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Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK
Correspondence
Jeffrey Green
jeff.green{at}sheffield.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Covert et al., 2004; Gonzalez et al., 2003; Kang et al., 2005; Partridge et al., 2006; Salmon et al., 2003). Under anaerobic conditions FNR contains a [4Fe–4S] cluster that promotes dimerization of the protein and enhances site-specific DNA binding (Lazazzera et al., 1996). In the presence of oxygen, the iron–sulphur clusters disassemble, leading to the formation of FNR monomers and subsequent inhibition of DNA binding, thereby switching off FNR-activated genes (Crack et al., 2004, 2006; Kiley & Beinert, 2003; Lazazzera et al., 1996; Sutton et al., 2004a, b). Upon binding of the active protein to DNA, FNR activates transcription by recruiting RNA polymerase, or can alternatively repress transcription by inhibiting the formation of productive promoter–RNA polymerase interactions (reviewed by Browning et al., 2002). Transcription activation is facilitated by the formation of specific protein–protein contacts between the activating regions (AR1, AR2 and AR3) of FNR and regions of RNA polymerase (Bell & Busby, 1994; Blake et al., 2002; Lamberg et al., 2002). The DNA sequence recognized by FNR consists of an inverted repeat with a consensus TTGATnnnnATCAA (Eiglmeier et al., 1989). There are two basic types of FNR-dependent promoter. Class I promoters possess FNR sites located close to –61.5, –71.5, –82.5 or –92.5 (Wing et al., 1995) that permit the formation of only one protein–protein contact, between AR1 of FNR and the C-terminal domain of the RNA polymerase 
-subunit. However, the more common architecture is that of the class II promoters, which have an FNR site centred at, or close to, –41.5 relative to the transcript start (Browning et al., 2002; Guest et al., 1996; Wing et al., 2000). This arrangement allows the formation of multiple protein–protein contacts between RNA polymerase and all three activating regions of FNR (Bell & Busby 1994; Blake et al., 2002; Li et al., 1998; Williams et al., 1997).
The FNR protein primarily coordinates gene regulation in remodelling central metabolism in response to changes in oxygen availability in many bacteria (Guest et al., 1996; Korner et al., 2003), but it also exerts effects over other systems, and new members of the FNR regulon are still being uncovered (Covert et al., 2004; Kang et al., 2005). For example, FNR has been implicated in the peroxide stress response as an anaerobic activator of cytochrome c peroxidase (CCP) in a number of bacteria including Neisseria gonorrhoeae, Paracoccus denitrificans and Pseudomonas stutzeri (Turner et al., 2003; van Spanning et al., 1997; Vollack et al., 1999). Furthermore, the FNR homologue, Anr, activates CCP expression in Pseudomonas aeruginosa (Zimmermann et al., 1991).
The primary role of CCP is thought to be in peroxide stress resistance, because of its ability to catalyse the conversion of hydrogen peroxide to water (Minard & McAlister-Henn, 2001; Seib et al., 2004, reviewed by Atack & Kelly, 2006). However, because typical bacterial CCP proteins are periplasmic dihaem-containing proteins that use a monohaem cytochrome c as an electron donor they could potentially allow the use of hydrogen peroxide as a terminal electron acceptor.
Here, a search for new members of the E. coli FNR regulon identified the yhjA gene as a likely candidate. The expression of this gene, which is predicted to encode a CCP, is shown to be driven from an FNR-dependent class II promoter that is also dependent on OxyR. The phenotype of the yhjA mutant indicates a role for YhjA in the peroxide stress response of E. coli.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. For 
-galactosidase assays, L broth (Sambrook & Russell, 2001) containing appropriate antibiotics was inoculated (1 : 50) from overnight cultures. Cultures were incubated under aerobic (250 ml conical flasks containing 10 ml medium shaken at 250 r.p.m.), micro-aerobic (250 ml conical flasks containing 150 ml medium shaken at 100 r.p.m.), or anaerobic (bottles filled to the neck, without shaking) conditions at 37 °C until an OD600 of 0.4–0.6 was reached, at which point -galactosidase activities were measured (Miller, 1972). Oxygen transfer rates were measured as described by Wainwright et al. (2005).
