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Journal of Bacteriology, May 2005, p. 2948-2956, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.2948-2956.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Bacillus subtilis Paraquat Resistance Is Directed by {sigma}M, an Extracytoplasmic Function Sigma Factor, and Is Conferred by YqjL and BcrC

Min Cao, Charles M. Moore, and John D. Helmann*

Department of Microbiology, Cornell University, Ithaca, New York 14853-8101

Received 23 August 2004/ Accepted 22 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function {sigma}M protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate {sigma}M-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacillus subtilis responds to the presence of oxidizing agents by activating complex adaptation mechanisms under the control of redox-sensitive regulatory proteins. Transcriptional profiling indicates that hydrogen peroxide activates a large stimulon, including genes controlled by the general stress {sigma} factor {sigma}B, the peroxide sensor PerR, and the organic peroxide sensor OhrR (15). Under some conditions, members of the Fur regulon are also induced by hydrogen peroxide (30). Many of these same regulons are induced by the superoxide generator paraquat (PQ), although it is difficult to know whether these responses are due to superoxide or subsequently produced hydrogen peroxide (30). Nitric oxide induces both the PerR and Fur regulons, particularly under anaerobic conditions (28), consistent with a direct nitrosylation of the Fe(II) corepressors (10). B. subtilis mounts a distinct, but overlapping, response to disulfide stress elicited by thiol-oxidizing agents such as diamide (23). The diamide stimulon includes both the PerR regulon and genes regulated by the novel thiol stress sensor Spx (31, 49).

Extracytoplasmic function (ECF) {sigma} factors have also been implicated in the regulation of oxidative stress responses. ECF {sigma} factors comprise a unique subgroup within the {sigma}70 family (14, 24) which, in general, controls genes related to cell envelope functions such as cell surface stress (Escherichia coli {sigma}E and B. subtilis {sigma}W and {sigma}M), secretion (Pseudomonas aeruginosa {sigma}E), and metal ion homeostasis (E. coli FecI, Pseudomonas fluorescens PbrA, and Ralstonia eutropha CnrH). However, there are exceptions: Streptomyces coelicolor {sigma}R and Mycobacterium tuberculosis {sigma}H respond to cytoplasmic disulfide stress (25, 34, 35, 43). In both cases, the {sigma} factor is negatively regulated by an anti-{sigma} factor (RsrA for {sigma}R and RshA for {sigma}H) containing a Zn(II)-binding motif (His-X-X-X-Cys-X-X-Cys, where X stands for any amino acid) near the N terminus. In RsrA, cysteines within this motif are part of a thiol-disulfide redox switch (34). Upon oxidation, RsrA releases {sigma}R, which in turn switches on the transcription of the thioredoxin operon trxBA. RsrA and related anti-{sigma} factors comprise the Zn-binding anti-{sigma} (ZAS) family (34).

B. subtilis encodes two members of the ZAS anti-{sigma} factors: RsiW and YlaD. RsiW serves as the anti-{sigma} factor for {sigma}W (40), while YlaD is a transmembrane protein of unknown function encoded by the same operon as the ECF sigma factor ({sigma}YlaC), although it does not appear to bind directly to {sigma}YlaC (47). The {sigma}W regulon has been extensively characterized and includes approximately 60 genes that function in a cell envelope stress response (5). There is no evidence, as yet, to link either {sigma}W or {sigma}YlaC to the regulation of oxidative stress response genes.

