# Regulation of Expression of Oxacillin-Inducible Methionine Sulfoxide Reductases inStaphylococcus aureus

Authors: Kyle R. Baum; Zulfiqar Ahmad; Vineet K. Singh
Journal: International Journal of Microbiology
 (2015)

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## Abstract

Cell wall-active antibiotics cause induction of a locus that leads to elevated synthesis of two methionine sulfoxide reductases (MsrA1 and MsrB) inStaphylococcus aureus. To understand the regulation of this locus, reporter strains were constructed by integrating a DNA fragment consisting of the msrA1/msrB promoter in front of a promoterless lacZ gene in the chromosome of wild-type and MsrA1-, MsrB-, MsrA1/MsrB-, and SigB-deficient methicillin-sensitive S. aureus strain SH1000 and methicillin-resistant S. aureus strain COL. These reporter strains were cultured in TSB and the cellular levels of β-galactosidase activity in these cultures were assayed during different growth phases. β-galactosidase activity assays demonstrated that the lack of MsrA1, MsrB, and SigB upregulated the msrA1/msrB promoter in S. aureus strain SH1000. In S. aureus strain COL, the highest level of β-galactosidase activity was observed under the conditions when both MsrA1 and MsrB proteins were absent. The data suggest that the msrA1/msrB locus, in part, is negatively regulated by MsrA1, MsrB, and SigB in S. aureus.


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## Body

## 1. Introduction

Staphylococcus aureus is part of the microbiome of roughly 30% of people who show no clinical symptoms [1]. It is an opportunistic human pathogen that can cause a wide variety of diseases and can involve any organ system in the human body. Diseases caused byS. aureus may include mild skin infections such as folliculitis and impetigo to fatal conditions such as pneumonia, osteomyelitis, and endocarditis [2]. Treatment ofS. aureus infections has become problematic as it has developed numerous mechanisms to become resistant to almost all known antibiotics [3, 4].It was previously reported that exposure ofS. aureus to oxacillin and other cell wall-active antibiotics increases the expression ofmsrA1 andmsrB both at the transcriptional and at the protein level [5, 6]. Pathogenic bacterial species are exposed to a variety of extremely potent reactive oxygen species (ROS) by the host phagocytic cells during the course of phagocytosis that are damaging to all cellular macromolecules. ROS can cause damage to proteins by the oxidation of sulfhydryl groups, reduction of disulfides, oxidative adduction of amino acid residues close to metal-binding sites, and peptide fragmentation [7]. In particular, ROS oxidize the sulfur atom of protein-bound methionine residues resulting in methionine sulfoxide (MetO) and loss of protein function. However, almost all biological species possess the ability to reduce oxidized methionines [8]. MsrA and MsrB proteins reduce S- and R-epimers of methionine sulfoxides (MetO), respectively [8].InS. aureus, genes encoding MsrA1 and MsrB are the first and second genes of a four-gene cluster that are cotranscribed [6]. A mutation in themsrA1 gene increased the susceptibility ofS. aureus to oxidative stress [6, 9]. More recently, it was shown that the MsrA1 protein was critical forS. aureus in establishing an infection in mice [10]. Interestingly, the MsrA1-deficientS. aureus was shown to possess an elevated level of MsrB [9] giving rise to the speculation of autoregulation of themsrA1/msrB locus. Additionally, sigma factor B (SigB) is an alternative sigma factor that is involved in regulating the expression of stress response genes inS. aureus [11]. Thus, it seems plausible that SigB may have a role in the regulation of themsrA1/msrB locus. Findings of this study provide evidence that themsrA1/msrB locus is negatively regulated by the products of this locus and SigB.


