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1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
2 Molecular Microbiology Group, Institute of Food Research, Norwich NR4 7UA, UK
3 Department of Molecular and Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX 75080, USA
Correspondence
David J. Richardson
d.richardson{at}uea.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Three supplementary figures showing the effects of NO on the aerobic and anaerobic growth curves, with glucose or glycerol as the carbon source, of the eight S. Typhimurium strains examined in this study are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Many bacteria are unable to withstand the combined actions of NO and superoxide, but a few, including Salmonella, are able to survive and eventually replicate within the macrophage (Vazquez-Torres & Fang, 2001). It has been shown that the combined actions of inducible nitric oxide synthase (iNOS) and phagocyte oxidase (Phox) are important in bacterial killing and clearance by macrophages (Shiloh et al., 1999). In addition, Salmonella possesses enzymes capable of metabolizing NO, both aerobically and anaerobically. Consequently, much research has been undertaken to understand the mechanisms of in vitro NO detoxification, but with some contrasting results.
The cytoplasmic flavohaemoglobin (HmpA) enzyme has a well characterized NO dioxygenase activity in both Escherichia coli (Gardner et al., 1998; Hausladen et al., 1998; Membrillo-Hernandez et al., 1999) and Salmonella (Crawford & Goldberg, 1998). However, the physiological significance of the oxygen-independent NO reductase activity of HmpA is not fully understood. Purified HmpA is able to reduce NO to nitrous oxide under anoxic conditions (Kim et al., 1999), and a Salmonella hmpA mutant was sensitive to the NO-releaser S-nitrosoglutathione (GSNO) when cultured in an anaerobic environment (Crawford & Goldberg, 1998). E. coli hmpA expression is induced anaerobically by different sources of NO, including acidified nitrite (Mukhopadhyay et al., 2004), GSNO (Flatley et al., 2005), an NO-saturated solution (Justino et al., 2005), and NO made as a by-product of nitrite respiration (Bodenmiller & Spiro, 2006). However, it has been argued that HmpA can only reduce NO anaerobically with a turnover number 100-fold less than that recorded for aerobic conditions (Mills et al., 2001). Furthermore, E. coli overexpressing hmpA have a high aerobic NO dioxygenase activity, but a very low NO reductase activity (Gardner & Gardner, 2002). Recent results have demonstrated some contribution of HmpA to Salmonella virulence in mice (Bang et al., 2006), and to survival within macrophages (Bang et al., 2006; Gilberthorpe et al., 2007; Stevanin et al., 2002), suggesting that this enzyme is important for successful infection.
The E. coli flavorubredoxin (NorV) is an oxygen-sensitive NO reductase, which contributes to NO detoxification under anaerobic conditions (Gomes et al. 2002; Gardner & Gardner, 2002; Gardner et al., 2002). The anaerobic growth of an E. coli norV mutant is impaired in the presence of NO under anaerobic conditions that require the activities of NO-sensitive enzymes (Gardner et al., 2002) and is sensitive to the NO+ donor sodium nitroprusside (SNP) (Hutchings et al., 2002). The expression of norV is upregulated upon the addition of a variety of NO sources (Flatley et al., 2005; Justino et al., 2005; Mukhopadhyay et al., 2004; Pullan et al., 2007), and this is dependent on its regulator, NorR (Mukhopadhyay et al., 2004; Hutchings et al., 2002). Furthermore, norV is upregulated in macrophage-internalized Salmonella at a time that corresponds to the NO burst (Eriksson et al., 2003), but the loss of NorV does not reduce the ability of E. coli to survive within macrophages (Pullan et al., 2007).
Cytochrome c nitrite reductase (NrfA), which catalyses the six-electron reduction of nitrite, is also thought to catalyse the anaerobic five-electron reduction of NO. Purified NrfA from E. coli (Costa et al., 1990) and Sulfurospirillum deleyianum (Stach et al., 2000) can reduce NO to ammonium, with rates comparable to those of NO reductases that belong to the family of bacterial respiratory NO reductases (Field et al., 2008). Interestingly, an E. coli nrfA mutant showed an increased anaerobic sensitivity towards NO (Poock et al., 2002). The nrf operon is regulated by the NO-sensitive repressor NsrR in E. coli (Filenko et al., 2007), which provides a possible mechanism for the upregulation of nrf expression by NO. However, other studies have found little increase in nrf expression upon the addition of NO (da Costa et al., 2003; Pullan et al., 2007), suggesting that it is not highly NO-responsive, at least under the conditions used in those assays.
