoxide reductase (Msr), referred to as MsrA and MsrB, have been reported. MsrA is specific for the S-MetO enantiomer, and the newly described MsrB is specific for R-MetO (Kryukov et al., 2002; Kumar et al., 2002
; Moskovitz et al., 2002
; Olry et al., 2002
; Skaar et al., 2002
). The msrA and msrB genes in several bacterial species are organized adjacent to each other and appear to be co-transcribed (Singh et al., 2001b
; Kryukov et al., 2002
). In addition, MsrA and MsrB are also often linked to each other forming two-domain fusion proteins, where MsrB is located either downstream or upstream of MsrA (Kryukov et al., 2002
). It is widely believed, based on the close proximity of these two genes and their similar enzymic activity but distinct substrate stereospecificity, that the proteins encoded by these two genes complement each other in protecting organisms from oxidative stress (Rodrigo et al., 2002
). Organisms with low Msr activity have indeed been demonstrated to show increased sensitivity to oxidative stress (Moskovitz et al., 1997
, 1998
; Skaar et al., 2002
; Stadtman et al., 2002
). In addition, Msr activity was found to be low in patients with Alzheimer's disease (Gabbita et al., 1999
) and, more recently, MsrA has been demonstrated to regulate the life span of mammals (Moskovitz et al., 2001
) and flies (Ruan et al., 2002
). More significantly, Msr proteins have been shown to regulate virulence in several bacteria (Hassouni et al., 1999
; Dhandayuthapani et al., 2001
; Olry et al., 2002
; Wizemann et al., 1996
).
The bacterium Staphylococcus aureus is a versatile pathogen that causes a variety of infections and has acquired resistance to almost all available antibiotics (Archer, 1998). In our study of the antibiotic stress response, we found that MsrA was significantly induced by treatment of S. aureus by wall-active antibiotics (Singh et al., 2001a
). Subsequent investigation revealed that msrA (now designated msrA1) and msrB (initially designated pilB) are the first and second genes of a four-gene polycistronic message (Singh et al., 2001b
). Insertional inactivation of msrA1 resulted in increased susceptibility of the mutant to H2O2, but not to the cell-wall-active antibiotic oxacillin. In addition, msrA1 has been identified as a S. aureus virulence gene in a murine model of bacteraemia using signature-tagged mutagenesis (Mei et al., 1997
). Besides MsrA1/MsrB, a second MsrA protein, designated MsrA2, has been demonstrated with Msr activity (Moskovitz et al., 2002
). The genome-wide search presents another gene in the S. aureus chromosome that encodes a protein (MsrA3) with considerable homology to MsrA1 and MsrA2. In this study, we have attempted to unravel the physiological significance of MsrA1, MsrA2 and MsrB homologues in S. aureus using genetic approaches. We report that, under the conditions tested here, the MsrA1/MsrB system is physiologically more significant in S. aureus than MsrA2.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Construction of an msrA1 msrA2 double knockout mutant in S. aureus.
Construction of an msrA1 knockout mutant of S. aureus has been described previously (Singh et al., 2001b). Construction of a double msrA1 msrA2 knockout mutant was achieved by transducing the mutation in the msrA2 gene to the msrA1 mutants of oxacillin-sensitive strain RN450 and oxacillin-resistant strain BB270 using phage-80
. Methicillin-resistant strain COL, used previously (Singh et al., 2001b
), could not be used in this study to construct a double mutant due to its intrinsic resistance to tetracycline.
Determination of Msr activity.
Liquid cultures of wild-type strain RN450, its msrA1 and msrA2 mutants and the msrA1 msrA2 double mutant were grown to an OD600 value of 0·3. The cultures were divided into two tubes at this density and one set was challenged with 1·2 µg oxacillin ml-1 for 2·5 h as described previously (Singh et al., 2001a). Cells were subsequently washed with buffer A' [20 mM Tris/HCl buffer (pH 7·5) containing 145 mM NaCl]. Cells from 5·0 ml of the cultures grown without oxacillin and 15·0 ml of the cultures grown with oxacillin were resuspended and lysed in 0·5 ml of buffer A' containing 0·25 mg lysostaphin ml-1 by incubation at 37 °C for 10 min. The lysed cells were sonicated briefly and centrifuged for 5 min at maximum speed in a refrigerated Eppendorf centrifuge at 4 °C. Total cellular Msr activity in the cell-free extract was determined using 1 mM Dabsyl-Met(O) and 20 mM DTT in 50 mM Tris/HCl (pH 7·5) and incubation at 37 °C for 30 min, as described previously (Moskovitz et al., 1997
).