## 2. Materials and Methods

### 2.1. Bacterial Strains, Antibiotics, and Growth Conditions

The bacterial strains used in this study are shown in Table1.S. aureus cultures were grown aerobically at 37°C in tryptic soy broth (TSB) in a shaking incubator (220 rpm) or on tryptic soy agar (TSA) by incubation for 24–48 h. Overnight cultures ofS. aureus reporter strains were prepared in the presence of erythromycin at 10 μg mL−1. Oligonucleotide primers used in this study were obtained from Eurofins and are shown in Table 2.Table 1
Bacterial strains used in this study.
Strains
Characteristics
Reference
SH1000
S. aureus strain 8325-4 with functional RsbU
[11]
COL
Homogeneous in methicillin-resistance expression
[37]
SH1000ΔmsrA1
msrA1 mutant of SH1000
[10]
SH1000ΔmsrB
msrB mutant of SH1000
[10]
SH1000ΔmsrA1-msrB
msrA1-msrB double mutant of SH1000
[10]
SH1000ΔsigB
sigB mutant of SH1000
[14]
COLΔmsrA1
msrA1 mutant of SH1000
[6]
COLΔmsrB
msrB mutant of SH1000
This study
COLΔmsrA1-msrB
msrA1-msrB double mutant of SH1000
This study
COLΔsigB
sigB mutant of SH1000
[38]
SH1000-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in SH1000 (ErmR)
[13]
SH1000ΔmsrA1-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrA1 mutant of SH1000 (KanR, ErmR)
This study
SH1000ΔmsrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrB mutant of SH1000 (KanR, ErmR)
This study
SH1000ΔmsrA1-msrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msr1-msrB mutant of SH1000 (KanR, ErmR)
This study
SH1000ΔsigB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in sigB mutant of SH1000 (KanR, ErmR)
This study
COL-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in COL (ErmR)
This study
COLΔmsrA1-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrA1 mutant of COL (KanR, ErmR)
This study
COLΔmsrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrB mutant of COL (KanR, ErmR)
This study
COLΔmsrA1-msrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrA1-msrB mutant of COL (KanR, ErmR)
This study
COLΔsigB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in sigB mutant of COL (KanR, ErmR)
This study
Ka
n
R: kanamycin resistant; ErmR: erythromycin resistant.Table 2
Oligonucleotide primers used in this study.
Oligo
Sequence (5′→3′)
P1
GCTAACGTCATTGAATATG
P2
GGAAGTAACCTCTGGATCA
P3
GTTACACAAGAAAACGGCA
P4
TCATCATCGTGTTTTGGG
P5
AGGATGTTTCTGGTGCATGG
P6
GACACAACTTCTCCTTCAGT
P7
CCTTTGAACGGAAGTTTGA
P8
TCTAATAGCAACCCACCT
P9
GCTAACGTCATTGAATATG
P10
GGATGGTTCGGATAATGC
P11
GATTGGGATCATAGCGTCA
P12
CTTCAGAGTTAATGGGACCA
P13
AGGCATCAAGTCAGTCGTATC
P14
GAAGTAACCTCTGGATCAAACG
P15
GGTATGGTAAGAACTGAAGTGC
P16
ATTGCAGCGGAATTGATACAG
P17
TCTCCAATTGCAGGACGTGT
P18
ACACTTCAAATCCTTCACCGTCT
P19
TCCACAAGTCGCACGTACAG
P20
GGAAGGCTTGCTACATCTAACG


### 2.2. Transduction ofmsrA1/msrB Promoter-lacZ intoS. aureus Strains

Construction ofmsrA1/msrB promoter-lacZ reporter strain has been previously described [6]. In this construct, a 1.3 kb DNA fragment starting 44 nucleotides downstream and going upstream of themsrA1 gene cloned in front of a promoterlesslacZ gene in the vector pAZ106 [12] was integrated in the chromosome ofS. aureus strain RN450 [5, 6]. ThemsrA1/msrB promoter-lacZ reporter was transduced into various strains ofS. aureus using a phage 80α transduction procedure. Strains used in this study were verified by PCR.


### 2.3. Determination of themsrA1/msrB Promoter Strength inS. aureus

To determine if themsrA1/msrB locus is autoregulated, the expression oflacZ from themsrA1/msrB promoter-lacZ fusion was investigated in MsrA1-, MsrB-, and MsrA1-MsrB-deficient strains ofS. aureus strains SH1000 and COL. In addition, SigB is a major regulator of stress response inS. aureus. Therefore, the strength of themsrA1/msrB promoter was also assessed in asigB mutant. Overnight cultures of these strains were diluted (1 : 100) and grown at 37°C with shaking. These cultures were grown to OD600 = 0.5 that was considered time 0 and the levels of β-galactosidase activity in these cultures were measured at different time points (0, 90, 180, 270, and 360 min) as an indicator of the strength of themsrA1/msrB promoter.


### 2.4. Expression ofmsrA1/msrB Promoter in the Presence of a Cell Wall-Active Antibiotic, Oxacillin

Previous studies [5, 6, 9, 10, 13] have shown that, in the presence of oxacillin, there is an increased production of MsrA1 and MsrB inS. aureus. To further investigate the regulation of themsrA1/msrB locus and to see if it can be magnified in the presence of oxacillin, overnight cultures of wild-type and the derivativemsrA1-msrB mutant ofS. aureus strain COL were diluted (1 : 100) in fresh TSB and grown to OD600 of 0.5. 10.0 mL of the culture was split into two 15 mL tubes. To one of the cultures, oxacillin was added to the final concentration of 1.0 mg mL−1. Both cultures with and without oxacillin were allowed to grow for an additional 2 h at 37°C with shaking. Bacterial cells were harvested by centrifugation and β-galactosidase activities in these cells were measured.


### 2.5. Measurement ofβ-Galactosidase Activity

The OD600 of the culture was determined as a measure of cell density and cells were subsequently collected by centrifugation. For precise optical density readings, cultures were diluted appropriately to bring density into measurable range. The cell pellet was used to measure β-galactosidase activity as described previously using O-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate [5, 6, 13].


