A bacterial hemerythrin-like protein MsmHr inhibits the SigF-dependent hydrogen peroxide response in mycobacteria

Hydrogen peroxide (H2O2) is one of a variety of reactive oxygen species (ROS) produced by aerobic organisms. Host production of toxic H2O2 in response to pathogen infection is an important classical innate defense mechanism against invading microbes. Understanding the mechanisms by which pathogens, in response to oxidative stress, mediate defense against toxic ROS, can reveal anti-microbial targets and shed light on pathogenic mechanisms. In this study, we provide evidence that a Mycobacterium smegmatis hemerythrin-like protein MSMEG_2415, designated MsmHr, is a H2O2-modulated repressor of the SigF-mediated response to H2O2. Circular dichroism and spectrophotometric analysis of MsmHr revealed properties characteristic of a typical hemerythrin-like protein. An msmHr knockout strain of M. smegmatis mc2155 (ΔmsmHr) was more resistant to H2O2 than its parental strain, and overexpression of MsmHr increased mycobacterial susceptibility to H2O2. Mutagenesis studies revealed that the hemerythrin domain of MsmHr is required for the regulation of the H2O2 response observed in the overexpression study. We show that MsmHr inhibits the expression of SigF (MSMEG_1804), an alternative sigma factor that plays an important role in bacterial oxidative stress responses, including those elicited by H2O2, thus providing a mechanistic link between ΔmsmHr and its enhanced resistance to H2O2. Together, these results strongly suggest that MsmHr is involved in the response of mycobacteria to H2O2 by negatively regulating a sigma factor, a function not previously described for hemerythrins.