MsrA- and MsrB-specific activities in the cell-free extracts of the wild-type and msrA mutants were determined using enantiomeric substrates R-MetO (specific for MsrB) and S-MetO (specific for MsrA), as described previously (Moskovitz et al., 2002). The MsrA activity contributed by each MsrA protein in the wild-type S. aureus strain RN450 was calculated by assigning the loss of MsrA activity in the MsrA1 mutant to MsrA1, and the loss of activity in the MsrA2 mutant to MsrA2. MsrA3 activity is representative of residual MsrA activity in the msrA1 msrA2 double mutant.
Determination of the sensitivity of msrA mutants to oxacillin and H2O2.
The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for the wild-type and different msrA mutant strains of S. aureus were determined as described previously (Pfeltz et al., 2000; Singh et al., 2001b
).
Complementation of the msrA1 msrA2 double mutant.
The double mutant was complemented with construct pCU1-msr1 as described previously (Singh et al., 2001b), resulting in strain MC3. To complement the double mutant with the msrA2 gene, an
2·5 kb fragment was PCR-amplified using primers P13 and P16. The resulting amplicon was cloned in pCU1, resulting in construct pCU1-msrA2, which was transferred to S. aureus RN4220 by electroporation and subsequently transduced into the double mutant, resulting in strain MC4. The wild-type strain, the msrA2 mutant and the double mutant were also transformed with the vector plasmid pCU1. The antibiotic chloramphenicol was present in TSB at a concentration of 10 µg ml-1 for all the strains used during the oxacillin or H2O2 MIC determinations.
Expression of msrA2 from the msrA1 promoter and vice versa.
An 1·3 kb fragment was PCR-amplified using primers P6 and P17. A 236 bp fragment starting 44 nt downstream of the msrA1 ORF and going upstream was excised from the amplicon by digestion with HindIII (site internal to the amplicon) and cloned in pCU1 at the XbaI and HindIII sites, resulting in pCU1-msrA1P. An
1·3 kb DNA fragment starting precisely from the msrA2 gene was PCR-amplified using primers P18 and P19 and the amplicon was cloned into the BamHI and EcoRI sites of the construct pCU1-msrA1P, resulting in pCU1-msrA1P-msrA2. This construct was transferred to S. aureus RN4220 by electroporation and subsequently transduced into the double mutant, resulting in strain MC5.
An 1·4 kb fragment starting 41 nt downstream of the msrA2 gene and going upstream was PCR-amplified using primers P20 and P21, and the amplicon was cloned into the XbaI and HindIII sites of pCU1, resulting in construct pCU1-msrA2P. An
0·7 kb DNA fragment starting precisely from the msrA1 gene was PCR-amplified using primers P22 and P23, and cloned into the BamHI and EcoRI sites of the construct pCU1-msrA2P, resulting in pCU1-msrA2P-msrA1. This construct was transferred to S. aureus RN4220 by electroporation and subsequently transduced into the double mutant, resulting in strain MC6.
MsrB expression in strains with msrB antisense RNA.
The antisense msrB fragment was cloned downstream of the msrA1 promoter in the construct pCU1-msrA1P. To accomplish this, the msrB ORF was PCR-amplified using primers P24 and P25, and the amplicon was cloned into the BamHI and EcoRI sites of construct pCU1-msrA1P, resulting in construct pCU1-msrA1P-antisense-msrB. This construct was transferred into the wild-type S. aureus strain RN450 and various msrA mutants.
Preparation of anti-MsrB antibodies.