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). Promoter regions of interest were amplified from the chromosome of MC1000 by PCR using the following oligonucleotides engineered to contain EcoRI/BamHI restriction sites: yjhB, JDP1 and JDP2; yfgF, JDP3 and 4; yhjA, JDP5 and 6; yecI, FyecI and RyecI; smf, Fsmf and Rsmf (Table 1
). The sequence of each fragment was verified by DNA sequencing after ligation into EcoRI/BamHI-digested pRS415 (Simons et al., 1987). Selected constructs were then transferred to 
RZ5 (Simons et al., 1987) and the resulting (promoter–lacZ) fusions were introduced in single copy into the attachment site of isogenic E. coli strains. Lysogeny status was verified using the method of Powell et al. (1994). Further transfer of the promoter fusions into different genetic backgrounds was achieved using bacteriophage P1vir-mediated transduction (Sambrook & Russell, 2001).
For 5' random amplification of cDNA ends (RACE)-PCR transcript mapping, RNA was isolated from anaerobic cultures of E. coli MC1000 using Qiagen RNeasy mini kits according to the manufacturer's instructions. The yhjA transcript start was identified using 2 µg E. coli RNA per RACE reaction according to the manufacturer's instructions (Roche) with oligonucleotides JDP7 and JDP8 (Table 1).
Site-directed mutagenesis of the consensus FNR site (TTGAT-N4-ATCAA to ATCAT-N4-ATGAT) and putative OxyR-binding site (ATAGgcacaggCTATcttattgATAGtTtatAttcAT to TATCgcacaggGATActtattgTATCtAtatTttcTA) was achieved using PCR and appropriate synthetic oligonucleotides, with all mutations confirmed by DNA sequencing.
Construction of mutants.
The yhjA gene of W3110 was disrupted by linear transformation based on the method of Yu et al. (2000). Oligonucleotides containing 3'-end sequences complementary to the first or last 20 bp of the chloramphenicol resistance cassette of plasmid pACYC184 (Martinez et al., 1988) and 5'-end sequences flanking yhjA were constructed. Linear DNA carrying the resistance cassette and flanking regions was generated by PCR. E. coli strain W3110 containing the plasmid pTP223 (TetR) (Poteete & Fenton, 1984), which carries the red recombinase genes under the control of an IPTG-inducible promoter, were grown overnight at 37 °C and diluted (1 : 100) in L broth containing tetracycline (25 µg ml–1) and IPTG (2 mM) and grown to an OD600 of 
0.3. Electrocompetent cells were prepared and transformed with approximately 5 µg of PCR product then recovered in 1 ml L broth for 1 h before plating on selective medium (CmR). The resulting colonies were immediately cured of pTP223 and mutations were screened by PCR and DNA sequencing. Further transfer of the mutations into clean genetic backgrounds was achieved using bacteriophage P1vir-mediated transduction (Sambrook & Russell, 2001).
The oxyR-deficient mutant for use in promoter assays was generated through P1vir-mediated transduction (Sambrook & Russell, 2001) of the mutation from GSO47 (Zheng et al., 1999) to MC1000, to generate JRG5393.
Overexpression and purification of OxyR and FNR*.
Overexpression of OxyR from MV247 was as described by Haagmans & van der Woude (2000) with modifications (Correnti et al., 2002). Solubilization was aided by the addition of 0.1 % Tween 20 to the bacterial pellet and incubation at room temperature for 30 min prior to sonication. Protein was purified from the cell-free extract using a HiTrap heparin column (Amersham) as described previously (Storz et al., 1990) with modifications according to Kullik et al. (1995). The final preparations contained 60 % OxyR as judged by Coomassie-stained SDS-polyacrylamide gels.
The FNR protein FNR-D154A (designated FNR*) retains DNA-binding activity under aerobic conditions (Ziegelhoffer & Kiley, 1995) and was isolated as described by Meng et al. (1998).
Gel retardation assays.
Radiolabelled promoter fragments (50 ng) were incubated with 6 µM FNR* and/or 0.06 µM OxyR in binding buffer (Correnti et al., 2002) for 30 min before separating the protein–DNA complexes from DNA on 6 % TBE-buffered polyacrylamide gels. After electrophoresis the gels were transferred to filter paper (3MM, Whatman) and dried for autoradiography.
Disc diffusion assays.