INTRODUCTION
Hydrogen peroxide (H 2 O 2 ) is a universal oxidative stress molecule produced by aerobic organisms from all three domains of life (Imlay, 2008). The production of H 2 O 2 by the host in response to pathogen infection is also an important innate defense mechanism (Fang, 2004). This ROS can be damaging via direct toxic effects or disruption of redox balance, the latter being critical for metabolic homeostasis and hence survival. An important intracellular pathogen, Mycobacterium tuberculosis has evolved many strategies to detoxify H 2 O 2 , some of which are unique to mycobacteria (Kumar et al., 2011;Trivedi et al., 2012). For example, M. tuberculosis, which lacks the conventional redox buffer glutathione, uses mycothiol (MSH), a lowmolecular-weight thiol that exists in millimolar quantities in the cytoplasm, to generate a reducing environment (Farhana et al., 2010); and MSH-deficient mycobacterial mutants are hypersusceptible to H 2 O 2 (Rawat et al., 2002). The precise mechanisms by which MSH protects against oxidative stress and redox imbalance remain to be determined. M. tuberculosis also lacks classic sensors such as OxyR and SoxR for the detection of redox signals (Imlay, 2013). The tubercle bacillus, however, expresses DosS and DosT (Kumar et al., 2007), two sensor histidine kinases whose heme iron plays a critical role in their response to the levels of O 2 , nitric oxide (NO) and carbon monoxide (CO). Interaction with these various gasses activates the kinase activity of DosS and DosT, relaying the signals to the response regulator DosR. WhiB3 is an Fe-S cluster transcription factor that controls the expression of the hypoxia regulon of M. tuberculosis . Indeed, accumulating evidence suggests that the M. tuberculosis WhiB family of Fe-S cluster proteins plays important roles in regulating a wide spectrum of microbial functions including responses to oxidative stress and virulence (Burian et al., 2012;Saini et al., 2012a). Together, the DosS-DosT/WhiB3 paradigm underscores the importance of iron-containing proteins in the response of M. tuberculosis to redox signals, including those imposed by reactive oxygen intermediates.
The non-heme, di-iron, O 2 -binding hemerythrin-like proteins are present in all domains of life (Bailly et al., 2008;French et al., 2008). Bioinformatics analyses have revealed over 400 hemerythrin-like proteins in available prokaryotic genomes (Bailly et al., 2008;French et al., 2008). These proteins harbor the conserved hemerythrin domain either singly or jointly with another distinct functional domain (Bailly et al., 2008;French et al., 2008). Experimental evidence suggests that they can function as oxygen sensors and reserves, as well as mediate the delivery and transport of this diatomic gas (French et al., 2008). Dcr (Desulfovibrio chemoreceptor) H was the first bacterial hemerythrin-like protein to be identified. It has been proposed that the C-terminal hemerythrin domain of DcrH, a member of the Dcr family of putative methyl-accepting chemotaxis proteins of the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris, functions to sense O 2 (Xiong et al., 2000); the signal thus generated is proposed to be transduced to the transmitting domains to mediate chemotaxis (Xiong et al., 2000). The first single-domain hemerythrin-like protein was identified in Methylococcus capsulatus. The expression of the M. capsulatus hemerythrin-like protein is enhanced significantly with increasing concentrations of copper, and is thought to be an oxygen carrier that supplies copper-containing methane monooxygenase with oxygen (Kao et al., 2004;Karlsen et al., 2005;Chen et al., 2012). Despite the wide distribution of hemerythrin-like proteins in a wide variety of bacterial species, including M. tuberculosis (Xiong et al., 2000;Karlsen et al., 2005;Isaza et al., 2006;Justino et al., 2007;Onoda et al., 2011;Schaller et al., 2012), functional characterization studies are scarce.
Transcriptional regulation is critical to bacterial survival in response to various stresses. Sigma factors are the primary transcriptional regulators of bacterial gene expression. M. tuberculosis has 13 sigma factors, and SigH, SigE, SigL, and SigF play important roles in ROS detoxification (Rodrigue et al., 2006). SigF, a highly conserved sigma factor in the genus Mycobacterium (Rodrigue et al., 2006), is highly induced by various environmental stresses and during stationary phase (Demaio et al., 1996). In M. smegmatis, sigF deletion also increases susceptibility to oxidative stress (Gebhard et al., 2008;Humpel et al., 2010). Recently, a genome-wide gene expression study (Humpel et al., 2010) and work from our laboratory  have provided evidence that SigF regulates the expression of oxidative stress defense genes such as katA, dps1, and sodA, but not katG and ahpC, genes that have been linked to mycobacterial resistance to isoniazid (Silva et al., 2003). These studies suggest that SigF-mediated resistance to H 2 O 2 is independent of KatG and AhpC. This notion is also supported by the fact that SigF-deficient mutants do not display decreased susceptibility to isoniazid (Demaio et al., 1996;Humpel et al., 2010). Regulation of SigF in mycobacteria is generally thought to be predominantly at the post-transcriptional level via the action of anti-sigma and anti-anti-sigma factors (Michele et al., 1999;Beaucher et al., 2002;Singh and Singh, 2008). SigF is transcribed from two promoters, P msmeg_1802 and P rbsw (Gebhard et al., 2008), and transcriptional reporter fusion studies have shown that promoter P msmeg_1802 responds to entry into the stationary phase and promoter P rbsw is inducible upon treatment with D-cycloserine (Gebhard et al., 2008).
In this study, to investigate the role of hemorythrin-like proteins in mycobacteria, we cloned, expressed, and characterized the M. smegmatis mc 2 155 strain MsmHr protein (encoded by msmeg2415). Using a genetic and biochemical approach, we show that (i) MsmHr displays circular dichroism (CD) and UVvis spectrophotometric features typical of a hemerythrin-like protein; (ii) relative to wild-type bacilli, the msmHr knockout strain ( msmHr) is more resistant to H 2 O 2 and mc 2 155 overexpressing MsmHr exhibits enhanced H 2 O 2 susceptibility; (iii) the H 2 O 2 response is dependent on the hemerythrin domain; (iv) MsmHr represses sigF transcription through the promoter P rbsw , and thus participates in regulating the SigF-mediated H 2 O 2 response. Our results indicate that MsmHr, the first mycobacterial hemorhythrin-like protein to be characterized, is involved in the H 2 O 2 response in mycobacteria and provide insight into its mechanism.