Here, we demonstrate that only sigM of the seven ECF {sigma} factors in B. subtilis contributes to oxidative stress resistance. A sigM null mutant had a dramatically increased sensitivity to PQ and structurally related superoxide-generating agents. This is consistent with previous results, demonstrating that the expression of {sigma}M is induced by PQ (44). Although PQ can increase the intracellular production of superoxide in many cell types, the mechanism and subcellular location of PQ stress in B. subtilis is largely unknown. We identify two {sigma}M-regulated proteins, YqjL (a putative hydrolase) and BcrC (a bacitracin resistance protein) (4), as major contributors to the {sigma}M-dependent resistance to PQ and structurally related superoxide-generating compounds.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, growth conditions, and chemicals. All B. subtilis strains used in this study are listed in Table 1. Bacteria were grown in Luria-Bertani (LB) medium or low-salt LB medium (LS) lacking added NaCl at 37°C with vigorous shaking. Antibiotics were added to the growth medium when appropriate at 100 µg/ml of ampicillin or 200 µg/ml of spectinomycin for E. coli and 100 µg/ml of spectinomycin, 10 µg/ml of kanamycin, 10 µg/ml of tetracycline, 10 µg/ml of chloramphenicol, 8 µg/ml of neomycin, or 1 µg/ml of erythromycin plus 25 µg/ml of lincomycin (for macrolide-lincomycin-streptogramin B [MLS] resistance) for B. subtilis.


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  		TABLE 1. Bacterial strains used in this study

 
All chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, Mo.). Paraquat dichloride (PQ), diquat dibromide monohydrate (DQ), ethyl viologen dibromide (EV), benzyl viologen (BV), pyrogallol, and mitomycin C were dissolved in distilled water. Plumbagin, menadione, and dipyridyl were dissolved in ethanol. Nitrofurantoin (7.6 mg) was dissolved in 14.9 ml of 95% ethanol. One gram of 4-nitroquinoline N-oxide (4NQO) was dissolved in 52.5 ml of acetone. One gram of 4, 4'-dimethylbiphenyl was dissolved in 5 ml of chloroform. Benzidine was dissolved in 10% acetic acid.

Construction of sigV, sigZ, ylaC, sigW ylaC, and sigX sigW sigM mutants. Primers 335 and 336 (all oligonucleotide primers are listed in Table 1S in the supplemental material; see "Supplemental figures and material" below) were used to amplify the sigV gene from B. subtilis strain CU1065 chromosomal DNA. The PCR fragment (about 900 bp, including the coding region of sigV as well as 200 bp of upstream sequence and 190 bp of downstream sequence) was digested with HindIII and EcoRI and ligated into pUC18 to generate plasmid pMC61. pMC61 was digested with SacI and HincII and ligated with a Kanr cassette (from pJM114 [36], which was digested with SacI and HincII) to generate pMC65. Thus, an internal fragment (~320 bp) of sigV was replaced by the Kanr cassette. pMC65 was linearized with ScaI and moved into B. subtilis strain CU1065 by transformation, selecting for Kanr, to generate strain HB0028. The Kanr cassette is oriented in the same direction as sigV.

Primers 325 and 327 were used to amplify the sigZ gene from B. subtilis strain CU1065 chromosomal DNA. The PCR fragment (about 950 bp, including the coding region of sigZ as well as 220 bp of upstream sequence and 200 bp of downstream sequence) was digested with HindIII and BamHI and ligated into pUC18 to generate plasmid pMC60. pMC60 was digested with StyI and XhoI (to release a 70-bp internal fragment) and ligated with the pJM114 Kanr cassette (digested with XbaI and XhoI) to generate pMC64. pMC64 was linearized with ScaI and moved into B. subtilis strain CU1065 by transformation, selecting for Kanr, to generate strain HB0032. The Kanr cassette is oriented in the same direction as sigZ.

Primers 328 and 330 were used to amplify the ylaC gene from B. subtilis strain CU1065 chromosomal DNA. The PCR fragment (about 800 bp, including the coding region of ylaC as well as 200 bp of upstream sequence and 80 bp of downstream sequence) was digested with HindIII and BamHI and ligated into pUC18 to generate plasmid pMC62. pMC62 was digested with StyI and HincII (releasing a 170-bp internal gene fragment) and ligated with the same Kanr cassette (digested with XbaI and HincII) to generate pMC66. pMC66 was linearized with ScaI and moved into B. subtilis strain CU1065 by transformation, selecting for Kanr, to generate strain HB0029. The Kanr cassette is oriented in the same direction as ylaC.

The sigW ylaC double mutant HB0139 was constructed by transforming chromosomal DNA from HB0029 (ylaC::kan) into HB0020 (sigW::MLS) and selecting for Kanr and MLS resistance. The sigX sigW sigM triple mutant HB0099 was constructed by moving chromosomal DNA from HB0020 (sigW::MLS) into HB0097 (sigXsigM) by transformation and selecting for Spcr, Kanr, and MLS resistance.