Soft-top agar (3 ml, 0.65 % agar) was mixed with an aliquot of a culture (200 µl, OD600 0.3) of the strain under investigation grown under anaerobic conditions. The mix was poured evenly onto L agar plates and, once set, a sterile antibiotic filter disc (6 mm) was placed in the centre of the plate. The indicated reagents were added to the centre of the disc and the plates were incubated under anaerobic conditions at 37 °C for 16 h before measuring the zone of growth inhibition around the disc.
Killing curve studies.
Bacterial cultures were grown to OD600 0.3 under anaerobic conditions and then challenged with a stress reagent. Aliquots were removed at t=0, t=20, t=40 and t=60 min and serially diluted in L broth before being plated onto L agar plates incubated under aerobic conditions. The numbers of viable bacteria were calculated from the numbers of c.f.u. after 16 h growth at 30 °C under aerobic conditions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The key features of this inverted repeat are the A–T base pairs at positions +7 and –7 (underlined), which allow discrimination between FNR sites and sites recognized by related proteins, and the G–C base pairs at +5 and –5 (bold), which form a core interaction with the DNA recognition helices of this family of transcription factors (Schultz et al., 1991; Spiro et al., 1990). The E. coli K-12 genome sequence was searched for the presence of consensus FNR sites (TTGATnnnnATCAA) using the search pattern tool in the Colibri database (http://genolist.Pasteur.fr/Colibri). This analysis yielded a total of 21 sites. Eliminating those located within coding regions, and those associated with prophages, left 12 consensus FNR sites, which were all centred within 345 bp of a start codon. Of the 12 transcriptional units associated with consensus FNR sites, seven (cydA, ndh, narK, narG, nirB, ydhY and yfiD) are known to be FNR-regulated (Green et al., 1998; Guest et al., 1996; Kang et al., 2005). The regulation of the remaining five genes (yecI, yfgF, yhjA, yjhB and smf) had not been investigated. To test whether the FNR sites upstream of yecI, yjhB, yfgF, yhjA and smf contributed to regulation of these genes, the corresponding promoter regions (e.g. PyecI–lacZ) were fused to lacZ in the reporter plasmid pRS415 and transferred into MC1000 (lac) and JRG1728 (fnr lac). Expression of yecI, yjhB and smf was not affected by the presence or absence of FNR under anaerobic conditions (Table 2), although expression of both yecI and smf was apparently lower in the absence of oxygen. However, expression of yfgF, and yhjA was enhanced under anaerobic conditions in an FNR-dependent manner (Table 2). Here the results of experiments to further characterize the regulation and function of yhjA are presented.
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32-fold induction under anaerobic compared to aerobic conditions (Table 3). This anaerobic activation was lowered to only 3-fold in the fnr mutant JRG1728 (fnr lac) (Table 3). Therefore, evidence for direct regulation by FNR was sought using gel retardation assays. To simplify the experiments an FNR protein (FNR-D154A, referred to as FNR*) that retains some DNA-binding activity in the presence of air was used (Ziegelhoffer & Kiley, 1995). The assays showed that FNR* was able to bind at the native yhjA promoter region, but not at the same promoter fragment with mutations in the predicted FNR site (Fig. 1, lanes 2 and 3). Furthermore, mutation of the FNR site essentially abolished FNR-mediated regulation (Table 3).
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). Thus, it was concluded that database searching had identified the yhjA gene as a member of the FNR regulon with an FNR-dependent class II promoter.
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). The 3' end of the potential OxyR site is located only 8 bp upstream of the 5' end of the FNR site (Fig. 2). The OxyR protein acts as a transcriptional regulator initiating, directly or indirectly, the synthesis of more than 40 gene products to counter the effects of peroxide stress, although transcription of some of these genes is also altered during exposure to heat shock or redox-cycling agents (Greenberg & Demple, 1989; Storz & Zheng, 2000; Walkup & Kogoma, 1989; Zheng et al., 2001).