For the production of anti-MsrB polyclonal antibodies, 100 µg of hexahistidine-tagged MsrB purified as described previously (Singh et al., 2001b) was emulsified with Freund's complete adjuvant and subsequently injected subcutaneously into New Zealand White female rabbits. After primary immunization, the animals were injected twice on days 14 and 21 with 100 µg of MsrB emulsified with Freund's incomplete adjuvant. Serum from the immunized rabbits was collected from the ear vein 7 days after the second booster injection and used for immunoblotting studies.
Western blot analysis.
Cell-free protein extracts from the S. aureus wild-type and msr mutants grown with and without oxacillin were prepared essentially as described previously (Singh et al., 2001a) and in the Determination of Msr activity' section of Methods. The cell-free protein extract (10 µg) from each sample was separated by SDS-PAGE (15 % gel) and the polypeptides were transferred onto nitrocellulose membranes (0·45 µm pore size) (Bio-Rad) by semi-dry electroblotting using a Tris/CAPS buffer system. The membranes were subsequently blocked with PBS (0·14 M NaCl, 0·003 M KCl, 0·002 M potassium phosphate, 0·01 M sodium phosphate, pH 7·2) containing 5 % non-fat milk prior to incubation with anti-rabbit MsrB polyclonal antibodies [1/1000 dilution in PBS/Tween 20 containing 1 % BSA (fraction V)] for 1 h. The unbound antibodies were washed twice with PBS/Tween 20 (0·1 %) and subsequently incubated with the rabbit polyclonal anti-HRP antibodies [1/1000 dilution in PBS/Tween 20 containing 1 % BSA (fraction V)]. Blots were washed twice with PBS/Tween 20 (0·1 %) for 10 min each and the MsrB polypeptides were visualized using the opti-4CN kit according to the manufacturer's instruction (Bio-Rad).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Mutation in the msrA genes and H2O2 tolerance
Present findings of increased total Msr activity in the MsrA1 mutants are inconsistent with our earlier determination of increased H2O2 sensitivity of these mutants (Singh et al., 2001b). Recently, Skaar et al. (2002)
reported a similar observation where an msr mutant of Neisseria gonorrhoeae had increased sensitivity to oxidative stress despite four times higher Msr activity than its parent strain. To explain this intriguing observation, Skaar et al. (2002)
proposed that, due to higher activity, the mutants were more capable of destroying the reactive oxygen intermediates through reversible methionine oxidation/reduction. This stops the mutants reaching the threshold needed to activate additional systems that might be involved in protection against oxidative damage in an organism. However, this view is inconsistent with previous findings where overexpression of MsrA activity in Saccharomyces cerevisiae and T cells provided the yeast and T cells with high resistance to oxidative stress (Moskovitz et al., 1998
). In addition, Staphylococcus aureus msrA1 mutants complemented with the wild-type msrA1 gene on a high-copy shuttle plasmid showed higher H2O2 resistance than even the wild-type cells (Singh et al., 2001b
). Moreover, the observed increase in cellular MsrB activity in the msrA1 mutants (Table 3
) apparently has not contributed to a better resistance of these mutants to oxidative stress conditions. Taking all these data together, it seems that the cellular capability of S-MetO reduction (carried out by MsrAs) is more prominent than the cellular R-MetO reduction (carried out by MsrB) in determining the resistance level to oxidative stress condition in S. aureus.
Of the other two MsrA proteins in staphylococci, only MsrA2 is significantly homologous to MsrA1 (49 % identity; 65 % similarity). MsrA3 shows only limited homology to MsrA1 (22 % identity; 33 % similarity) and MsrA2 (25 % identity; 39 % similarity). Amino acid sequence analysis further indicates that whereas MsrA1 and MsrA2 possess the stretch of five amino acids near the N terminus (GCFWC) that has been determined to be critical for MsrA enzymic activity (Moskovitz et al., 2000), this is changed to GCLWG in MsrA3. However, since most of the MsrA3 active site is preserved (except for the F to L substitution), it is most likely that the MsrA activity of this protein is intact. In addition, the genes encoding these three MsrA proteins show nucleotide sequences different to each other which eliminates the likelihood of any of these genes being the result of the duplication of a single gene in S. aureus during the course of evolution.