Construction of null mutants in yrhJ, ydaH, ypbG, yraA, radA, yacK, radC, and yqjL. Long-flanking homology PCR was used as described previously (45) to generate allelic replacement mutants for each gene or group of genes. In brief, approximately 1,000-bp genomic regions flanking the gene(s) to be deleted were amplified from CU1065 chromosomal DNA by PCR. Drug resistance cassettes were amplified by PCR from pGEM-cat-3Zf(+) (48) (cat) or pDG1513 (tet) (12).

For each mutant construction, equal amounts (approximately 200 to 300 ng) of purified upstream flanking fragment, downstream flanking fragment, and the corresponding drug resistance cassette were used in a joint PCR procedure using the HotStarTaq master mix kit (QIAGEN). The resulting PCR products were purified, and B. subtilis wild-type strain CU1065 was transformed selecting for the corresponding antibiotic resistance. The generated mutant strains are listed in Table 1. A yqjL bcrC double mutant strain (HB0154) was constructed by transformation of the bcrC mutant strain (HB0106) to Tetr using yqjL chromosomal DNA.

Overproduction of SodA, BcrC, and YqjL in B. subtilis using a xylose-inducible system for complementation tests. The sodA open reading frame was PCR amplified with primers 900 and 901, digested with BamHI and EcoRI, and cloned into pXT, a derivative of pDG1731 that places the inserted gene under control of the inducible PxylA promoter and can be integrated into the thrC locus (3). The resulting plasmid was designated pMC121 (PxylA-sodA). pMC121 was linearized with ScaI and ectopically integrated into B. subtilis strains HB0141 (sodA::cat) and HB0031 (sigM::kan) at the thrC locus by transformation with selection for Spcr. The transformants were screened for MLS sensitivity (indicative of a double-crossover event) and threonine auxotrophy. The resulting strains were designated HB0144 (sodA, with PxylA-sodA at the thrC locus) and HB0145 (sigM, with PxylA-sodA at the thrC locus).

Primer sets 732-919 and 1668-1669 were used to amplify the bcrC and yqjL open reading frames, respectively. The fragments were first cloned into pCR2.1-TOPO vector (Invitrogen) and digested with EcoRI. The resulting fragments were cloned into pXT at the EcoRI site. Plasmids with correct orientation were selected and designated pMC122 (PxylA-yqjL) and pMC123 (PxylA-bcrC). The same procedures as described above were used to introduce the plasmid into the thrC locus of corresponding mutant strains. For overproduction, xylose was added to the growth medium to a final concentration of 1%.

Construction of PyqjL-cat-lacZ transcriptional fusion. The yqjL promoter region was amplified from B. subtilis chromosomal DNA by PCR using primers 552 and 553. The resulting fragment was digested with HindIII and BamHI and cloned into pJPM122 (42) to generate plasmid pMC88 (PyqjL-cat-lacZ). The sequence of the promoter region was verified by DNA sequencing (Cornell University BioResources facility). Plasmid pMC88, carrying the promoter fusion was linearized with ScaI and introduced into the modified SPß prophage of B. subtilis strain ZB307A (50) by a double-crossover recombination with selection for Neo resistance. This SPß prophage is a temperature-sensitive lysogen, and plasmid integration results in the generation of promoter region cat-lacZ operon fusions in which both chloramphenicol resistance and ß-galactosidase can be used as reporters for promoter activity (42). SPß lysates were prepared by heat induction as described previously (42) and used to transduce CU1065 and HB0031 (sigM::kan) to generate strains HB0183 and HB0184.

Zone of inhibition assay. B. subtilis strains were grown at 37°C overnight in LB or LS medium, diluted 1:100 into fresh LB or LS medium, and grown at 37°C with shaking. At mid-logarithmic phase, 20 µl of culture was mixed with 2 ml of 0.7% soft LB or LS medium agar and poured onto the bottom agar. After cooling, filter paper disks (6-mm diameter) carrying chemicals to be tested were placed on the top of the agar and the plates were incubated at 37°C overnight. The next day, the diameters of the inhibition zones were measured.