Despite many reports on the proteins involved in NO detoxification, there are still inconsistencies. Previous research on single norV and hmpA mutants has shown differing levels of bacterial growth inhibition by an anaerobic NO source (Gilberthorpe et al., 2007; Hutchings et al., 2002; Justino et al., 2005). The cause of these discrepancies could be twofold: first, the type of growth medium used; and second, the source of NO used. As well as releasing NO, some chemical sources also release other toxic compounds, such as cyanide (released by SNP), though they have the advantage of releasing NO over a prolonged period. The use of NO gas is desirable, since no other compounds are released, but has the disadvantage that the NO can potentially be degraded quickly in the presence of oxygen.
In this study we aimed to discover the contribution that NrfA and NorV make to the detoxification of NO by Salmonella under anaerobic growth conditions. We chose two types of growth medium, one of which promoted fermentative growth and has been reported to induce low levels of NrfA activity in E. coli [minimal glucose medium (MGM)], and the other of which promoted nitrate-respiratory growth and induced high levels of NrfA activity in E. coli [minimal glycerol nitrate fumarate (GNF) medium] (Darwin et al. 1993). We found that HmpA, NorV and NrfA can all contribute to NO detoxification in particular environments, but that the anaerobic detoxification of NO required the combination of the NorV and NrfA enzymes. We suggest that Salmonella has evolved multiple NO-detoxification mechanisms to resist killing by NO under a wide range of environmental conditions.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were constructed using the method of Datsenko & Wanner (2000). For general culture, bacteria were grown aerobically at 37 °C with shaking at 200 r.p.m. in 50 ml LB medium in a 250 ml conical flask with 20 µg chloramphenicol ml–1 (final concentration) and/or 50 µg kanamycin ml–1 (final concentration) added where appropriate. For experimental analysis, bacteria were grown in a minimal salts medium (Pope & Cole, 1984) supplemented with either 55 mM glucose and 0.01 % (w/v) casamino acids (MGM) or with 40 mM glycerol, 22 mM sodium nitrate, 40 mM sodium fumarate and 0.01 % (w/v) casamino acids (GNF).
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Anaerobic growth.
Either 1 ml fresh MGM or 1 ml GNF medium, in a 1 ml cuvette, was inoculated with 5 % (v/v) of an overnight anaerobic culture grown in the same medium. Once inoculated, the cuvette was sealed and flushed with oxygen-free nitrogen to rid the system of oxygen. The cuvettes were incubated at 37 °C with periodic mixing to prevent settling. Growth was monitored by measuring the OD600. Once the cultures had reached an OD600 of 0.1, NO was added to a final concentration of 40 µM and growth was continued at 37 °C.
Aerobic growth.
A flask containing 50 ml fresh MGM was inoculated with 1 % (v/v) of an overnight culture grown in the same medium. Cultures were incubated at 37 °C with shaking and growth was monitored by measuring the OD600. Once the culture reached an OD600 of 0.1, NO was added to a final concentration of 200 µM and monitoring of growth was continued.
Viability counts.
Duplicate cultures of wild-type S. Typhimurium SL1344 and each mutant were grown anaerobically in MGM at 37 °C. Once the OD600 reached 0.1, 40 µM NO was added to one culture and growth was continued. At 4 h post NO addition, samples were taken from each culture, which were serially diluted and plated onto LB agar containing the appropriate antibiotic(s). Following overnight growth at 37 °C, the number of c.f.u. was determined. The percentage survival was calculated using cultures that had not been challenged with NO as the control.
Nitrite reductase assay.
Cultures were harvested and the periplasmic fraction was isolated as described previously (Poock et al., 2002). Periplasmic fractions (0.5 ml) in 10 mM phosphate buffer (pH 7.5) and 1 mM (final concentration) methyl viologen were added to a 1 ml cuvette. The cuvette was sealed and the headspace degassed with oxygen-free nitrogen for 10 min. Methyl viologen was reduced by the addition of a few microlitres of degassed 1 mM sodium dithionite. Once the A600 was stable between 1.5 and 2 units, potassium nitrite was added to a final concentration of 10 mM to begin the reaction. This was followed by a decrease in A600 and the specific activity was calculated from this rate, using a molar absorption coefficient of 13 mM–1 cm–1 for reduced methyl viologen.
NO consumption assays.