In this study, we have further investigated the significance of MsrA1 and MsrA2 in relation to the oxidative stress response in S. aureus. The H2O2 tolerance of the msrA2 mutant was comparable to that of the wild-type parent (Table 4), which is consistent with no significant decrease in the MsrA activity in the MsrA2 mutant. In addition, there was no further increase in the susceptibility to oxidative stress when a mutation in the msrA2 gene was transduced into the msrA1 mutant to generate an msrA1 msrA2 double mutant (Table 3
). Furthermore, in complementation assays with the msrA1 gene on a shuttle plasmid, tolerance of the msrA1 msrA2 double mutants to H2O2 oxidative stress was restored (strain MC3) (Table 3
). However, when it was complemented with the msrA2 gene, the double mutant remained H2O2-susceptible (strain MC4) (Table 4
).
|
The msrA1 msrA2 double mutant is not an MsrB mutant
In Western analysis, rabbit MsrB polyclonal antibodies reacted positively with the protein extracts of the msrA1 mutants and the msrA1 msrA2 double mutants, indicating that the msrA1 mutants were indeed leaky for the synthesis of MsrB (Fig. 1a, lanes 3, 4, 7 and 8). To rule out the possibility of msrB transcription from the promoter of the kanamycin-resistance gene used to insertionally inactivate msrA1, we sequenced the plasmid-borne DNA fragment used during the msrA1 mutant construction and found that the kanamycin-gene promoter was in the opposite orientation to the msrA1/msrB promoter. In addition, in Northern analysis of the RNA extracted from the double mutant, we found a 3·8 kb band hybridizing when the msrA1 gene was used as a probe, unlike the 2·4 kb band found in the RNA from the wild-type cells (Fig. 2
a). However, when the kanamycin-resistance cassette was used as a probe with RNA extracted from the msrA1 mutant grown without and with oxacillin, we observed two bands (3·8 and 1·4 kb, respectively) (Fig. 2b
). In addition, the intensity of the 3·8 kb band was higher in the mutant cells exposed to oxacillin compared to mutant bacteria grown without oxacillin (Fig. 2b
). Northern-generated data provide evidence that, in the msrA1 mutant and the msrA1 msrA2 double mutant, the msrA1 promoter was able to direct increased transcription of a larger message leading to elevated synthesis of MsrB as observed in the Western analysis (Fig. 1a
, lanes 3, 4, 7 and 8). Also, as apparent from the Western blot, in the msrA1 mutant or the msrA1 msrA2 double mutant but not in the msrA2 mutant, MsrB synthesis was higher compared to the wild-type bacterium under normal physiological conditions of growth [Fig. 1a
, lanes 1 (wild-type), 3 and 7 (MsrA1 mutants)]. Increased MsrB levels in MsrA1 mutants suggest that the expression of msrB and msrA1 is dependent on the cellular levels of MsrA/MsrB, but the possibility of a larger msrB message being more stable and leading to increased synthesis of MsrB can not be ruled out.
|
|
Growth and oxacillin-resistance phenotypes of the MsrA1/MsrA2 mutants
Further characterization of the msrA2 mutant revealed its growth kinetics were comparable to those of the wild-type bacterium under normal physiological and various stress conditions including the presence of H2O2 (data not shown). However, unlike MsrA2 mutants, MsrA1 mutants showed slower growth kinetics under normal physiological conditions (Fig. 3). Another feature of the msrA1 mutant was its colony size. The colony size of the msrA1 mutant of all the S. aureus strains used was smaller than that of the wild-type. One can argue that the differences in the colony size might be due to slower growth of the msrA1 mutant. To confirm this observation, we plated cells from the mid-exponential phase (OD600 0·8) from the wild-type RN450 and its msrA1 mutant. On average, the msrA1 mutant had 1·4 times more colony-forming units than the wild-type at this density. Similar observations were recorded with strains BB270 and COL and their msrA1 mutants. This phenotype of smaller colony size was reversed in the msrA1 mutants complemented with the intact gene on a shuttle plasmid. This observation probably implies that the lack of MsrA1 activity in S. aureus not only slows growth but also limits the size of the bacterium.