Gradient plate assays. B. subtilis strains were grown in LB or LS medium at 37°C overnight, diluted 1:100 into LB or LS medium, and grown at 37°C with shaking. Mid-logarithmic-growth-phase cultures were adjusted to an optical density at 600 nm (OD600) of 0.3 by dilution with prewarmed LB or LS and streaked onto gradient agar plates using a sterile Dacron swab applicator. Plates were prepared using Omnitray single-well microtiter plates (Nalge Nunc International) and poured to a thickness of 0.8 cm. The initially poured agar wedge (34 ml) contained the test compound and was allowed to solidify for 30 min prior to the addition of a second agar wedge (34 ml). Plates were allowed to equilibrate for 2 h at room temperature prior to inoculation and overnight incubation (37°C).

SOD activity assay. Overnight B. subtilis cultures (wild type, sigM, and sodA mutants) were diluted 1:100 into LB medium and grown at 37°C. Cell samples were collected at early, mid-, and late log growth phase and washed in Tris-HCl buffer (50 mM Tris-HCl, pH 9.0, 5% glycerol, 30 µM dithiothreitol). Cell pellets were resuspended in Tris-HCl buffer and lysed using sonication and lysozyme. The total protein concentration was determined by the Bradford dye-binding assay (Bio-Rad). Total protein (30 µg) was applied onto a 10% native polyacrylamide gel electrophoresis gel. Bands representing superoxide dismutase (SOD) activity were visualized by negative staining using the nitroblue tetrazolium method (2).

Paraquat induction and ß-galactosidase assay. Cells from overnight cultures were inoculated into fresh LB medium and grown to an OD600 of 0.2. The culture was split into two aliquots, one of which was challenged by the addition of paraquat to a final concentration of 80 µM. The culture was returned to 37°C, and cell samples were collected every 15 min. The ß-galactosidase activity of each sample was measured according to the method of Miller (26).

Supplemental figures and material. A complete listing of the primers used during strain construction (Table 1S) and supplemental Fig. 1S are available at http://www.micro.cornell.edu/faculty.JHelmann.html.



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  		FIG. 1. Role of {sigma}M in resistance to paraquat. (A) Sensitivity of the sigM mutant to PQ but not to diamide (left). Lawns of wild type (wt) and sigW were generated by overlaying LB agar plates with 2 ml of soft LB agar containing 200 µl of mid-log-phase (OD600 = 0.4) cultures. Five microliters of 0.5 M PQ or 1 M diamide was spotted on the filter paper disk and added to the plate. The plates were scanned after overnight incubation at 37°C. PQ resistance of the sigM mutant was completely restored by overexpression of sigM under a xylose-inducible system (right). Xylose (1% [wt/vol]) was added to the cell culture and was present in the soft LB agar for induction. (B) The sigM mutant was sensitive to PQ but not to structurally different redox cycling compounds. Five microliters of 0.5 M PQ, 1% menadione, 400 mg/ml pyrogallol, or 100 mM plumbagin was spotted on the filter disk, respectively. (C) The chemical structures of paraquat, menadione, pyrogallol, and plumbagin.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The {sigma}M ECF {sigma} factor contributes to oxidative stress resistance. We tested the sensitivity of mutant strains altered in each of the seven ECF {sigma} factors to hydrogen peroxide (8.8 M), tert-butyl hydroperoxide (7.77 M), cumene hydroperoxide (0.53 M), diamide (1 M), and PQ (0.5 M) using zone of inhibition experiments. Only sigM affected oxidative stress resistance and only to PQ (Fig. 1A). Moreover, induction of sigM from a xylose-inducible promoter (PxylA) completely restored PQ resistance to the sigM mutant (Fig. 1A). We conclude that {sigma}M contributes specifically to superoxide resistance and not to redox stress resistance in general. In contrast, no increased sensitivity to any of these reagents was observed for the sigW, ylaC, or sigW ylaC mutant despite the fact that it is these {sigma} factors that are regulated by ZAS-type anti-{sigma} factors.