Following growth in MGM, 50 ml bacterial culture was harvested by centrifugation at 10 000 g for 25 min. The resulting pellet was washed twice in fresh MGM and the final pellet was resuspended in 250 µl fresh MGM to give a final density of approximately 1 g ml–1. A 1.8 ml volume of fresh MGM was added to the chamber of a Clark-type oxygen electrode (Hansatech Instruments). Once the signal was stable, 100 µl of bacterial suspension was added. Following the removal of oxygen by bacterial respiration, 200 nmol NO was added, and the rate of NO consumption was followed on the trace.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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60 min. In cultures to which NO was added (to a final concentration of 40 µM) a short 50 min period of growth inhibition was observed (see Table 2, Fig. 1 for four examples of growth curves, and Supplementary Fig. S1 for all eight growth curves). Under these conditions, single hmpA and nrfA mutants did not show an increased sensitivity to NO compared with the wild-type (Table 2). However, the norV mutant showed a longer period of growth inhibition (100 min; Table 2, Fig. 1). These results suggest a key role for NorV-dependent NO detoxification under these anaerobic growth conditions, but do not reveal a clear role for either NrfA or HmpA. However, the norV strain does recover from NO addition, suggesting that another enzyme is capable of detoxifying the NO, possibly NrfA, HmpA or both.
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), but the norV hmpA mutant had a substantially increased growth inhibition period of 160 min (Table 2), revealing the importance of NorV and HmpA for NO detoxification under these growth conditions. Most significantly, the norV nrfA double mutant showed a severe growth impediment upon NO addition, and did not recover (Table 2, Fig. 1; note that growth curves were monitored for a further 24 h beyond the time shown in Fig. 1 and recovery was still not observed). These data suggest that NorV and NrfA are the most important enzymes for anaerobic detoxification of NO during growth in MGM. In the absence of NorV and NrfA, HmpA is unable to protect Salmonella under the conditions tested, and apparently no other system can facilitate growth recovery. This was confirmed by the essentially identical phenotypes following challenge with NO that were shown by the norV nrfA hmpA triple mutant and the norV nrfA double mutant (Table 2).
To assess the effect of NO on cell viability and to distinguish between bacteriostasis and toxicity, growth experiments were conducted in exactly the same manner as those described above, but NO was added to cultures at a range of different concentrations. Cultures were sampled after 240 min and viable counts determined. The results showed a concentration of NO that caused a 50 % decrease in cell viability (I50) of between 40 and 60 µM for wild-type cultures and the norV, nrfA, hmpA, norV hmpA and nrfA hmpA mutants, but this is decreased to 5 µM for the norV nrfA mutant and the triple mutant (Table 3). This hypersensitivity to NO underlies the inability of the norV nrfA and triple mutant cultures to recover from the 40 µM NO pulses administered in the experiments depicted in Fig. 1 and Table 2, since cell killing occurred at this level of NO (Fig. 2). By contrast, the wild-type and the norV, nrfA, hmpA, norV hmpA and nrfA hmpA mutants retained 50–80 % viability at this NO concentration (Fig. 2), and the cultures were able to recover after a transient stasis (Table 2).
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; Hutchings et al., 2002) that suggest that NorV is important in anaerobic NO detoxification, and that HmpA is not needed for protection against anaerobic NO attack. They also support the results of Poock et al. (2002), in suggesting that NrfA plays a role in anaerobic NO detoxification in E. coli. However, the results disagree with other reports which argue that HmpA is important for anaerobic NO detoxification in E. coli (Justino et al., 2005) and S. Typhimurium (Crawford & Goldberg, 1998; Gilberthorpe et al., 2007). This variation may reflect differences in growth conditions and/or the source of NO. Some of these studies relied upon GSNO or SNP as the NO source, which were not used here due to the release of other toxic compounds.
Correlation of cellular NO reduction capacity with nitrite reductase activity
To gain further insight into the contribution of NorV, NrfA and HmpA to NO consumption during anaerobic growth on MGM, cells were removed from the cultures at various times after NO addition and the specific anaerobic NO consumption activity was measured. Relatively low NO consumption rates were measured before NO addition (Fig. 3a). This cellular activity increased over the next 180 min: after the cultures had recovered from the transient NO-induced, decreased growth rate, the specific NO reduction capacity was approximately eightfold greater than that prior to NO addition. This increase presumably reflects the NO-inducible synthesis or activation of enzymic systems capable of anaerobic NO reduction. The final specific NO reduction activity of the nrfA strain was only 20 % of that of the wild-type, indicating a significant contribution of NrfA activity to the overall rate of NO consumption.