### 2.6. Quantitative Real-Time PCR (qRT-PCR) Assays

qRT-PCR assays were used to verify induced expression of the genes of themsrA1/msrB locus under oxacillin stress and to validate thelacZ reporter expression data insigB mutants. Cultures ofS. aureus strain COL were grown to OD600 = 0.3 and divided into two tubes. One tube was stressed with oxacillin at a concentration of 1.0 mg mL−1 for 2 h. Total RNA was extracted from these oxacillin stressed and control cultures as described previously [14]. For the validation oflacZ data, the wild-type andsigB mutant strains ofS. aureus were allowed to grow for 90 min and 6 h after reaching the OD600 = 0.5 and total RNA from these cultures were extracted. cDNA from DNase treated 0.5 μg of total RNA was synthesized in a 20 μL reverse transcription reaction containing random hexamers and SuperScript III reverse transcriptase (Invitrogen). All real-time PCR reactions were carried out with Bio-Rad iCycler (iQ5 system). The transcript level ofmsrA1 was quantified using primers P13 and P14, that ofmsrB was quantified using P15 and P16, and that of the gene encoding the IIa(PTS) was quantified using primers P17 and P18. Transcript levels of genes were normalized to DNA gyrase mRNA using primers P19 and P20 based on a previous report [15, 16]. Changes in gene expression were calculated using the formula 2-ΔΔCq as described [17].


### 2.7. Statistical Analysis

All results are reported as the mean ± SE of at least three independent experiments. Data were analyzed with Student’st-test using R Studio for Windows (version 0.98.1103, 3.1.3). Statistical significance was set at p≤0.05.


## 2.1. Bacterial Strains, Antibiotics, and Growth Conditions

The bacterial strains used in this study are shown in Table1.S. aureus cultures were grown aerobically at 37°C in tryptic soy broth (TSB) in a shaking incubator (220 rpm) or on tryptic soy agar (TSA) by incubation for 24–48 h. Overnight cultures ofS. aureus reporter strains were prepared in the presence of erythromycin at 10 μg mL−1. Oligonucleotide primers used in this study were obtained from Eurofins and are shown in Table 2.Table 1
Bacterial strains used in this study.
Strains
Characteristics
Reference
SH1000
S. aureus strain 8325-4 with functional RsbU
[11]
COL
Homogeneous in methicillin-resistance expression
[37]
SH1000ΔmsrA1
msrA1 mutant of SH1000
[10]
SH1000ΔmsrB
msrB mutant of SH1000
[10]
SH1000ΔmsrA1-msrB
msrA1-msrB double mutant of SH1000
[10]
SH1000ΔsigB
sigB mutant of SH1000
[14]
COLΔmsrA1
msrA1 mutant of SH1000
[6]
COLΔmsrB
msrB mutant of SH1000
This study
COLΔmsrA1-msrB
msrA1-msrB double mutant of SH1000
This study
COLΔsigB
sigB mutant of SH1000
[38]
SH1000-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in SH1000 (ErmR)
[13]
SH1000ΔmsrA1-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrA1 mutant of SH1000 (KanR, ErmR)
This study
SH1000ΔmsrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrB mutant of SH1000 (KanR, ErmR)
This study
SH1000ΔmsrA1-msrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msr1-msrB mutant of SH1000 (KanR, ErmR)
This study
SH1000ΔsigB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in sigB mutant of SH1000 (KanR, ErmR)
This study
COL-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in COL (ErmR)
This study
COLΔmsrA1-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrA1 mutant of COL (KanR, ErmR)
This study
COLΔmsrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrB mutant of COL (KanR, ErmR)
This study
COLΔmsrA1-msrB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in msrA1-msrB mutant of COL (KanR, ErmR)
This study
COLΔsigB-(A1/B)P-lacZ
msrA1/msrB promoter-lacZ fusion in sigB mutant of COL (KanR, ErmR)
This study
Ka
n
R: kanamycin resistant; ErmR: erythromycin resistant.Table 2
Oligonucleotide primers used in this study.
Oligo
Sequence (5′→3′)
P1
GCTAACGTCATTGAATATG
P2
GGAAGTAACCTCTGGATCA
P3
GTTACACAAGAAAACGGCA
P4
TCATCATCGTGTTTTGGG
P5
AGGATGTTTCTGGTGCATGG
P6
GACACAACTTCTCCTTCAGT
P7
CCTTTGAACGGAAGTTTGA
P8
TCTAATAGCAACCCACCT
P9
GCTAACGTCATTGAATATG
P10
GGATGGTTCGGATAATGC
P11
GATTGGGATCATAGCGTCA
P12
CTTCAGAGTTAATGGGACCA
P13
AGGCATCAAGTCAGTCGTATC
P14
GAAGTAACCTCTGGATCAAACG
P15
GGTATGGTAAGAACTGAAGTGC
P16
ATTGCAGCGGAATTGATACAG
P17
TCTCCAATTGCAGGACGTGT
P18
ACACTTCAAATCCTTCACCGTCT
P19
TCCACAAGTCGCACGTACAG
P20
GGAAGGCTTGCTACATCTAACG


## 2.2. Transduction ofmsrA1/msrB Promoter-lacZ intoS. aureus Strains

Construction ofmsrA1/msrB promoter-lacZ reporter strain has been previously described [6]. In this construct, a 1.3 kb DNA fragment starting 44 nucleotides downstream and going upstream of themsrA1 gene cloned in front of a promoterlesslacZ gene in the vector pAZ106 [12] was integrated in the chromosome ofS. aureus strain RN450 [5, 6]. ThemsrA1/msrB promoter-lacZ reporter was transduced into various strains ofS. aureus using a phage 80α transduction procedure. Strains used in this study were verified by PCR.