GENERATION OF KNOCKOUT MUTANT STRAINS, COMPLEMENTATION STRAINS AND OVEREXPRESSION STRAINS
The msmHr (msmeg_2415) and sigF (msmg_1804) deletion mutants were generated via a specialized transducing phage delivery system as previously described (Bardarov et al., 2002). The 5 -flanking region of msmHr was amplified by polymerase chain reaction (PCR) with the 2415LL/2415LR primer pair and the 3 -flanking region of msmHr was amplified with the 2415RL/2415RR primer pair (all primers are listed in Supplemental Table S2. The flanking regions of sigF were generated by amplifying the upstream and downstream regions of sigF using the 1804LL/1804LR and 1804RL/1804RR primer pairs, respectively. Amplified fragments were ligated with plasmid p0004S, digested with PflMI (msmHr) or AlwnI (sigF), and allelicexchange plasmids thus constructed were digested with PacI, and then ligated with PacI-digested phAE159. Phage packaging was performed using a MaxPlax packaging extract (Epicenter Biotechnologies, USA) to yield the knockout phages for msmHr (phAE-msmHr) and sigF (phAE-sigF). Specialized transduction was carried out as described previously (Bardarov et al., 2002). The knockout clones were screened by PCR using the primer pairs 2415InL/2415InR, 2415LLL/IL(R) and IR(F)/2415RRR for msmHr and 1804InL/1804InR, 1804LLL/IL(R), and IR(F)/1804RRR for sigF. Primer positions with respect to the appropriate genes are shown in Figure 2A and Figure S1. No msmHr or sigF mRNA was detected in the corresponding deletion strains by qRT-PCR using the appropriate primer pairs (Figure 2A and Figure S1). Complementation strains were constructed as described previously (Stover et al., 1991). Briefly, the full-length sequence of msmHr or sigF amplified from M. smegmatis genomic DNA was cloned into the integrating vector pMV361 (Stover et al., 1991) and the resultant plasmids were electroporated into the corresponding knockout strains to yield C-msmHr ( msmHr::P hsp60 -msmHr) and C-sigF ( sigF::P hsp60 -sigF). To over-express msmHr, the msmHr fragment was subcloned into pMV261 (Stover et al., 1991) to yield pMV261-msmHr for transformation into M. smegmatis (O-msmHr).

DETERMINATION OF SURVIVAL PHENOTYPES UNDER STRESS
Early phase cultures (OD 600 = 0.3) of all tested strains were serially diluted (1:10) and spotted (3 μl) onto solid 7H10 medium supplemented with ADS enrichment and stress-inducing chemical agents (20 μM streptonigrin, 250 μM NaNO 2 ) or subjected to low-pH stress (7H9 supplemented with ADS enrichment and 0.05% Tween 80, pH 5.5). For peroxide stress, early-phase cultures were treated with 5 mM H 2 O 2 for 3 h, serially diluted (1:10) and spotted (3 μl) onto solid 7H10 medium supplemented with 10% ADS. The optical density at 600 nm (OD 600 ) was measured at the indicated times in the presence of various stresses. Survival under heat shock stress at 50 • C was determined by the number of colony forming units during the time indicated. Statistical analyses were performed using unpaired two-tailed t-tests. P-values are only shown where significant differences were found. * P < 0.05 and * * P < 0.01.

SPECTROPHOTOMETRIC AND CIRCULAR DICHROIC (CD) ANALYSIS OF MsmHr
Purified MsmHr was diluted in 20 mM Tris-Cl buffer (pH 7.5). Deoxy samples were obtained by adding a 10-fold molar excess of Na 2 S 2 O 4 to MsmHr. UV-Vis spectrophotometric spectra were obtained in 1 mm path length quartz cuvettes on a UV-2802H UV-Vis spectrophotometer (Unico Shanghai Instruments Co., Ltd., China). Spectra of deoxy-MsmHr were collected in an anaerobic incubator (Shanghai Yuejin Medicial Instruments Co., Ltd, China). CD measurements were performed using a Chirascan Circular Dichroism Spectrometer (Applied Photophysics Ltd. UK). The analysis software provided with the instrument was used for analysis of the results.

DETERMINATION OF THE MINIMUM INHIBITORY CONCENTRATION OF ISONIAZID IN M. SMEGMATIS
The susceptibility of M. smegmatis to isoniazid (INH) was determined using the broth microdilution method (Wallace et al., 1986). After two-fold dilutions of INH in 7H9 supplemented with ADS enrichment and 0.5% (v/v) glycerol, 40-μl aliquots were mixed with 40 μl of M. smegmatis suspension (10 5 cells/ml) and deposited into wells of 96-well microtiter plates. The highest concentration of INH was 100 μg/ml. Plates were incubated at 37 • C for 2 days and OD 600 values of cultures were then measured using a microplate reader (FLUOstar OPTIMA, BMG Labtech). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug that inhibited the visible bacterial growth of M. smegmatis after a 2-day incubation (OD 600 < 0.05). INH susceptibility tests were repeated at least 3 times.