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). This increase was not detected in the nrfA mutant, confirming that NrfA was responsible for most of the measurable nitrite reductase activity. Residual activity in the nrfA mutant could be due to non-specific nitrite reactions of other enzymes. These results suggest that NrfA accounts for most of the NO reduction capacity that is switched on following addition of NO to the bacterial cultures. Consistent with this view, a large increase in both nitrite reductase and NO reduction activity was still observed in the norV strain following NO addition. In the hmpA strain the final nitrite reductase activity and the NO reduction activity were both around 50 % of that of the wild-type (Fig. 3). The reason for this lower activity in the hmpA strain is not yet known, but the ratio of NO reduction activity to nitrite reductase activity remained similar to that of the wild-type. Accordingly, we suggest that most of the NO consumption activity of wild-type S. Typhimurium can be ascribed to NrfA. However, in the nrfA hmpA mutant a clear increase in NO reduction capacity did occur following NO addition, although the final level was only 20 % of that of wild-type cells. This probably reflects NorV activity, and is similar to the residual capacity measured in the nrfA single mutant. Whilst no significant decrease in NO reduction capacity was measured in norV cells, the high background activity of NrfA would make it difficult to measure a 20 % contribution by NorV.
Anaerobic growth of S. Typhimurium mutants and sensitivity to NO under NrfA-inducing conditions
The experiments described above suggested that NrfA contributed to the anaerobic detoxification of NO by S. Typhimurium. To develop this finding, further experiments were conducted under conditions that have been reported to induce NrfA in E. coli, namely anaerobic growth in minimal medium with glycerol as a carbon source and nitrate and fumarate as respiratory electron acceptors (GNF medium; Darwin et al., 1993). The activity of S. Typhimurium nitrite reductase was assayed and compared following anaerobic growth in GNF or MGM medium. Nitrite reductase activity was increased by growth in GNF [170±15 nmol min–1 (mg protein)–1] by 40-fold compared to MGM [4±2 nmol min–1 (mg protein)–1]. Similar results were observed for E. coli, with nrfA expression being maximal during growth in GNF medium, and the addition of glucose causing repression of NrfA (Darwin et al., 1993).
In MGM-grown S. Typhimurium cells the anaerobic NO consumption rate was 500±60 nmol NO min–1 (mg cells)–1. Growth in GNF medium increased this rate by almost sevenfold to 3400±150 nmol NO min–1 (mg cells)–1. Thus, there is a good correlation between the increase in nitrite reductase activity and the rate of NO consumption under GNF growth conditions.
To determine whether high levels of NrfA affected the ability to detoxify NO, the growth experiments conducted with MGM were repeated under NrfA-inducing conditions, in GNF medium. A key difference was observed: in contrast to the observations in the MGM growth experiments, wild-type Salmonella and the norV and hmpA mutants grown in GNF medium did not exhibit significant growth inhibition following addition of the NO (Table 4, Fig. 4; see Supplementary Fig. S2 for all eight growth curves). However, the nrfA mutant displayed a growth inhibition of 80 min (Table 4). These data show that in wild-type S. Typhimurium, a high nitrite reductase activity causes rapid NO detoxification and protects against growth inhibition following NO addition. However, NrfA is less able to protect in the absence of NorV or HmpA, since a double norV hmpA mutant showed a long period of growth inhibition (125 min) that was absent in the single norV and hmpA mutants (Table 4). As with the MGM cultures, the norV nrfA double mutant stopped growing immediately upon NO addition and was unable to recover (Fig. 4), confirming that HmpA alone was unable to protect S. Typhimurium against anaerobic NO addition under the conditions tested here. These results demonstrate that NrfA can detoxify NO, and that prior synthesis of NrfA offers immediate protection to S. Typhimurium upon exposure to the cytotoxin.
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, Fig. 5; see Supplementary Fig. S3 for all eight growth curves). A higher concentration of NO (200 µM) was used in these experiments, since NO is labile in the presence of oxygen and has a short lifespan. To establish a function for each enzyme, the inhibition of wild-type cultures was used as a reference, and any role for HmpA, NorV or NrfA was indicated by longer inhibition periods. Even though norV expression is highly upregulated by aerobic NO exposure (Bang et al., 2006), both the norV and nrfA mutants were unaffected by addition of NO in comparison to the wild-type strain. However, strains with a hmpA mutation showed increased sensitivity to NO (Table 5), and were unable to recover to the same OD600 as the control cultures, agreeing with the well-documented findings that HmpA detoxifies NO aerobically (Crawford & Goldberg, 1998; Gardner & Gardner, 2002).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gardner & Gardner, 2002). The ability of NrfA and NorV to contribute to anaerobic NO detoxification has a precedent in E. coli, but has not been observed in a pathogenic bacterium. Mutation of the nrfA or norV gene increases the sensitivity of E. coli towards NO under anaerobic conditions (Poock et al., 2002; Gardner et al., 2002; Gomes et al., 2002; Hutchings et al., 2002). The combined action of NrfA and NorV under these conditions provides distinct advantages to the organism. Periplasmic NrfA would metabolize NO prior to its entering the cell. This would reduce the amount of intracellular NO, and any that entered would be reduced by the cytoplasmic NorV. Furthermore, NrfA is part of the electron-transport chain, and NO detoxification would conserve energy, in contrast to the NADH-dependent activities of NorV and HmpA.