Previous studies have demonstrated that the {sigma}M regulon has some overlap with regulons controlled by {sigma}X and {sigma}W (4, 5, 27, 33). However, analysis of sigX sigM and sigW sigM double mutants and a sigX sigW sigM triple mutant did not reveal an increase in PQ sensitivity relative to the sigM single mutant.

The sigM mutant is only sensitive to superoxide-generating reagents with a dipyridyl ring. The sigM mutant, while sensitive to PQ, did not have an increased sensitivity to the structurally distinct superoxide generators menadione, pyrogallol, or plumbagin (Fig. 1B). Whereas PQ is a dication with a 4, 4'-dipyridyl ring, menadione, pyrogallol, and plumbagin are structurally distinct (Fig. 1C). The correlation with chemical properties was reinforced by the finding that the sigM mutant was more sensitive than the wild type to EV and BV (both with a 4, 4'-dipyridyl ring) and DQ (containing a 2, 2'-dipyridyl ring), all of which are also cationic compounds (Table 2). In contrast, the sigM mutant was not more sensitive to compounds with pyridine or dipyridyl rings that do not generate superoxide radicals (pyridine, 2, 2'-dipyridyl, and 4, 4'-dipyridyl), benzidine and 4, 4'-bimethylbiphenyl, which are structurally similar to PQ but have a biphenyl ring and do not generate superoxide radicals, or nitrofurantoin, a structurally distinct superoxide generator (Table 2).


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  		TABLE 2. Chemical sensitivity of wild-type and mutant B. subtilis strains

 
Since PQ, EV, BV, and DQ are all superoxide generators, we hypothesized that their toxicity was linked to the production of reactive oxygen species. Consistent with this hypothesis, the sigM mutant and wild type displayed greatly reduced, but equal, sensitivity to these compounds in zone of inhibition assays under anaerobic conditions (data not shown). This indicates that one role of {sigma}M is to protect cells against the ability of these compounds to trigger oxidative stress.

Superoxide radicals cause damage to proteins, DNA, and lipids. As the {sigma}M regulon includes several putative DNA repair proteins (e.g., RadA, RadC, and YpbG), we tested whether the sigM mutant was also sensitive to other DNA-damaging agents. However, no significant differences were observed between the wild type and the sigM mutant in sensitivity to mitomycin C, 4NQO, or methyl methanesulfonate (Table 2).

PQ sensitivity of the sigM mutant is not due to a decrease in superoxide dismutase activity. In B. subtilis, only one detectable superoxide dismutase (SodA) is found in both vegetative cells and spores (21). The factors controlling expression of sodA are not well defined, but sodA is not thought to be part of the {sigma}M regulon. Moreover, a sodA mutant was sensitive not only to PQ but also to menadione, pyrogallol, and plumbagin (data not shown). This suggests that the effect of the {sigma}M mutation is unlikely to be due to effects on sodA expression. Indeed, induction of sigM from the xylose-inducible PxylA promoter completely restored PQ resistance to the sigM mutant (Fig. 1A) but not to the sodA mutant (data not shown). Conversely, induction of sodA from PxylA could fully restore PQ resistance to the sodA mutant but not to the sigM mutant (data not shown). In addition, we directly measured SOD activity on a native polyacrylamide gel by negative staining using the nitroblue tetrazolium method (2). Only one active band, in approximately equal intensities, was seen in both the wild-type and the sigM mutant strains at all growth phases (Fig. 2). This band corresponds to SodA, as it disappeared in the sodA mutant.



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  		FIG. 2. {sigma}M does not affect SOD activity in B. subtilis. Wild-type (lanes 1), sigM (lanes 2), and sodA (lanes 3) mutant cells were harvested during early log, mid-log, and transition (late log/onset of stationary) phases. Total protein (30 µg) was applied to a 10% native polyacrylamide gel electrophoresis gel. Bands representing SOD activity were visualized by negative staining using the nitroblue tetrazolium method (2).