RNA ISOLATION, RT-PCR AND QUANTITATIVE PCR
Log phase cultures (OD 600 = 0.8-1.0) of all tested strains were diluted 1:100 in 7H9 media supplemented with ADS enrichment, 0.5% (v/v) glycerol and 0.05% (v/v) Tween 80. Strains were cultured until the OD 600 reached 0.3 and then divided into control and treatment groups. In the treatment group, the cells were treated with 5 mM H 2 O 2 for 30 min, and then collected by centrifugation at 12,000 g. Bacterial pellets were resuspended in TRIzol (Invitrogen, USA) and RNA was purified according to the manufacturer's instructions. cDNA was synthesized using the SuperScriptTM III First-Strand Synthesis System (Invitrogen, USA). Quantitative real-time PCR (qRT-PCR) was performed in a Bio-Rad iCycler using 2x SYBR real-time PCR pre-mix (Takara Biotechnology Inc., Japan). The following cycling program was used: 95 • C for 1.5 min followed by 40 cycles of 95 • C for 10 s, 60 • C for 15 s, and 72 • C for 15 s, followed for 72 • C for 6 min. M. smemgatis rpoD encoding RNA polymerase sigma factor SigA was selected as a reference to normalize gene expression. The 2 − CT method was used (Livak and Schmittgen, 2001) to evaluate the relative gene expression in different strains and/or different treatments. Primers used are listed in Table S2.

CONSTRUCTION OF msmHr POINT MUTANTS
Mutation of specific amino acids was incorporated into the hemerythrin-like domain of msmHr in pMV261-msmHr by mismatched PCR primers (

MsmHr IS A BACTERIAL HEMERYTHRIN-LIKE PROTEIN
Detoxification strategies for scavenging host immune defense system-derived H 2 O 2 are important for the intracellular survival of mycobacterial pathogens. Expression of Fe-related proteins, such as the Fe-S cluster transcription factor WhiB3, and hemeiron sensors DosS and DosT, is an important strategy for regulating redox balance . Hemerythrin proteins are iron-binding proteins known to be involved in oxygen transport and storage, but their biological functions in mycobacteria have yet to be elucidated. Three proteins in M. smegmatis, MsmHr, Msmeg_3312 and Msmeg_6612, are predicted to be hemerythrinlike proteins, however, as preliminary experiments indicated that only MsmHr is related to the H 2 O 2 response (data not shown), we focused our attention on MsmHr. We first constructed a multiple alignment of MsmHr with other hemerythrin-like proteins from different bacterial species (Figure 1A). Residues H24, H57, E61, H66, H88, H121, and E126 (numbering based on the MsmHr sequence) matched the characteristic motifs H. . . HxxxE . . . HxxxH. . . HxxxxD/E of hemerythrin domains. The secondary structure predicted by SWISS-MODEL (http://swissmodel.expasy.org/) (Guex and Peitsch, 1997;Schwede et al., 2003;Arnold et al., 2006;Kiefer et al., 2009) suggested that MsmHr has a typical hemerythrin structure with four α-helices (residues 16-40, 41-68, 80-103, and 104-132) (Figure 1B).
To confirm the predicted helical structure of MsmHr, we purified MsmHr-His 6 protein from E. coli. Circular dichroism (CD) spectra of the E. coli-purified MsmHr showed two minima at 208 and 222 nm (Figure 1C). Such a pattern, which is characteristic of protein α-helical structures, is typical of the circular dichroism spectra of previously analyzed bacterial hemerythrins (Wirstam et al., 2003). We also performed a CD analysis of gelactin AAL (Agrocybe aegerita lectin, PDB 2ZGU), a protein which consists mainly of β-sheets. The CD spectra showed only one minima at 210 nm (Supplemental Figure S3). The UV-visible absorption spectra of MsmHr showed peaks at 327 and 376 nm, a pattern ascribable to the di-iron-center of hemerythrin-like proteins (Karlsen et al., 2005). Absorbance peaks were abrogated upon reduction with Na 2 S 2 O 4 to generate the deoxy form by removing oxygen (Figure 1D). Since MsmHr has no Trp and only one Phe, UV/Vis spectrophotometric analysis of the purified protein did not detect absorbance at 280 nm (Figure 1D inset). Taken together, these bioinformatics, CD and spectrophotometric results strongly suggest that MsmHr is a hemerythrin-like protein.