This study has focused on the role of the NorV, NrfA and HmpA enzymes in the survival of S. Typhimurium after NO exposure under fermentative and nitrate-respiratory growth conditions, and was designed to assess the differences that arise from the presence of high or low endogenous levels of NrfA. It is interesting to consider how this fundamental laboratory study relates to pathogenic bacteria in the outside world. Salmonellae are likely to encounter NO in many different environments, including soil, the gastrointestinal tract, the bloodstream and the macrophage. We speculate that the capacity to express norV, nrfA and hmpA under different conditions provides the metabolic flexibility to protect Salmonella against this cytotoxin in a wide range of oxic and anoxic environments containing different carbon and energy sources and respiratory electron acceptors. Our data suggest that NrfA is not a vital component of the immediate response to NO, but would be important under conditions where NrfA has already been activated. A nrfA mutant is slightly attenuated in a mouse infection model (Bang et al., 2006), suggesting a role for this enzyme in either NO detoxification or anaerobic respiration, both of which have been shown to be important for survival of S. enterica serovar Typhi in epithelial cells (Contreras et al., 1997).
HmpA was not required for S. Typhimurium to survive exposure to NO under the anoxic conditions used in this in vitro study, and only played a minor role in anaerobic NO detoxification; this was consistent with some E. coli studies (Gardner & Gardner, 2002), but not with others (Crawford & Goldberg, 1998). These discrepancies may reflect the growth conditions used, or whether nitrosated compounds or NO gas were used in the experiments as a source of NO, or both. Earlier research (Gardner & Gardner, 2002) showed that HmpA was unable to reduce compounds such as GSNO and acidified nitrite directly under in vitro experimental conditions, and so the apparent detoxification effects that were reported earlier may not be due to NO reduction. Our study uses NO, which is the ideal substrate for the study of NO reduction.
A growing body of evidence suggests that HmpA is important for successful Salmonella infection in mice, and for survival of both Salmonella and E. coli within macrophages (Bang et al., 2006; Gilberthorpe et al., 2007; Stevanin et al., 2002, 2007). Most of this research has focused upon replication inside macrophages grown ex vivo, for which HmpA is undoubtedly vital for the survival of Salmonella. Overall, the fact that multiple enzymes are capable of protecting Salmonella from NO confirms the importance of NO detoxification for Salmonella, and reflects the variety of environmental conditions that are experienced by this bacterium before and during the course of infection. Some of our data strongly indicate that yet more NO-detoxification systems remain to be identified. For example, it is surprising that: (i) the growth of a double hmp nrfA mutant on GNF medium was not inhibited by NO, whereas growth of the single nrfA mutant was inhibited by NO (Table 4); and (ii) recovery from NO addition during aerobic growth of the hmp norV, hmp nrfA and hmp norV nrfA strains was quicker than that of the hmp strain (Table 5). Both observations point towards additional NO-detoxification systems being synthesized in the absence of Hmp, and this will be investigated in future studies. Multiple enzymes are also used to survive oxidative stress (Farr & Kogoma, 1991), and it is likely that the availability of different NO-detoxification strategies for different growth environments is one of the secrets of the success of Salmonella as a pathogen.
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ACKNOWLEDGEMENTS |
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Edited by: S. C. Andrews
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 22 October 2007;
revised 11 January 2008;
accepted 14 January 2008.
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) and presence (
) of 40 µM NO. NO was added at the time indicated by the arrow and the data are representative of three independent experiments. Representative growth curves for all seven mutants are shown in Supplementary Fig. S1.
, nrfA strain; 
, hmpA strain; 
, norV hmpA strain; 
, nrfA hmpA strain; 
, norV nrfA strain; 
, norV nrfA hmpA strain.