## 2.3. Determination of themsrA1/msrB Promoter Strength inS. aureus

To determine if themsrA1/msrB locus is autoregulated, the expression oflacZ from themsrA1/msrB promoter-lacZ fusion was investigated in MsrA1-, MsrB-, and MsrA1-MsrB-deficient strains ofS. aureus strains SH1000 and COL. In addition, SigB is a major regulator of stress response inS. aureus. Therefore, the strength of themsrA1/msrB promoter was also assessed in asigB mutant. Overnight cultures of these strains were diluted (1 : 100) and grown at 37°C with shaking. These cultures were grown to OD600 = 0.5 that was considered time 0 and the levels of β-galactosidase activity in these cultures were measured at different time points (0, 90, 180, 270, and 360 min) as an indicator of the strength of themsrA1/msrB promoter.


## 2.4. Expression ofmsrA1/msrB Promoter in the Presence of a Cell Wall-Active Antibiotic, Oxacillin

Previous studies [5, 6, 9, 10, 13] have shown that, in the presence of oxacillin, there is an increased production of MsrA1 and MsrB inS. aureus. To further investigate the regulation of themsrA1/msrB locus and to see if it can be magnified in the presence of oxacillin, overnight cultures of wild-type and the derivativemsrA1-msrB mutant ofS. aureus strain COL were diluted (1 : 100) in fresh TSB and grown to OD600 of 0.5. 10.0 mL of the culture was split into two 15 mL tubes. To one of the cultures, oxacillin was added to the final concentration of 1.0 mg mL−1. Both cultures with and without oxacillin were allowed to grow for an additional 2 h at 37°C with shaking. Bacterial cells were harvested by centrifugation and β-galactosidase activities in these cells were measured.


## 2.5. Measurement ofβ-Galactosidase Activity

The OD600 of the culture was determined as a measure of cell density and cells were subsequently collected by centrifugation. For precise optical density readings, cultures were diluted appropriately to bring density into measurable range. The cell pellet was used to measure β-galactosidase activity as described previously using O-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate [5, 6, 13].


## 2.6. Quantitative Real-Time PCR (qRT-PCR) Assays

qRT-PCR assays were used to verify induced expression of the genes of themsrA1/msrB locus under oxacillin stress and to validate thelacZ reporter expression data insigB mutants. Cultures ofS. aureus strain COL were grown to OD600 = 0.3 and divided into two tubes. One tube was stressed with oxacillin at a concentration of 1.0 mg mL−1 for 2 h. Total RNA was extracted from these oxacillin stressed and control cultures as described previously [14]. For the validation oflacZ data, the wild-type andsigB mutant strains ofS. aureus were allowed to grow for 90 min and 6 h after reaching the OD600 = 0.5 and total RNA from these cultures were extracted. cDNA from DNase treated 0.5 μg of total RNA was synthesized in a 20 μL reverse transcription reaction containing random hexamers and SuperScript III reverse transcriptase (Invitrogen). All real-time PCR reactions were carried out with Bio-Rad iCycler (iQ5 system). The transcript level ofmsrA1 was quantified using primers P13 and P14, that ofmsrB was quantified using P15 and P16, and that of the gene encoding the IIa(PTS) was quantified using primers P17 and P18. Transcript levels of genes were normalized to DNA gyrase mRNA using primers P19 and P20 based on a previous report [15, 16]. Changes in gene expression were calculated using the formula 2-ΔΔCq as described [17].


## 2.7. Statistical Analysis

All results are reported as the mean ± SE of at least three independent experiments. Data were analyzed with Student’st-test using R Studio for Windows (version 0.98.1103, 3.1.3). Statistical significance was set at p≤0.05.


## 3. Results

### 3.1. Construction ofmsrA1/msrB Promoter-lacZ Reporter in Wild-Type andmsrA1,msrB,msrA1-msrB, andsigB Mutants ofS. aureus

Previously createdmsrA1,msrB,msrA1-msrB, andsigB knockout mutants ofS. aureus strain SH1000 [6, 9, 10] were transduced in the methicillin-resistantS. aureus strain COL. These mutants and the presence ofmecA gene in these strains were verified by PCR (see Supplemental Figures S1-S2 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/617925). ThemsrA1/msrB promoter-lacZ fusion was subsequently integrated into the chromosome of these mutant strains using a bacteriophage transduction procedure. Overall, fivemsrA1/msrB promoter-lacZ reporter strains were created in methicillin-resistant as well as methicillin-sensitiveS. aureus backgrounds. Proper integration of themsrA1/msrB promoter-lacZ fusion was also confirmed by PCR (Supplemental Figure S3).


