Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790, USA1
Laboratory of Biochemistry, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20982, USA2
Author for correspondence: Radheshyam K. Jayaswal. Tel: +1 309 438 5128. Fax: +1 309 438 3722. e-mail: drjay{at}ilstu.edu
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ABSTRACT |
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Keywords: PilB, oxidative stress, MsrA, antibiotic stress
Abbreviations: MBC, minimal bactericidal concentration; Met(O), methionine sulfoxide
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INTRODUCTION |
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Interestingly, increased expression of MsrA has been observed under different environmental conditions and studies on msrA mutants in various organisms have provided evidence that MsrA functions beyond its role in oxidative stress. In Escherichia coli, MsrA synthesis was increased about threefold in stationary phase (Moskovitz et al., 1995 ), suggesting it to be a stationary-phase response protein. In addition, an increase in pH of the growth medium has been reported to cause increased msrA expression in Streptococcus gordonii (Vriesema et al., 2000
). MsrA synthesis was also induced in a rabbit model of infective endocarditis (Kili et al., 1999
). The MsrA proteins have been shown to contribute to the maintenance of adhesins in three major pathogens, viz., Streptococcus pneumoniae, Neisseria gonorrhoeae and E. coli (Wizemann et al., 1996
), and to be a virulence determinant of the plant pathogen Erwinia chrysanthemi (Hassouni et al., 1999
).
Although MsrA was determined to have a role in the virulence of Staphylococcus aureus (Mei et al., 1997 ), its precise role in this bacterium has not been elucidated. We recently identified a S. aureus MsrA homologue to be induced in response to cell-wall-active antibiotics (Singh et al., 2001
). In this paper, we report on the cloning and molecular characterization of the chromosomal locus that encodes S. aureus MsrA. Northern blot analysis indicates that the S. aureus msrA gene is transcribed as a polycistronic message with three additional downstream genes. An insertional msrA knockout mutation increased the sensitivity of the bacterium to oxidative stress conditions.
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METHODS |
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RNA analysis.
To confirm oxacillin-induced synthesis of MsrA overnight cultures of S. aureus were diluted 100-fold in 25·0 ml of fresh TSB and grown to an OD600 of 0·3. At this point, 1·2 µg ml-1 oxacillin (a concentration that was found to be optimum for MsrA induction in our previous study; Singh et al., 2001 ) was added and the cells were allowed to grow for 1 h. Fifteen millimolar H2O2 was added to one of the S. aureus RN450 cultures to generate conditions comparable to oxacillin stress, as indicated by the degree of growth inhibition. The culture grown for 1 h without the addition of H2O2 or oxacillin after reaching a culture density of 0·3 at OD600 was used as a control. Cells were harvested by centrifugation, resuspended in 20 mM Tris/HCl buffer (pH 7·5) with 145 mM NaCl, and were lysed by the addition of 50 µg lysostaphin for 10 min at room temperature. Total RNA was subsequently isolated using a Qiagen RNeasy mini kit. Equal amounts of RNA (10 µg) from each sample were run on a 1·2% denaturing agarose gel and transferred to a nylon membrane. The construct pRSETa-msrA was digested with BamHI and HindIII; the msrA gene fragment was gel-purified, radiolabelled using the Prime-a-Gene labelling system (Promega) in the presence of [
-32P]dCTP (specific activity >3000 Ci mmol-1 [111000 Bq], ICN Pharmaceuticals) and used to probe the membrane.
Determination of msrA promoter activity under different environmental conditions.
Construction of an msrA promoter::lacZ reporter strain has been previously described (Singh et al., 2001 ). In this construct, a 1·3 kb DNA fragment starting 44 nt downstream and going upstream of the msrA gene was cloned in the correct orientation in front of the promoterless lacZ gene of the vector pAZ106 (Chan et al., 1998
) and subsequently fused in the chromosome of S. aureus RN450. ß-Galactosidase activity in the reporter strain (control and oxacillin-treated) was assayed using ONPG as the substrate (Miller, 1972
).