MsmHr IS INVOLVED IN THE H 2 O 2 STRESS RESPONSE
To define the biological functions of MsmHr, a deletion mutant, msmHr, was generated by specialized transduction (Bardarov et al., 2002) (Figure 2A). The loss of msmHr was confirmed by PCR (Figure 2A) and no msmHr mRNA was detected in msmHr ( Figure 2B). To determine if deletion of msmHr had polar effects on the msmeg_2414 and msmeg_2416 genes, their mRNA levels were compared in the wild type mc 2 155 and msmHr strains by RT-PCR. Statistically significant differences in msmeg_2414 and msmeg_2416 mRNA were not detected (Figure 2B), indicating that msmHr knockout did not affect the transcriptional levels of msmeg_2414 and msmeg_2416.
We then compared the growth rates of the M. smegmatis parental strain mc 2 155 with the MsmHr-deficient mutant msmHr in both 7H9 rich medium and Sauton's minimal medium. No growth abnormalities were detected in msmHr relative to mc 2 155, demonstrating that MsmHr does not influence M. smegmatis growth in rich (7H9) or minimal medium (Sauton) (Figure 3A). To identify possible biological roles of MsmHr, the growth kinetics and survival of msmHr were examined under various stress conditions, including NO, hypoxia, H 2 O 2 , heat shock, and acidic pH. No growth defects in msmHr were detected under the conditions tested, with the exception of H 2 O 2 stress ( Figure 3B). msmHr exhibited mild H 2 O 2 resistance compared to the wild type mc 2 155 strain after treatment with 5 mM H 2 O 2 for 3 h, while no difference in the growth of the two strains was observed under non-H 2 O 2 treatment conditions. In addition, the resistance phenotype was abrogated in the complemented strain, C-msmHr ( Figure 3B). Moreover, overexpression of msmHr in mc 2 155 (O-msmHr) was associated with H 2 O 2 susceptibility after treatment with 5 mM H 2 O 2 for 3 h (Figure 3B). Differences in mRNA levels of msmeg_2414 and msmeg_2416 in wild type mc 2 155 and msmHr were not detected after treatment with H 2 O 2 ( Figure 2B). A statistically significant reduction in msmHr mRNA levels was found between mc 2 155 in the presence and absence of H 2 O 2 ( Figure 2B). Taken together, our data suggest that msmHr plays an inhibitory role in the response to H 2 O 2 stress.

THE HEMERYTHRIN-LIKE DOMAINS OF MsmHr ARE REQUIRED FOR THE RESPONSE TO H 2 O 2 STRESS
To evaluate the potential roles of the hemerythrin-like domain of MsmHr in the H 2 O 2 response, we constructed 3 mutants, H1M, H2M, and H3M, in which the respective conserved amino acid motifs HxxxEE, HxxxQQ, and HxxxEE were all mutated to LxxxAA ( Figure 4A). As overexpression of msmHr showed higher susceptibility to H 2 O 2 , overexpression vector pMV261 containing msmHr alleles harboring the point mutations H1M, H2M or H3M was introduced into mc 2 155 which was then assessed for PCR products for the upstream and downstream regions of msmHr were amplified using the primer pairs 2415LLL/IL(R) and 2415RRR/IR(F), respectively. (B) Effect of msmHr deletion on msmeg_2414 and msmeg_2416 expression. Quantitative real-time PCR (qRT-PCR) analysis of msmHr cluster transcription. The primer pairs 2414qF/2414qR and 2416qF/2416qR were used for qRT-PCR. Results are shown as the means ± standard deviations of three replicates ( * P < 0.05).
resistance to H 2 O 2 . All strains overexpressing the corresponding H1M, H2M or H3M mutant msmHr proteins behaved like mc 2 155. By contrast, strains containing intact msmHr proteins were susceptible to H 2 O 2 ( Figure 4B). This result indicates that the hemerythrin-like domain of MsmHr is required for H 2 O 2 susceptibility.