### 3.2. Regulation ofmsrA1/msrB Locus inS. aureus

Previously, we reported higher MsrB levels in MsrA1-deficientS. aureus cells [9, 10]. This led to the speculation that themsrA1/msrB locus may in part be regulated by the products of this locus. To investigate this possibility, the level of β-galactosidase was measured in MsrA1-, MsrB-, and MsrA1-MsrB-deficient strains ofS. aureus. β-galactosidase activity levels were higher in these strains compared to the activity level in the wild-typeS. aureus strain SH1000 (Figure 1). ThemsrA1/msrB promoter-lacZ reporter was also studied in the methicillin-resistant strain COL. Overall, the expression oflacZ was lower in methicillin-resistantS. aureus compared to the methicillin-sensitiveS. aureus (Figures 1(b) and 2(b)). In addition, β-galactosidase activity comparison revealed that only themsrA1-msrB double mutant strains had higher activity levels compared to the wild-type COL at the various time points (Figure 2(b)). In the individualmsrA1 ormsrB mutant strains, a significant increase in β-galactosidase activity was not observed compared to wild-typeS. aureus COL (Figure 2(b)).Figure 1
Regulation ofmsrA1/msrB locus in a methicillin-sensitiveS. aureus strain SH1000. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured in wild-typeS. aureus strain SH1000 (open triangles) and its derivativesmsrA1 (open circles),msrB (closed triangles), andmsrA1-msrB (open square) mutants during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (SE) (∗ significant at p≤0.05).
(a)
(b)Figure 2
Regulation ofmsrA1/msrB locus in a methicillin-resistantS. aureus strain COL. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured in wild-typeS. aureus strain COL (open triangles) and its derivativesmsrA1 (open circles),msrB (closed triangles), andmsrA1-msrB (open square) mutants during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).
(a)
(b)


### 3.3. Role of SigB in the Regulation ofmsrA1/msrB Locus inS. aureus

Measurement ofβ-galactosidase activity demonstrated that there was increased expression oflacZ from themsrA1/msrB promoter whenS. aureus was deficient of SigB in strain SH1000 (Figure 3(b)). However, inS. aureus COL, no such increase in the expression oflacZ was observed from themsrA1/msrBpromoter under SigB-deficient conditions compared to the wild-type strain (Figure 4(b)). In qRT-PCR assays, a relatively higher level ofmsrA1 transcripts was observed insigB mutant ofS. aureus strain SH1000 compared to the wild-type strain (Table 3). However, this increase inmsrA1 gene expression was not evident in thesigB mutant ofS. aureus strain COL (Table 3) supporting the findings of themsrA1/msrB promoter-lacZ data insigB mutant strains.Table 3
Expression levels ofmsrA1 in sigB mutants relative to wild-type S. aureus strains SH1000 and COL.
Strain
Fold increase in expression
90 min
6 h
SH1000ΔsigB1.30
3.16
COLΔsigB
0.98
1.52
Values indicate averages of three independent experiments.Figure 3
Regulation ofmsrA1/msrB locus in a methicillin-sensitiveS. aureus strain SH1000 by SigB. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured in wild-typeS. aureus strain SH1000 (open triangles) and its derivativesigB mutant (closed squares) during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).
(a)
(b)Figure 4
Regulation ofmsrA1/msrB locus in a methicillin-resistantS. aureus strain COL by SigB. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured inS. aureus strain COL (open triangles) and its derivativesigB mutant (closed squares) during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).
(a)
(b)


### 3.4. Induction ofmsrA1/msrB Locus in the Presence of Cell Wall-Active Antibiotic, Oxacillin

Previous studies have shown that themsrA1/msrB locus is induced by the cell wall-active antibiotics, oxacillin, vancomycin, and D-cycloserine, in a methicillin-sensitiveS. aureus strain [5]. In a later study, while themsrA1/msrB locus remained inducible in the presence of D-cycloserine and vancomycin, no induction of this locus was noted in the presence of oxacillin, when similar experiments were carried out in a methicillin-resistantS. aureus strain COL [18]. However, in our experiments, a significantly increased β-galactosidase activity clearly indicates a significant induction ofmsrA1/msrB locus in the presence of oxacillin, even in a methicillin-resistantS. aureus (Figure 5). We also investigated the expression of the downstream genes ofmsrA1 locus in qRT-PCR assays. We determined that the oxacillin stress dramatically induced the expression ofmsrA1,msrB, and the gene encoding IIa(PTS) (Table 4). The expression level of the fourth gene of this locus was not investigated due to its very small size. This finding further supports our previous observation of cotranscription of the four genes of themsrA1/msrB locus [5, 6].Table 4
Induced expression ofmsrA1/msrB locus genes in S. aureus strain COL under oxacillin stress.
Gene
Fold increase in expression under oxacillin stress
msrA1
22.9
msrB
18.97
IIa(PTS)
13.45
Values indicate averages of three independent experiments.Figure 5
Analysis ofmsrA1/msrB promoter-lacZ fusion inS. aureus COL in response to oxacillin. Bacterial culture was grown in TSB to OD600 = 0.3 and then exposed to oxacillin (0.4 and 1.0 mg mL−1, resp.) for 2 h. Subsequently, cells were collected via centrifugation and the β-galactosidase activity was determined. Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).