Primer extension reaction.
The primer extension reaction was carried out as described previously (Xiong & Jayaswal, 1998 ). Total RNA was isolated from the oxacillin-stressed cells of S. aureus and 5·0 µg of this RNA was reverse-transcribed using primer P1 (5'-CACCAGAAACATCCTCCTGC-3') in the presence of radiolabelled dATP (
-32P, specific activity >3000 Ci mmol-1). A DNA sequencing ladder was generated using a 70770 Sequenase DNA sequencing kit (version 2.0; USB), primer P1 and an appropriate DNA fragment; the ladder was electrophoresed alongside the product of the primer extension reaction to determine the position of the transcription start site.
Overexpression and purification of S. aureus MsrA and PilB for determination of methionine sulfoxide reductase activity.
The msrA ORF was PCR amplified using S. aureus RN450 genomic DNA as the template, a 5' sense primer (P2) containing a BamHI site (underlined) (5'-GGATCCATGACAAAAGAATATGCAAC-3') and a 3' reverse complement primer (P3) containing a HindIII site (underlined) (5'-AAGCTTAGCATTTTGATTCCCCCAATGTG-3'). The amplified msrA gene was cloned in-frame at the BamHI and HindIII sites of the overexpression vector pRSETa (Invitrogen). E. coli strain BLR(DE3)pLysS (Novagen) was subsequently transformed with the construct pRSETa-msrA. The resulting transformants were grown in LB containing ampicillin (50 µg ml-1), chloramphenicol (30 µg ml-1), and tetracycline (12 µg ml-1) to an OD600 of 0·4 and induced for the synthesis of His-tagged MsrA by the addition of 2·5 mM IPTG for 2·5 h. The induced culture was harvested, resuspended in 50 mM Tris/HCl buffer (pH 7·5), sonicated, and centrifuged. The supernatant was applied to a nickel-charged-agarose-affinity-column and eluted with 400 mM imidazole using the Xpress Purification system (Invitrogen). Fractions containing the overexpressed His-tagged MsrA were pooled, dialysed, and concentrated against 50 mM Tris/HCl, 50 mM NaCl, 1 mM DTT, pH 7·5. The methionine sulfoxide reducatse activity of the purified protein was assayed using 1 mM Dabsyl-Met(O) and 20 mM DTT in 50 mM Tris/HCl (pH 7·5) following incubation at 37 °C for 30 min, as described by Moskovitz et al. (1997) . The pilB ORF was amplified using S. aureus RN450 genomic DNA as the template, a 5' sense primer (P4) containing a BamHI site (underlined) (5'-GGATCCATGCTTAAAAAAGATAAA-3'), and a 3' reverse complement primer (P5) containing a HindIII site (underlined) (5'-AAGCTTCTTATCAAAATGTGATATTA-3'). The amplified pilB gene was cloned in-frame at BamHI and HindIII sites of pRSETa, overexpressed, purified, and assayed for methionine sulfoxide reductase activity essentially as described above for MsrA.
Construction of an msrA knockout mutant in S. aureus.
To create an msrA null mutant, primers P1 and P6 (5'-AAACCACTACCGAATCGTCG-3') were used to amplify a 1·3 kb DNA fragment, using S. aureus RN450 chromosomal DNA as the template, which represented the left flanking region of the msrA gene starting 44 nt downstream of the msrA start codon. Primers P7 (5'-CAGGAGGATGTTTCTGGTGC-3') and P8 (5'-GCCGCCACTTTCTTTAGGTC-3') were used to amplify an 831 bp fragment which represented the right flanking region of the msrA gene starting 25 nt downstream of the msrA start codon. These two fragments were ligated together in vector pTZ18R (Mead et al., 1986 ) resulting in the construct pTZ-msr, which simultaneously generated a unique EcoRI restriction site between the ligated fragments. A 1·5 kb kanamycin gene cassette was inserted into this EcoRI site, resulting in the construct pTZ-MK. The vector pTZ18R cannot replicate in Gram-positive bacteria; thus pTZ-MK was used as a suicidal construct to transform S. aureus RN4220 cells by electroporation (Schenk & Laddaga, 1992
). Selection of the transformants on kanamycin plates led to the integration of the entire construct into the chromosome. Phage-80
was propagated on these transformants and used to resolve the mutation in the msrA gene in the S. aureus strains RN450 and COL by performing transductional outcrosses, as described by Novick et al. (1986)
.