To investigate whether OxyR contributes to the regulation of yhjA, the PyhjA–lacZ fusion was transferred to an oxyR mutant. The anaerobic induction of yhjA expression observed previously was significantly lowered (Table 3). Furthermore, mutation of the OxyR site essentially abolished OxyR-mediated regulation (Table 3). These data suggest that OxyR is required for expression of yhjA. Therefore, evidence for direct regulation of yhjA expression was sought using gel retardation assays. These showed that, like FNR, OxyR was able to bind at the unaltered yhjA promoter region, but not at the same promoter fragment carrying mutations in the predicted OxyR site (Fig. 1, lanes 4 and 5). Gel retardation assays in which the yhjA promoter was incubated with increasing amounts of OxyR revealed an apparent Kd of 10 nM for OxyR binding (not shown). Moreover, in gel retardation assays in which both FNR* and OxyR were present, the migration of the labelled yhjA promoter was slower than in the presence of either FNR* or OxyR alone, suggesting that both proteins occupy the yhjA promoter simultaneously (Fig. 1, lane 6).
Thus, the regulation of yhjA is interesting because transcription appears to be dependent on two transcription factors that respond to seemingly incompatible signals, i.e. lack of oxygen and peroxide stress. Nevertheless, during micro-aerobic growth oxidative stress might be encountered whilst some FNR activity is retained. However, yhjA expression was only partially activated under micro-aerobic (150 ml culture volumes shaken at 100 r.p.m., equivalent to an oxygen transfer rate of 12 µmol min–1 l–1) compared to anaerobic conditions (Table 3), indicating that these particular micro-aerobic conditions are not optimal for yhjA expression.
A yhjA mutant exhibits increased sensitivity to exogenous peroxides
The gene encoding the predicted CCP of E. coli, yhjA, was expected to contribute to the peroxide stress response. Therefore the sensitivity of a yhjA mutant to hydrogen peroxide and organic peroxides during anaerobic growth was tested. Initial disc diffusion assays indicated that the mutant was more sensitive than the parent to hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide (not shown). This enhanced sensitivity was confirmed in killing assays using cultures grown under anaerobic conditions to OD600 0.3 before exposure to the peroxide stress reagents for a total of 20, 40 and 60 min (Fig. 3). Complementation of the yhjA peroxide-sensitive phenotype by supplying a wild-type copy of yhjA in trans was not possible, because all attempts to create plasmids that expressed the YhjA protein in an active form were unsuccessful (not shown). Therefore, to ensure that the observed phenotype was caused by disruption of yhjA and not by point mutations located elsewhere in the genome, the lesion was transferred by P1 transduction to a clean genetic background, and the observations reported above were confirmed with the new mutant, although this approach does not exclude the possibility of polar effects on neighbouring genes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, the YhjA protein is somewhat atypical. Generally bacterial CCP proteins possess two haem cofactors, the first (high-potential) site accepts electrons from the donating cytochrome c before transferring them to the second (low-potential) site, often referred to as the peroxidatic site, where hydrogen peroxide is reduced to water (reviewed by Atack & Kelly, 2006). Most physiological and biochemical studies of bacterial CCP proteins have been of these typical dihaem proteins, including those of Methylococcus capsulatus (Zahn et al., 1997), Neisseria gonorrhoeae (Turner et al., 2003), Nitrosomonas europaea (Arciero & Hooper, 1994), Paracoccus denitrificans (Gilmour et al., 1993), Paracoccus pantotrophus (Goodhew et al., 1990), Pseudomonas aeruginosa (Ellfolk & Soininen, 1970; Fülöp et al., 2001), Pseudomonas nautica (Alves et al., 1999) and Rhodobacter capsulatus (Hanlon et al., 1992). However, the YhjA amino acid sequence reveals the presence of a third haem-binding motif. The additional haem-binding site is located in an N-terminal extension of 80 amino acid residues, which is absent from the dihaem CCP proteins. The exact role of this extended domain and the third haem is unclear, because the trihaem CCP proteins are poorly characterized. However, it has been suggested that the trihaem CCP proteins may have evolved by fusion of a monohaem cytochrome c gene to a dihaem ccp gene, consequently bypassing the need for a separate donor system (Atack & Kelly, 2006). Another distinction between CCP proteins is their cellular location, being either soluble periplasmic proteins or anchored to the membrane as lipoproteins. Cellular location is controlled by the presence of N-terminal amino acid sequences that are recognized by the Sec secretion pathway (Braun & Wu, 1993) and either type I or type II signal peptidases, the former generating a soluble periplasmic protein, whilst the latter leads to anchoring as a lipoprotein. Thus, the CCP of N. gonorrhoeae possesses a signal peptidase II recognition sequence and is a lipoprotein (Turner et al., 2003). Similarly, YhjA also possesses a potential target for signal peptidase II cleavage (VAIC), suggesting it is likely to be a lipoprotein, perhaps accounting for the lack of success in attempts to create expression plasmids that yielded functional protein.