### 3.5. Expression ofmsrA1/msrB Locus in MsrA1-MsrB-DeficientS. aureus Strain COL in the Presence of Oxacillin

While studying the regulation of themsrA1/msrB locus in a methicillin-resistantS. aureus strain COL, oxacillin was added during the growth of themsrA1/msrB promoter-lacZ reporter to investigate any magnification of the regulation. In these studies, while an increasedlacZ expression was observed in wild-typeS. aureus strain COL after oxacillin treatment, a more dramatic increase in thelacZ expression in response to oxacillin was seen in MsrA1-MsrB-deficient COL (Figure 6).Figure 6
Analysis ofmsrA1/msrB promoter-lacZ fusion in wild-type andmsrA1-msrB double mutant ofS. aureus COL in response to oxacillin. At OD600 = 0.5, cells were treated with oxacillin for 2 h and the β-galactosidase activity levels were measured. Values indicate averages of data from at least three independent experiments ± standard error (∗ significant between samples with and without oxacillin at p≤0.05; † significant between oxacillin treated wild-type and the oxacillin treatedmsrA1-msrB double mutant at p≤0.05).


## 3.1. Construction ofmsrA1/msrB Promoter-lacZ Reporter in Wild-Type andmsrA1,msrB,msrA1-msrB, andsigB Mutants ofS. aureus

Previously createdmsrA1,msrB,msrA1-msrB, andsigB knockout mutants ofS. aureus strain SH1000 [6, 9, 10] were transduced in the methicillin-resistantS. aureus strain COL. These mutants and the presence ofmecA gene in these strains were verified by PCR (see Supplemental Figures S1-S2 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/617925). ThemsrA1/msrB promoter-lacZ fusion was subsequently integrated into the chromosome of these mutant strains using a bacteriophage transduction procedure. Overall, fivemsrA1/msrB promoter-lacZ reporter strains were created in methicillin-resistant as well as methicillin-sensitiveS. aureus backgrounds. Proper integration of themsrA1/msrB promoter-lacZ fusion was also confirmed by PCR (Supplemental Figure S3).


## 3.2. Regulation ofmsrA1/msrB Locus inS. aureus

Previously, we reported higher MsrB levels in MsrA1-deficientS. aureus cells [9, 10]. This led to the speculation that themsrA1/msrB locus may in part be regulated by the products of this locus. To investigate this possibility, the level of β-galactosidase was measured in MsrA1-, MsrB-, and MsrA1-MsrB-deficient strains ofS. aureus. β-galactosidase activity levels were higher in these strains compared to the activity level in the wild-typeS. aureus strain SH1000 (Figure 1). ThemsrA1/msrB promoter-lacZ reporter was also studied in the methicillin-resistant strain COL. Overall, the expression oflacZ was lower in methicillin-resistantS. aureus compared to the methicillin-sensitiveS. aureus (Figures 1(b) and 2(b)). In addition, β-galactosidase activity comparison revealed that only themsrA1-msrB double mutant strains had higher activity levels compared to the wild-type COL at the various time points (Figure 2(b)). In the individualmsrA1 ormsrB mutant strains, a significant increase in β-galactosidase activity was not observed compared to wild-typeS. aureus COL (Figure 2(b)).Figure 1
Regulation ofmsrA1/msrB locus in a methicillin-sensitiveS. aureus strain SH1000. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured in wild-typeS. aureus strain SH1000 (open triangles) and its derivativesmsrA1 (open circles),msrB (closed triangles), andmsrA1-msrB (open square) mutants during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (SE) (∗ significant at p≤0.05).
(a)
(b)Figure 2
Regulation ofmsrA1/msrB locus in a methicillin-resistantS. aureus strain COL. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured in wild-typeS. aureus strain COL (open triangles) and its derivativesmsrA1 (open circles),msrB (closed triangles), andmsrA1-msrB (open square) mutants during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).
(a)
(b)


## 3.3. Role of SigB in the Regulation ofmsrA1/msrB Locus inS. aureus

Measurement ofβ-galactosidase activity demonstrated that there was increased expression oflacZ from themsrA1/msrB promoter whenS. aureus was deficient of SigB in strain SH1000 (Figure 3(b)). However, inS. aureus COL, no such increase in the expression oflacZ was observed from themsrA1/msrBpromoter under SigB-deficient conditions compared to the wild-type strain (Figure 4(b)). In qRT-PCR assays, a relatively higher level ofmsrA1 transcripts was observed insigB mutant ofS. aureus strain SH1000 compared to the wild-type strain (Table 3). However, this increase inmsrA1 gene expression was not evident in thesigB mutant ofS. aureus strain COL (Table 3) supporting the findings of themsrA1/msrB promoter-lacZ data insigB mutant strains.Table 3
Expression levels ofmsrA1 in sigB mutants relative to wild-type S. aureus strains SH1000 and COL.
Strain
Fold increase in expression
90 min
6 h
SH1000ΔsigB1.30
3.16
COLΔsigB
0.98
1.52
Values indicate averages of three independent experiments.Figure 3
Regulation ofmsrA1/msrB locus in a methicillin-sensitiveS. aureus strain SH1000 by SigB. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured in wild-typeS. aureus strain SH1000 (open triangles) and its derivativesigB mutant (closed squares) during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).
(a)
(b)Figure 4
Regulation ofmsrA1/msrB locus in a methicillin-resistantS. aureus strain COL by SigB. ThemsrA1/msrB promoter-lacZ reporter strains were cultured in TSB and growth was measured as OD600 (a). β-galactosidase activity levels were measured inS. aureus strain COL (open triangles) and its derivativesigB mutant (closed squares) during different stages of growth (b). Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).
(a)
(b)