MsmHr REPRESSES sigF EXPRESSION THROUGH THE PROMOTER P rbsw
There are two independent oxidative stress pathways in mycobacteria: the KatG-, isoniazid (INH)-related pathway and the SigFrelated, INH-unrelated related pathway (Gebhard et al., 2008;Wu et al., 2012). INH is an important first-line anti-mycobacterial pro-drug and is activated by the bacterial catalase-peroxide enzyme encoded by katG (msmeg_3461). Resistance to H 2 O 2 has been shown to correlate with susceptibility to INH (Bulatovic et al., 2002). To determine which pathway MsmHr is involved in, we measured the minimum inhibitory concentration (MIC) of INH against msmHr and wild type mc 2 155. No difference in the MICs of these two strains was detected (3.125 mg/L in both msmHr and mc 2 155), suggesting that MsmHr is not involved in the INH-related H 2 O 2 response. We then examined whether MsmHr is involved in the SigF-mediated H 2 O 2 response. As previous studies have shown that sigF is transcribed from two promoters, we constructed two vectors with the two promoter regions, P msmeg_1802 and P rbsw , fused to lacZ (P msmeg_1802 -lacZ and P rbsw -lacZ, respectively) (Humpel et al., 2010). We measured the indicated promoter activities at the early logarithmic phase: β-galactosidase activity associated with P msmHr -lacZ was 6.9 ± 0.2 MU in mc 2 155 and 7.6 ± 0.1 MU in msmHr, indicating that MsmHr does not self-regulate at the transcriptional level. Wild type mc 2 155 harboring P msmeg_1802 -lacZ had a β-galactosidase activity of 3.6 ± 0.3 MU, while the activity of msmHr harboring P msmeg_1802 -lacZ was 3.7 ± 0.2 MU, indicating that MsmHr does not influence the promoter activity of P msmeg_1802 (Figure 5B left panel). In contrast, msmHr harboring P rbsw -lacZ had a significantly higher β-galactosidase activity (15.7 ± 1.9 MU) than that of wild type mc 2 155 (Figure 5B right panel). We also measured the mRNA levels of sigF in mc 2 155, msmHr and its complementary strain C-msmHr. Consistent with results for the promoter, the knockout msmHr led to a 1.9 ± 0.2 fold increase relative to the mRNA level of sigF to wild type mc 2 155, while levels of sigF mRNA were not significantly different ( Figure 5C). Taken together, our results suggest that MsmHr affects the mRNA level of sigF via P rbsw .

MsmHr IS NECESSARY FOR THE SigF MEDIATED H 2 O 2 RESPONSE
The above results indicate that MsmHr suppresses sigF expression at the early logarithmic phase (Figure 5). We next examined the influence of MsmHr on the SigF-mediated H 2 O 2 pathway. We compared the mRNA level of sigF between mc 2 155, msmHr and the msmHr complementary strain C-msmHr after 5 mM H 2 O 2 treatment for 30 min. As shown in Figure 5C, sigF mRNA was induced by H 2 O 2 in mc 2 155, while induction of sigF mRNA by H 2 O 2 was not detected in msmHr. The increase in sigF mRNA induced by H 2 O 2 was restored in C-msmHr. These  In addition, we used qRT-PCR to measure the mRNA levels of redox-related genes in both mc 2 155 and msmHr in response to H 2 O 2 treatment, using rpoD mRNA as an internal invariant control (Table S3). We then chose the high H 2 O 2 -induced genes msmeg_4753 and msmeg_1782, which belong to the SigF regulon, to evaluate their mRNA level in response to H 2 O 2 treatment in mc 2 155, sigF, msmHr and C-msmHr. The level of msmeg_4753 mRNA increased 5.6 ± 0.9 fold in mc 2 155 after treatment with H 2 O 2 , but induction of msmeg_4753 was abrogated in msmHr and sigF after treatment with H 2 O 2 ( Figure 6B). In C-msmHr, an increase in msmeg_4753 RNA was observed in response to H 2 O 2 ( Figure 6B). The level of Msmeg_1782 mRNA increased two-fold in both mc 2 155 and C-msmHr when treated with H 2 O 2 , while no changes in mRNA level were observed in response to H 2 O 2 in msmHr and sigF ( Figure 6A). Taken together, this data indicates that MsmHr is required for the SigF-mediated H 2 O 2 response.