Determination of the sensitivity of the msrA mutant to oxacillin and H2O2.
The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for the S. aureus strains were determined as described by Pfeltz et al. (2000 ) using 96-well microtitre plates with serial dilutions of oxacillin (1000 to 50 µg ml-1) and H2O2 (12 to 0·125 mM) in TSB. The overnight bacterial cultures were diluted to a final concentration of 1x106 cfu ml-1 in each well and the plates were incubated at 37 °C for 48 h. In H2O2 sensitivity studies, the msrA mutant was complemented in trans with the msrA gene on the shuttle vector pCU1 (Augustin et al., 1992
). To clone the entire msrA gene into pCU1, a 1110 bp fragment was amplified using the primers P8 and P9 (5'-CAAATCCATTGGTGTCGC-3') starting 254 bp upstream of the msrA start codon, thus representing the entire msrA gene. This construct, pCU-msr1, was transferred to S. aureus RN4220 by electroporation and subsequently transduced into the msrA mutant strain resulting in strain MC1. The msrA mutant was also complemented with a 2767 bp PCR-amplified fragment and cloned in the vector pCU1 using primers P9 and P10 (5'-CGTCTTTTTCTTCACTACCTC-3'), again starting 254 bp upstream of the msrA start codon, resulting in strain MC2. To maintain identical conditions during the H2O2 tolerance study, the wild-type and the msrA mutant were transformed with the empty plasmid pCU1. Subsequently, the parent and the mutant transformed with pCU1, MC1 and MC2 were diluted to 2·5x107 cfu ml-1 from their respective overnight cultures in 100 ml fresh TSB containing 10 µg chloramphenicol ml-1 and 8 mM H2O2. The number of c.f.u. in each culture was determined with respect to time.
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RESULTS |
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Analysis of msrA expression
The expression of the msrA gene was studied at the transcriptional level to complement the observation of induced MsrA synthesis in oxacillin-stressed S. aureus cells (Singh et al., 2001 ). In Northern blot analysis, only a faint band corresponding to the msrA transcript could be detected in the lanes representing RNA from the non-stressed control or H2O2-treated RN450 cells (Fig. 1
, lanes 1 and 2). However, the level of msrA transcript in the RNA from oxacillin-treated RN450 cells was higher, as a significantly darker band was observed (Fig. 1
, lane 3). The size of the msrA transcript on the Northern blot was determined to be
2·3 kb (Fig. 1
), which is considerably larger than the 531 nt msrA ORF. Determination of ß-galactosidase activity in an msrA promoter::lacZ reporter strain revealed a significant increase in ß-galactosidase activity when actively growing cultures of this reporter strain were exposed to oxacillin (Fig. 2a
, b
); however, other stress conditions had no appreciable effect when applied for 1 h (Fig. 2b
). These studies also revealed that under normal physiological conditions the msrA promoter is maximally active during late exponential phase (Fig. 2a
). In addition, the oxacillin-induced msrA promoter activity was apparent in growing cells from early exponential to stationary phase (Fig. 2a
). Detection of no apparent ß-galactosidase activity in the reporter strain exposed to 46 °C might be due to the inactivation of the enzyme at this elevated temperature, rather than to suppression of msrA promoter activity (Fig. 2b
).