 
YqjL and BcrC are implicated in paraquat resistance. To identify genes responsible for resistance to PQ, we generated null mutants in several {sigma}M-regulated genes including radA, yacK, radC, ypbG, ydaH, yqjL, yrhJ, and yraA (1, 44). In addition, we previously generated a null mutant for bcrC, a gene controlled by both {sigma}M and {sigma}X (4). Three of these genes (radA, radC, and ypbG) encode putative DNA repair proteins, yacK encodes a DNA-binding protein, ydaH encodes a membrane protein with unknown function, and yqjL encodes a member of the {alpha}/ß hydrolase family. YrhJ is a cytochrome P450/NADPH reductase, and YraA is 61% identical to the B. subtilis general stress protein YfkM. Of these nine mutant strains, only the yqjL mutant displayed an increased sensitivity comparable to that seen in the sigM mutant on LB plates containing 30 µM PQ (Fig. 3 and data not shown). Although a bcrC mutant did not display a significantly increased PQ sensitivity, the yqjL bcrC double mutant was more sensitive than either single mutant and comparable to the sigM mutant (Fig. 3 and 4). Overexpression of yqjL, under the control of a xylose-inducible promoter (PxylA), restored PQ resistance to both the yqjL and the sigM mutants (Fig. 4), but overexpression of bcrC could not restore the PQ resistance of the sigM mutant. These results support the notion that YqjL is the primary {sigma}M-dependent determinant of PQ resistance.



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  		FIG. 3. The yqjL and bcrC genes affect growth on plates containing PQ. Dilutions (1, 10–1, 10–2, 10–3, 10–4, and 10–5) of log-phase cell cultures (OD600 = 0.4) were spotted on LB and LB plus 30 µM PQ plates and incubated at 37°C overnight. This experiment was repeated several times with various concentrations of PQ, and similar results were observed. wt, wild type.

 


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  		FIG. 4. Both YqjL and BcrC contribute to protection against PQ. The sensitivity of each indicated strain was measured by zone of inhibition assays. The PQ resistance of the sigM mutant and the yqjL mutant was restored by overexpression of yqjL under control of a xylose-inducible promoter (PxylA). Xylose (1% [wt/vol]) was added (+) to the culture and in the soft LB agar for induction. The error bars indicate the standard deviations of the results from replicate samples. wt, wild type.

 
Since the disk diffusion assay requires growth in the presence of the superoxide generator, it does not distinguish between bactericidal and bacteriostatic effects. To monitor bactericidal effects, we added PQ to mid-logarithmic-phase cells and monitored both growth and viability. PQ resulted in a cessation of growth after approximately 20 min for both the wild type and the single and double yqjL bcrC mutants (Fig. 5A). Moreover, the rate of loss of viability (as measured by spotting dilutions of the culture onto LB plates) was indistinguishable between the four tested strains (Fig. 5B and data not shown). In all cases, there was approximately 99% killing by 80 min after addition. The difference between the PQ-sensitive phenotype observed in the plate-based growth assay and the lack of effect noted in these viability measurements may indicate that PQ has a bacteriostatic effect that is monitored on solid media, that the surface- and liquid-grown cells are physiological different, or both.



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  		FIG. 5. YqjL does not protect against the bactericidal effects of PQ. (A) The sensitivity of CU1065 and yqjL mutant cells was monitored in LB broth in the presence and absence of 1 mM PQ added at an OD600 of 0.3. PQ quickly resulted in bacteriostasis of both CU1065 and the yqjL mutant. Growth was monitored by OD600. (B) Viability was monitored by spotting 5 µl of a 10-fold dilution series on LB plates 20, 80, and 120 min after PQ addition to monitor cell killing. PQ addition resulted in significant killing in both the CU1065 and yqjL cultures as observed in the 80- and 120-min samples.

 
The toxicity of PQ and EV is exacerbated by low NaCl concentrations. In E. coli, high-salt concentrations, such as found in LB (over 170 mM), decrease PQ sensitivity relative to low-salt culture medium, perhaps by inhibition of uptake (22). Similarly, B. subtilis and the sigM mutant were more sensitive to PQ and EV (but not to BV or DQ) on LS medium than on LB (Table 2). Using the disk diffusion assay, we demonstrated that both yqjL and bcrC contribute to resistance to PQ, EV, BV, and DQ (Table 2). Like the sigM mutant, the sensitivity of these mutants to PQ and EV was higher on LS than on LB. Under these low-salt conditions, a PQ-sensitive phenotype for the bcrC single mutant is also readily apparent (Table 2).