DISCUSSION
In this study, we have identified a mycobacterial hemerythrinlike protein MsmHr, which regulates sigF expression via promoter P rbsw and is necessary for the SigF-mediated H 2 O 2 response. To our knowledge, MsmHr is the first hemerythrin-like protein to be characterized in mycobacteria.
Specific roles for hemerythrin-like proteins are just beginning to be characterized (Xiong et al., 2000;Justino et al., 2007;Schaller et al., 2012). On the basis of their sequences, hemerythrin-like proteins have been postulated to have diverse physiological functions related to oxygen and/or iron (Bailly et al., 2008;French et al., 2008). For example, the E. coli hemerythrin-like protein YtfE confers protection against both NO and H 2 O 2 stresses (Justino et al., 2005(Justino et al., , 2007. Here, however, we did not observe any growth differences between mc 2 155 and msmHr under NO stress, suggesting that the functions of hemerythrin-like proteins in mycobacteria might be distinct from those in E. coli. The variation in biological functions of hemerythrin-like proteins might be due to different selective evolutionary environments (Saini et al., 2012b;Martin-Duran et al., 2013).
No difference in sensitivity to INH was observed between the wild-type mc 2 155 and msmHr strains, suggesting that MsmHr is not involved in the INH-related oxidative stress response pathway but rather in an alternative SigF-related H 2 O 2 pathway (Gebhard et al., 2008;Wu et al., 2012). It will be interesting to explore why mycobacteria use two-independent H 2 O 2 scavenging pathways and which sensors trigger each of these signaling pathways. Recent reports show that a hemerythrin-like domain  of FBXL5 can sense if endogenous iron is limiting and respond to iron stress in the mammalian system (Salahudeen et al., 2009;Vashisht et al., 2009). The correlation between iron-and oxygenbinding MsmHr and SigF-dependent H 2 O 2 responses needs to be further explored. The complexity of the transcription regulatory network allows for efficient and prompt change in levels of gene transcription in response to environmental changes. The protein encoded by mycobacterial sigF has closest homology to Streptomyces coelicolor SigF, Bacillus subtilis SigF and B. subtilis SigB (Demaio et al., 1996(Demaio et al., , 1997Gebhard et al., 2008). In B. subtilis, sigB is activated upon entry into the stationary phase and by environmental stresses such as heat, oxidative stress and hyper osmosis. The transcription of sigB has been shown to be controlled by two promoters (Wise and Price, 1995). Similarly, SigF may be a potential general stress regulator; SigF is not only activated upon entry into the stationary phase, but is also induced by environmental stresses such as heat shock, acidic pH and oxidative stress (Wise and Price, 1995;Gebhard et al., 2008). Expression of sigF has been shown to be regulated by two promoters, P msmeg_1802 and P rbsw . While promoter P msmeg_1802 is known to respond to entry into the stationary phase (Gebhard et al., 2008), the role of P rbsw is less well understood. In this study, comparisons of the activity of the promoters of sigF and the mRNA level of sigF in the wild type mc 2 155 and mutant msmHr strains (Figure 5) showed that MsmHr regulates sigF expression via the P rbsw promoter.
We show here that MsmHr is essential for the SigF-mediated H 2 O 2 response (Figures 5, 6). MsmHr hinders sigF promoter activation and inhibits sigF transcription during normal growth. When msmHr is deleted, inhibition of sigF is abrogated, and sigF maintains a higher transcript level. The transcription level of sigF did not vary in response to H 2 O 2 treatment in mutant msmHr strains, possibly because the mRNA level of sigF was maintained at a higher level in msmHr (Figure 5). We measured mRNA level changes of members of the SigF-regulon, msmeg_4753 and msmeg_1782, before, between, and after treatment with H 2 O 2 . Our data show that in wild type mc 2 155, mRNA levels of both msmeg_4753 and msmeg_1782 increased in response to H 2 O 2 treatment, but no specific response to H 2 O 2 was observed in sigF and msmHr (Figure 6), suggesting that MsmHr is required for induction of msmeg_4753 and msmeg_1782 transcription in response to H 2 O 2 . The presence of inhibitory protein MsmHr suggests that the transcriptional regulation of SigF itself and the SigF regulon, or at least part of the SigF regulon, is based on the balance between the activation and inhibition of the H 2 O 2 response. In msmHr, the inhibition of sigF is abrogated and sigF maintains high transcript levels. The expression of one of the SigF regulon genes, msmeg_1782, is high in msmHr during normal growth compared with mc 2 155, but decreases in response to H 2 O 2 treatment. This is consistent with the finding that MsmHr is necessary for the SigF-dependent H 2 O 2 pathway and that the response of sigF to H 2 O 2 is also abrogated in the absence of msmHr.
In summary, we have identified a mycobacterial hemerythrinlike protein that negatively regulates SigF via the P rbsw promoter in response to oxidative stress.

ACKNOWLEDGMENTS
We thank Defeng Li for the gift of the protein gelactin AAL. This work was supported by the Ministry of Science and Technology of China (2014CB744402 and 2012CB518700).