|
In conclusion, in this study we have characterized the msrA1, msrA2 and msrB genes encoding Msr activity in S. aureus with respect to their oxacillin inducibility, H2O2 susceptibility and promoter strengths. Our data suggest that the MsrA1/MsrB system in S. aureus is physiologically more significant than other Msr proteins in this bacterium. The complexities of these Msr proteins as a whole as well as their individual significance in staphylococcal physiology need further investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Archer, G. L. (1998). Staphylococcus aureus: a well-armed pathogen. Clin Infect Dis 26, 11791181.[Medline]
Augustin, J., Rosenstein, R., Wieland, B., Schneider, U., Schnell, N., Engelke, G., Entian, K. D. & Götz, F. (1992). Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur J Biochem 204, 11491154.[Abstract]
Brot, N. & Weissbach, H. (1981). Chemistry and biology of Escherichia coli ribosomal protein L12. Mol Cell Biochem 36, 4763.[Medline]
Brot, N., Weissbach, L., Werth, J. & Weissbach, H. (1981). Enzymatic reduction of protein-bound methionine sulfoxide. Proc Natl Acad Sci U S A 78, 21552158.[Abstract]
Carp, H., Miller, F., Hoidal, J. R. & Janoff, A. (1982). Potential mechanism of emphysema: 1-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity. Proc Natl Acad Sci U S A 79, 20412045.[Abstract]
Dhandayuthapani, S., Blaylock, M. W., Bebear, C. M., Rasmussen, W. G. & Baseman, J. B. (2001). Peptide methionine sulfoxide reductase (MsrA) is a virulence determinant in Mycoplasma genitalium. J Bacteriol 183, 56455650.
Gabbita, S. P., Aksenov, M. Y., Lovell, M. A. & Markesbery, W. R. (1999). Decrease in peptide methionine sulfoxide reductase in Alzheimer's disease brain. J Neurochem 73, 16601666.[CrossRef][Medline]
Garner, M. H. & Spector, A. (1980). Selective oxidation of cysteine and methionine in normal and senile cataractous lenses. Proc Natl Acad Sci U S A 77, 12741277.[Abstract]
Gustafson, J. E., Berger-Bächi, B., Strassle, A. & Wilkinson, B. J. (1992). Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob Agents Chemother 36, 566572.[Abstract]
Hassouni, M. E., Chambost, J. P., Expert, D., Van Gijsegem, F. & Barras, F. (1999). The minimal gene set member msrA, encoding peptide methionine sulfoxide reductase, is a virulence determinant of the plant pathogen Erwinia chrysanthemi. Proc Natl Acad Sci U S A 96, 887892.
Hoshi, T. & Heinemann, S. (2001). Regulation of cell function by methionine oxidation and reduction. J Physiol 531, 111.
Kreiswirth, B. N., Lofdahl, S., Betley, M. J., O'Reilly, M., Schlievert, P. M., Bergdoll, M. S. & Novick, R. P. (1983). The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305, 709712.[Medline]
Kryukov, G. V., Kumar, R. A., Koc, A., Sun, Z. & Gladyshev, V. N. (2002). Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase. Proc Natl Acad Sci U S A 99, 42454250.
Kumar, R. A., Koc, A., Cerny, R. L. & Gladyshev, V. N. (2002). Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase. J Biol Chem 277, 3752737535.
Levine, R. L., Moskovitz, J. & Stadtman, E. R. (2000). Oxidation of methionine in proteins: roles in antioxidant defense and cellular regulation. IUBMB Life 50, 301307.[CrossRef][Medline]
Mead, D. A., Szczesna-Skorupa, E. & Kemper, B. (1986). Single-stranded DNA blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng 1, 6774.[Abstract]
Mei, J. M., Nourbakhsh, F., Ford, C. W. & Holden, D. W. (1997). Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol Microbiol 26, 399407.[Medline]
Moskovitz, J., Berlett, B. S., Poston, J. M. & Stadtman, E. R. (1997). The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo. Proc Natl Acad Sci U S A 94, 95859589.