YqjL and BcrC together account for the sensitivity of the sigM mutant. The PQ sensitivity of the yqjL bcrC double mutant was greater than either single mutant as measured by both disk diffusion (Fig. 4) and cell plating assays (Fig. 3). To extend these results to other stress agents and take advantage of the observation that the apparent role of bcrC is enhanced under low-salt conditions, we used a gradient plate analysis to compare yqjL and bcrC single and double mutants to the wild type (Table 3). Although the effect of the bcrC mutation alone is only apparent with PQ (and only under low-salt conditions), the additive effect of the bcrC and yqjL mutations is apparent for all four tested compounds, at least under low-salt conditions. For example, both the sigM and bcrC yqjL double mutant grow across 2.6 cm of the gradient plate, whereas the bcrC and yqjL single mutants grow 9.0 and 4.7 cm, respectively.


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  		TABLE 3. Resistance to superoxide-generating compounds as measured by a solid medium gradient plate assay

 
Expression of yqjL is {sigma}M dependent and is inducible by PQ. We used a PyqjL-cat-lacZ reporter fusion, integrated ectopically at SPß, to measure expression in both wild-type and sigM mutant strains. Expression was generally low in the wild-type strain, with maximum activity during early to mid-log growth phase and then decreasing during late log phase (Fig. 6A), a pattern similar to the expression of sigM (18). The expression was reduced to the background level in the sigM mutant strain (Fig. 6A). Addition of 80 µM PQ to the growth medium led to a strong induction of yqjL expression (Fig. 5A) but only after a 30-min delay. A similar delayed response to PQ is observed for the autoregulatory sigM promoter (Fig. 6B) (PM-cat-lacZ), as originally reported by Thackray and Moir (44). Note that both the basal and induced expression from these promoters is completely dependent on {sigma}M. Although the cloned sigM regulatory region also contains a {sigma}A-dependent promoter described previously (18), expression is entirely {sigma}M dependent in our construct (4).



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  		FIG. 6. PQ elicits induction of both yqjL and sigM. Induction of PyqjL-cat-lacZ (A) and PM-cat-lacZ (B) in LB medium by 80 µM PQ. ß-Galactosidase activity (filled symbols) and cell density (OD600, open symbols) are shown for the wild-type strains in the absence (squares) and presence (diamonds) of PQ. Expression is greatly reduced when the fusions are in the sigM mutant background in either the absence (circles) or presence (triangles) of PQ (symbols along the x axis). Where indicated, PQ was applied when the cell density (OD600) reached 0.2 (0 min). The results shown are representative of several replicates, although in some cases, the magnitude of induction was somewhat less (~2- to 3-fold). The lysis observed in this experiment at the late time points in panel A was not seen in other replicates.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that a {sigma}M null mutant is more sensitive to a subfamily of superoxide-generating reagents related to PQ but not to other superoxide generators such as menadione, pyrogallol, or plumbagin. This sensitivity was not due to a decrease in SodA activity because, unlike the sigM mutant, the sodA mutant is sensitive to all tested superoxide generators and the same level of SOD activity was detected in both the wild type and the sigM mutant.

PQ resistance is most frequently mediated by an efflux mechanism. For example, Salmonella enterica serovar Typhimurium has two efflux pumps (SmvA and YddG plus OmpD) that are both required for efficient export of PQ (39). SmvA is similar to the MvrA protein of E. coli (17, 29). A similar efflux system has been identified in S. coelicolor encoded by the pqrAB operon (9). The PqrA protein is a predicted transcriptional repressor, while PqrB is an MvrA/SmvA ortholog. B. subtilis contains a similarly organized operon (yvkBA) that may also play a role in PQ resistance (9); however, the yvkBA operon was not identified in global surveys of B. subtilis genes inducible by PQ or other oxidative stress agents (15, 23, 30), nor has expression of yvkBA been linked to {sigma}M (1, 44).