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Growth kinetics were determined for all four strains in liquid cultures containing 8 mM H2O2. The mutant bacteria were almost completely killed under these conditions, whereas the parent bacteria were able to grow after a lag phase (Fig. 4). However, the two complemented clones responded more efficiently and were able to grow more rapidly than the parent (Fig. 4
). A similar response was observed in liquid culture of these strains in the presence of 8 mM methyl viologen (paraquat), where the growth of the mutant was slower compared to that of the parent and the two msrA-complemented strains (data not shown).
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DISCUSSION |
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The active sites of the MsrA enzymes of E. coli and Bos taurus have been mapped and shown to possess an identical active site, GCFWG (Lowther et al., 2000 ; Moskovitz et al., 2000
). Comparative analysis of the amino acid sequences indicates that in the case of S. aureus MsrA the characteristic MsrA active site differs at one amino acid: GCFWC in place of GCFWG. However, purified S. aureus MsrA was shown to possess methionine sulfoxide reductase activity, which confirms the identity of the oxacillin-induced protein as an authentic MsrA, despite a modified active site. Although the active site of the MsrA in B. subtilis has not been mapped, amino acid analysis indicates that the active site of B. subtilis MsrA (Hayes et al., 1998
) is also GCFWC. In this context, very high methionine sulfoxide reductase activity for the protein product of the second gene of the msrA operon is quite interesting as the deduced protein shows >50% homology to the family of PilB transcriptional repressors, as per the database entry. However, the precise role of these proteins in the regulation of bacterial transcription is not yet known. More significantly, this PilB homologue was over 50% homologous to the C-terminus of S. gordonii (Vriesema et al., 2000
) and S. pneumoniae (Wizemann et al., 1996
) MsrA proteins, as well as those of several other bacterial species. In addition, NCBI database searches indicate that in numerous bacteria the gene encoding this protein is present just downstream of the msrA gene, in an operon-like organization. This indicates that the presence of a pilB-like gene (along with msrA) is conserved across many bacterial species, either as a separate gene entity or as an integral part of a larger msrA. However, to date, none of the PilB proteins that are expressed separately have been reported to possess MsrA-like functions. Interestingly, in the case of S. aureus, this protein shows 28-fold higher methionine sulfoxide reductase activity, but it lacks the characteristic active site GCFWG of the MsrA proteins. This indicates that the methionine sulfoxide reductase activity of PilB occurs via different, unknown mechanisms.
We previously observed that cell-wall-active antibiotics induced the S. aureus msrA operon (Singh et al., 2001 ). Studies employing a reporter strain with the msrA promoter cloned upstream of a promoterless lacZ gene and fused in the chromosome of the S. aureus strain RN450 revealed that the msrA promoter was not inducible by environmental stresses other than oxacillin. Increased expression of msrA has been reported upon an increase in pH of the growth medium in S. gordonii (Vriesema et al., 2000
); however, we did not see any significant increase in the msrA promoter activity in response to pH upshift or downshift in this study. Maximum msrA promoter activity in late-exponential and stationary phase is consistent with the observation of a threefold increase in the production of MsrA during this stage of growth in E. coli (Moskovitz et al., 1995
). Furthermore, as in E. coli, H2O2 did not induce the expression of the S. aureus msrA at the transcriptional level. This suggests that msrA expression is not under the control of the oxyR regulon that controls the oxidative stress response in bacteria (Moskovitz et al., 1995
). Although S. aureus does not have an OxyR homologue it does have proteins like PerR, which perform similar functions such as regulating the transcription of the gene encoding oxidative stress resistance proteins (Horsburgh et al., 2001
).