## 3.4. Induction ofmsrA1/msrB Locus in the Presence of Cell Wall-Active Antibiotic, Oxacillin

Previous studies have shown that themsrA1/msrB locus is induced by the cell wall-active antibiotics, oxacillin, vancomycin, and D-cycloserine, in a methicillin-sensitiveS. aureus strain [5]. In a later study, while themsrA1/msrB locus remained inducible in the presence of D-cycloserine and vancomycin, no induction of this locus was noted in the presence of oxacillin, when similar experiments were carried out in a methicillin-resistantS. aureus strain COL [18]. However, in our experiments, a significantly increased β-galactosidase activity clearly indicates a significant induction ofmsrA1/msrB locus in the presence of oxacillin, even in a methicillin-resistantS. aureus (Figure 5). We also investigated the expression of the downstream genes ofmsrA1 locus in qRT-PCR assays. We determined that the oxacillin stress dramatically induced the expression ofmsrA1,msrB, and the gene encoding IIa(PTS) (Table 4). The expression level of the fourth gene of this locus was not investigated due to its very small size. This finding further supports our previous observation of cotranscription of the four genes of themsrA1/msrB locus [5, 6].Table 4
Induced expression ofmsrA1/msrB locus genes in S. aureus strain COL under oxacillin stress.
Gene
Fold increase in expression under oxacillin stress
msrA1
22.9
msrB
18.97
IIa(PTS)
13.45
Values indicate averages of three independent experiments.Figure 5
Analysis ofmsrA1/msrB promoter-lacZ fusion inS. aureus COL in response to oxacillin. Bacterial culture was grown in TSB to OD600 = 0.3 and then exposed to oxacillin (0.4 and 1.0 mg mL−1, resp.) for 2 h. Subsequently, cells were collected via centrifugation and the β-galactosidase activity was determined. Values indicate averages of data from at least three independent experiments ± standard error (∗ significant at p≤0.05).


## 3.5. Expression ofmsrA1/msrB Locus in MsrA1-MsrB-DeficientS. aureus Strain COL in the Presence of Oxacillin

While studying the regulation of themsrA1/msrB locus in a methicillin-resistantS. aureus strain COL, oxacillin was added during the growth of themsrA1/msrB promoter-lacZ reporter to investigate any magnification of the regulation. In these studies, while an increasedlacZ expression was observed in wild-typeS. aureus strain COL after oxacillin treatment, a more dramatic increase in thelacZ expression in response to oxacillin was seen in MsrA1-MsrB-deficient COL (Figure 6).Figure 6
Analysis ofmsrA1/msrB promoter-lacZ fusion in wild-type andmsrA1-msrB double mutant ofS. aureus COL in response to oxacillin. At OD600 = 0.5, cells were treated with oxacillin for 2 h and the β-galactosidase activity levels were measured. Values indicate averages of data from at least three independent experiments ± standard error (∗ significant between samples with and without oxacillin at p≤0.05; † significant between oxacillin treated wild-type and the oxacillin treatedmsrA1-msrB double mutant at p≤0.05).