To identify genes that may play a role in PQ resistance, we analyzed null mutants defective for each of nine different {sigma}M-dependent genes. These genes, chosen from the list of genes previously shown to be {sigma}M dependent (4, 44) or found to be strongly induced upon induction of {sigma}M in microarray studies (1), included bcrC, radA, yacK, radC, ypbG, ydaH, yrhJ, yraA, and yqjL. Among these genes, only yqjL and bcrC significantly affected PQ resistance. Expression of YqjL from a xylose-inducible promoter complemented the PQ sensitivity of the sigM mutant, suggesting that this protein is the primary, {sigma}M-dependent defense against PQ. However, the BcrC bacitracin resistance protein (4) also plays a role in PQ resistance.

The yqjL gene was originally thought to be transcribed by either or both {sigma}W and {sigma}X, two other B. subtilis ECF {sigma} factors (5). A GAAAC-N17-CGTC sequence 34 bp upstream of yqjL functions as a promoter for both the {sigma}W holoenzyme (as seen in in vitro runoff transcription experiments) and {sigma}X holoenzyme (observed in runoff transcription/macroarray analysis [ROMA] experiments) (5). Induction of yqjL was also noted during the characterization of the vancomycin stimulon (7). However, induction was independent of {sigma}W, consistent with the notion that yqjL may be regulated primarily by {sigma}M, which controls several genes inducible by cell wall biosynthesis inhibitors (4, 7, 44).

The mechanism by which YqjL confers paraquat resistance remains unclear. YqjL has similarity to members of the {alpha}/ß hydrolase family protein, suggesting a possible role in the degradation of paraquat. The {alpha}/ß hydrolase fold is common to a number of hydrolytic enzymes of widely differing phylogenetic origin and catalytic function (16). One example is TodF of Pseudomonas putida, which is involved in toluene degradation (41). This suggests that YqjL may participate in the degradation of dipyridyl-containing compounds. Indeed, biodegradation of PQ by other soil bacteria, including Streptomyces and Nocardia spp., has been observed, but the molecular mechanism has not been characterized (32). Using a spectrophotometric assay, we observed little if any degradation of 50 µM PQ present in the culture medium over a 24-h period of growth of either wild-type or sigM mutant strains (see Fig. 1S in the supplemental material). Moreover, incubation with purified YqjL protein in vitro did not reduce the toxicity of PQ. Determining the mechanism of action of YqjL will clearly require further study. Similarly, the role of BcrC in PQ resistance is not yet clear. BcrC is a predicted membrane permease (although it also has similarity to the PAP2 family of acid phosphatases) and contributes to bacitracin resistance (4, 13, 33, 37, 38). BcrC could be involved in the efflux of either PQ or a PQ degradation product.

Interestingly, yqjM, encoding a flavoprotein xenobiotic reductase of the Old Yellow Enzyme family (46), is encoded about 113 bp upstream of yqjL. Expression of yqjM is activated by hydrogen peroxide and paraquat, as determined using both DNA microarray technology (30) and immunological analysis of YqjM protein levels (11), indicating its potential role in defending against oxidative stress. Members of this enzyme family reduce a wide range of compounds containing {alpha}/ß-unsaturated aldehydes and ketones (46). There is a transcription terminator between these two genes; thus, it is unlikely that expression of yqjL is due to readthrough from yqjM. Moreover, yqjL was not identified as PQ inducible under the conditions used for the DNA microarray studies (10-min exposure). Conversely, only yqjL (and not yqjM) was found to be induced by expression of {sigma}M (1). The independent regulation of these two enzymes suggests that they may function in distinct pathways for the protection against redox active compounds.


   ACKNOWLEDGMENTS
 
We thank Ahmed Gaballa and Jin-Won Lee for helpful discussions during this study.

This work was supported by grant GM47446 from the NIH.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101. Phone: (607) 255-6570. Fax: (607) 255-3904. E-mail: jdh9{at}cornell.edu. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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Journal of Bacteriology, May 2005, p. 2948-2956, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.2948-2956.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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