Interestingly, oxacillin induced the expression of S. aureus msrA but a mutation in the msrA gene did not affect the oxacillin MIC either in an oxacillin-sensitive or in an oxacillin-resistant staphylococcal strain. However, the msrA mutant showed higher sensitivity to H2O2 compared to the wild-type parent in the MIC determinations, although there was no appreciable difference in the growth kinetics of the mutant and the parent in liquid culture containing 4 mM H2O2 or paraquat. The increased sensitivity of the mutant was clearly observed in the presence of 8 mM H2O2, a concentration considered lethal in most other bacteria. At this concentration S. aureus was able to grow after a considerably longer lag; however, the msrA mutant failed to grow at all. For Northern blot experiments 15 mM H2O2 was added to actively growing cultures (OD600 of 0·3) where the bacteria showed higher tolerance, which is believed to be due to increased cell density. In these H2O2 tolerance studies the msrA mutant, complemented either with the msrA gene alone or with the entire msrA operon, responded more efficiently than even the wild-type parent. This is probably because of the presence of msrA on a high copy plasmid. The oxidative stress tolerance of msrA mutants of E. coli and B. subtilis were comparable to their respective wild-type parents in liquid cultures (Moskovitz et al., 1995 ; Hayes et al., 1998
). In E. coli increased sensitivity to H2O2 could be seen in msrA mutants only when grown on agar (Moskovitz et al., 1995
). The authors concluded that the unaffected oxidative stress tolerance of the mutant in liquid culture was probably due to the overriding effects of other protective mechanisms (Hayes et al., 1998
). However, the role of MsrA in oxidative stress is visible in liquid culture, which may suggest that MsrA is more important in oxidative stress resistance in S. aureus than it is in E. coli. In addition, a recent GenBank search reveals the presence of another locus in the S. aureus genome that encodes an MsrA-like protein (GI no. 12656489). This protein has also been identified in whole-genome sequence analyses of two methicillin-resistant S. aureus strains (GI no. 13701158) (Kuroda et al., 2001
). The protein is 50% homologous to the MsrA reported in the present study and contains the MsrA active site GCFWC; however, the genes encoding these two staphylococcal MsrA proteins share no homology with each other at the nucleotide level. It is also quite likely that this additional MsrA protein accounts for the partial tolerance of our S. aureus msrA mutant to H2O2, but the bacterium may need both msrA genes for full-scale recovery under more acute oxidative stress conditions.
A mutant with reduced adherence to type II lung epithelial and human vein endothelial cells was identified in S. pneumoniae. The protein encoded by the disrupted gene displayed >40% identity with the MsrA of E. coli and >50% identity with the C-terminal region of gonococcal PilB (Taha et al., 1988 , 1992
; Wizemann et al., 1996
). In addition, an Erwinia chrysanthemi msrA mutant was less motile on solid medium (Hassouni et al., 1999
). A role for MsrA has also been observed in the expression of adhesins in S. pneumoniae, N. gonorrhoeae and E. coli (Wizemann et al., 1996
). It has been argued that MsrA has a special role in repairing surface layers (Hassouni et al., 1999
). However, as MsrA is cytoplasmic and the pilins or adhesins are located outside the cell, the authors speculate that MsrA also functions as a repair system and as a regulator for the production of the extracellular appendices (Wizemann et al., 1996
). As MsrA and PilB have been speculated to repair the cell surface (Wizemann et al., 1996
), induction of the msrA operon by antibiotics that interfere with cell-wall synthesis might be an attempt by the bacterium to protect its cell surface. However, the roles of MsrA and PilB in the oxidative stress response and cell surface repair need further investigation. Experiments are currently under way to determine the precise roles of the individual ORFs of the msrA locus.
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ACKNOWLEDGEMENTS |
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This work was supported by a postdoctoral fellowship from the American Heart Association Midwest Affiliate to V.K.S.; grant 1 R15 AI43027-01 from the National Institutes of Health to B.J.W; and a grant-in-aid from the American Heart Association Midwest Affiliate to R.K.J.
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Received 18 April 2001;
revised 8 July 2001;
accepted 31 July 2001.