## 4. Discussion

Cell wall-active antibiotics have been used extensively for the treatment of infections caused by bacterial pathogens.S. aureus is a major human pathogen and is resistant to most commonly available antibiotics. Interestingly, cell wall-active antibiotics cause induction of a locus inS. aureus that leads to elevated synthesis of two methionine sulfoxide reductases (MsrA1 and MsrB) [5, 6]. These enzymes reduce methionine sulfoxide and play important roles in maintaining protein integrity and function particularly under oxidative stress. These two proteins have also been shown to have roles in the virulence of bacterial pathogens [19–23]. Msr-deficient bacterial mutants show a reduction in the ability to adhere to eukaryotic cells and are thus less likely to establish an inflection [21, 22, 24, 25]. It is speculated that the lack of the Msr enzymes compromises the integrity of the bacterial surface proteins responsible for adherence to eukaryotic cells. Reduced Msr activity decreases bacterial survival inside the phagocytic cells [20]. In addition to increased levels of MsrA1 and MsrB specifically in response to cell wall-active antibiotics, these proteins inS. aureus have been shown to play roles in the survival of bacterial cells under oxidative stress as well as in mice [6, 10].We previously demonstrated that when themsrA1 gene is deleted inS. aureus, there is an increase in MsrB synthesis suggesting a possible role in the regulation of this locus [9]. Findings of this study suggest that, in a methicillin-sensitiveS. aureus strain SH1000, MsrA1 and MsrB individually can downregulate themsrA1/msrB locus. However, in methicillin-resistantS. aureus strain COL, MsrA1 and MsrB both are needed to downregulate the expression of themsrA1/msrB locus. It is speculated that themsrA1/msrB locus, to some extent, is differentially regulated between methicillin-resistant and methicillin-sensitiveS. aureus strains. It is not uncommon to observe a differential gene expression pattern between differentS. aureus strains. It has been demonstrated that the growth of methicillin-resistantS. aureus is slower than that of methicillin-sensitiveS. aureus in the lag phase but not during the exponential phase and that the alterations in virulence between these two strains may at least partially be due to the growth rate differences [26]. Deletion of a gene encoding nitric oxide synthase (NOS) in a methicillin-resistantS. aureus reduced virulence as seen by decreased bacterial survival and smaller abscess formation [27]. However, NOS was shown to have a limited role in a methicillin-sensitiveS. aureus [28]. Expression of genes encoding staphylococcal superantigen-like (SSL) proteins also varies betweenS. aureus strains [29, 30]. Significant differences were also noted between the protein profiles of the methicillin-resistant and methicillin-sensitiveS. aureus strains exposed to Triton X-100 [31].It is well established that themsrA1/msrB locus is selectively induced in the presence of cell wall-active antibiotics [5]. These antibiotics interfere with the bacterial cell wall synthesis and, as a result, the cells become fragile and susceptible to lysis. Expression ofmsrA1/msrB locus is not induced by antibiotics that target other bacterial metabolic pathways [5]. In a previous report, it was shown that themsrA1/msrB locus was not induced by the presence of oxacillin but was induced by the presence of D-cycloserine and vancomycin in a methicillin-resistantS. aureus [18]. However, data from our study provide clear evidence that oxacillin does in fact induce themsrA1/msrB locus in a methicillin-resistant background ofS. aureus. The previous report [18] did not observe any induction because the bacterialcells were not exposed to a high enough concentration to impose antibiotic stress in a methicillin-resistantS. aureus strain. Furthermore, we explored the induction ofmsrA1/msrB genes inmsrA1-msrB double mutant in methicillin-resistant strain COL. An increase in induction of themsrA1/msrB locus was further magnified inmsrA1-msrB double mutant exposed to oxacillin compared to the wild-typeS. aureus COL in response to oxacillin. This further confirms the notion of downregulation of themsrA1/msrB locus by MsrA1 and MsrB and this is more likely an indirect effect. This speculation of an indirect regulation is based on the fact that, after conducting a protein domain search (http://prosite.expasy.org/), no specific DNA-binding domain was observed in MsrA1 and MsrB proteins. It is possible that the MsrA1 and MsrB enzymes are critical in maintaining the integrity of a cytoplasmic transcriptional regulator that is involved in the regulation of expression of this locus.In recent years, regulation ofmsrA andmsrB has been studied extensively across multiple species; however, none have shown that MsrA or MsrB directly or indirectly regulates its own expression. It has been demonstrated that RynB regulates the synthesis ofEscherichia coli MsrB but not MsrA by binding to the 5′ untranslated region ofmsrB mRNA and interfering with its binding to the ribosome [32]. Nitric oxide, which is induced inUlva fasciata upon exposure to light, upregulates the expression ofmsr genes in the intertidal macroalga [33]. InSaccharomyces cerevisiae, calcium phospholipid binding protein (CPBP) interacts with themsrA promoter and enhances its expression [34]. InBacillus subtilis, a transcriptional regulator, Spx, is shown to significantly upregulate the expression ofmsrA andmsrB [35]. Spx also upregulatesmsrA1 expression inS. aureus. Teicoplanin inducesmsrA1/msrB expression inS. aureus. However, inS. aureus spx mutant, teicoplanin exposure resulted in no significant induction of this locus, whereas, in thespx mutant strain complemented with the wild-typespx gene,msrA1/msrB induction in response to teicoplanin exposure was restored [36]. Additionally, in thespx mutant, basalmsrA1 mRNA was significantly lower thanspx complemented strain [36].SigB is the alternative sigma factor inS. aureus that plays a role in the regulation of expression of stress responsive genes inS. aureus [11]. In addition, SigB is also associated with the regulation of expression of the virulence genes inS. aureus [11]. In a previous report, the level of expression ofmsrA1/msrB locus was investigated between RN450 (SigB−) and SH1000 (SigB+) [18]. It was shown that, inS. aureus strain SH1000,msrA1/msrB expression was 30% more induced than inS. aureus strain RN450 in the presence of oxacillin [18]. In contrast, our study shows that SigB in fact downregulates the expression ofmsrA1/msrB locus inS. aureus in the methicillin-sensitiveS. aureus strain SH1000 and plays no role in the regulation of this locus in methicillin-resistant strain COL.In summary, this study provides evidence that the expression of themsrA1/msrB locus is enhanced whenS. aureus is deficient in MsrA1, MsrB, or both in a methicillin-sensitiveS. aureus. However, in methicillin-resistantS. aureus, increased expression of themsrA1/msrB locus was apparent only when the bacterial cells were deficient in both MsrA1 and MsrB. In addition, SigB also in part downregulates the expression of this locus in methicillin-sensitiveS. aureus but not in methicillin-resistantS. aureus.



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