Moskovitz, J., Flescher, E., Berlett, B. S., Azare, J., Poston, J. M. & Stadtman, E. R. (1998). Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress. Proc Natl Acad Sci U S A 95, 1407114075.
Moskovitz, J., Poston, J. M., Berlett, B. S., Nosworthy, N. J., Szczepanowski, R. & Stadtman, E. R. (2000). Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity. J Biol Chem 275, 1416714172.
Moskovitz, J., Bar-Noy, S., Williams, W. M., Requena, J., Berlett, B. S. & Stadtman, E. R. (2001). Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc Natl Acad Sci U S A 98, 1292012925.
Moskovitz, J., Singh, V. K., Requena, J., Wilkinson, B. J., Jayaswal, R. K. & Stadtman, E. R. (2002). Purification and characterization of methionine sulfoxide reductases from mouse and Staphylococcus aureus and their substrate stereospecificity. Biochem Biophys Res Commun 290, 6265.[CrossRef][Medline]
Novick, R. P. (1991). Genetic systems in staphylococci. Methods Enzymol 202, 587636.
Novick, R. P., Edelman, I. & Lofdahl, S. (1986). Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes. J Mol Biol 192, 209220.[Medline]
Olry, A., Boschi-Muller, S., Marraud, M., Sanglier-Cianferani, S., Van Dorsselear, A. & Branlant, G. (2002). Characterization of the methionine sulfoxide reductase activities of PILB, a probable virulence factor from Neisseria meningitidis. J Biol Chem 277, 1201612022.
Pfeltz, R. F., Singh, V. K., Schmidt, J. L., Batten, M. A., Baranyk, C. S., Nadakavukaren, M. J., Jayaswal, R. K. & Wilkinson, B. J. (2000). Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds. Antimicrob Agents Chemother 44, 294303.
Rodrigo, M. J., Moskovitz, J., Salamini, F. & Bartels, D. (2002). Reverse genetic approaches in plants and yeast suggest a role for novel, evolutionarily conserved, selenoprotein-related genes in oxidative stress defense. Mol Genet Genomics 267, 613621.[CrossRef][Medline]
Ruan, H., Tang, X. D., Chen, M. L. & 11 other authors (2002). High-quality life extension by the enzyme peptide methionine sulfoxide reductase. Proc Natl Acad Sci U S A 99, 27482753.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schenk, S. & Laddaga, R. A. (1992). Improved methods for electroporation of Staphylococcus aureus. FEMS Microbiol Lett 94, 133138.[CrossRef]
Singh, V. K., Jayaswal, R. K. & Wilkinson, B. J. (2001a). Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiol Lett 199, 7994.[CrossRef][Medline]
Singh, V. K., Moskovitz, J., Wilkinson, B. J. & Jayaswal, R. K. (2001b). Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin. Microbiology 147, 30373045.
Skaar, E. P., Tobiason, D. M., Quick, J., Judd, R. C., Weissbach, H., Etienne, F., Brot, N. & Seifert, H. S. (2002). The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species. Proc Natl Acad Sci U S A 99, 1010810113.
Stadtman, E. R. (2001). Protein oxidation in aging and age-related diseases. Ann N Y Acad Sci 928, 2238.
Stadtman, E. R., Moskovitz, J., Berlett, B. S. & Levine, R. L. (2002). Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism. Mol Cell Biochem 234235, 39.[CrossRef]
Sutherland, R. & Rollinson, G. N. (1964). Characteristics of methicillin resistant staphylococci. J Bacteriol 87, 887889.
Truscott, R. J. & Augusteyn, R. C. (1977). Oxidative changes in human lens proteins during senile nuclear cataract formation. Biochim Biophys Acta 492, 4352.[Medline]
Wizemann, T. M., Moskovitz, J., Pearce, B. J., Cundell, D., Arvidson, C. G., So, M., Weissbach, H., Brot, N. & Masure, H. R. (1996). Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens. Proc Natl Acad Sci U S A 93, 79857990.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103119.[CrossRef][Medline]
Received 26 April 2003;
revised 13 June 2003;
accepted 13 June 2003.