The peroxide-sensitive phenotype of the yhjA mutant reported here is consistent with the assignment of YhjA as a CCP. Whilst bacterial CCP proteins are known to detoxify hydrogen peroxide, there is evidence that they also work with organic peroxides. For example, a Bacteroides fragilis ccp mutant exhibits enhanced sensitivity to organic peroxides (t-butyl hydroperoxide and cumene hydroperoxide) compared with hydrogen peroxide (Herren et al., 2003). Likewise, the E. coli yhjA mutant was sensitive to organic peroxides and hydrogen peroxide, and the differences in sensitivity to these compounds might reflect the relative contribution of YhjA to their detoxification. Thus, the bioinformatic and phenotypic analyses are consistent with the assignment of the E. coli YhjA protein as a trihaem CCP.
The regulation of yhjA expression is interesting, because transcription is dependent on two transcription factors, FNR and OxyR, that respond to two seemingly incompatible signals, i.e. oxygen starvation and peroxide stress. It is often under conditions of high aeration that bacteria are exposed to hydrogen peroxide (Seaver & Imlay, 2004), leading to activation of OxyR. However, these same conditions inactivate FNR (Becker et al., 1996). It is possible that OxyR might be activated by nitrosative stress, as suggested by Kim et al. (2002), but analysis of the yhjA mutant phenotype did not support this idea. This raises the possibility that both oxidized and reduced OxyR could be functional at the yhjA promoter, because both forms of OxyR are able to bind at some promoters. Oxidized OxyR recognizes ATAGnT elements in four adjacent major grooves on one face of the DNA helix (Toledano et al., 1994). In contrast, reduced OxyR contacts ATAGnT elements in two pairs of major grooves separated by one turn of the DNA helix (Toledano et al., 1994). The DNA sequence of the oxyRS promoter region is such that both these patterns of binding are possible, and thus OxyR regulates its own expression, when reduced and oxidized, simply by repositioning itself on the DNA. At OxyR-activated promoters, such as ahpC, dps, gorA and katG, the ATAGnT elements are arranged such that only oxidized OxyR binds with significant affinity (Toledano et al., 1994). The yhjA OxyR site resembles those of OxyR-activated promoters, lacking an additional ATAGnT element. Moreover, the fourth OxyR contact point in yhjA does not obviously resemble both ATAGnT and AnCTAT elements, which is necessary to allow redox-state-responsive repositioning of OxyR at oxyRS (Toledano et al., 1994). Furthermore, because the yhjA OxyR and FNR sites are so close, any additional downstream OxyR contacts will overlap the FNR site and are likely to prevent FNR binding. Thus, the architecture of the yhjA promoter, the phenotype of the yhjA mutant and the predicted function of the YhjA protein suggest that transcription from the yhjA promoter is likely to be restricted to specific environmental conditions in which the bacteria are exposed to peroxides, but oxygen is limited such that some FNR activity is retained. The presence of a consensus FNR site in the yhjA promoter is consistent with this idea, because a consensus site is likely to be occupied in preference to other sites when the amount of active FNR is limited. The ability of FNR, located in a typical class II position, to only weakly activate expression of yhjA in the absence of OxyR suggests that the role of OxyR might be to counteract the effects of an as-yet-unidentified regulator that inhibits the action of FNR. The extensive mutagenesis of the promoter to inactivate the OxyR site is likely to have disrupted other regulatory elements in this region (–94 to –57), but this was not sufficient to enhance FNR-mediated yhjA expression (Table 3), and suggests that any such regulatory element is located upstream (–127 to –94) of the OxyR site.
The regulatory combination of OxyR and FNR might not be restricted to the yhjA promoter. The katG gene encodes hydroperoxidase I, which acts as a dual-function catalase and peroxidase with a proven role in the peroxide stress response. Expression of katG is activated by OxyR binding at a site centred at –52 relative to the transcript start. In addition, there are two credible FNR sites located at –96.5 and –112.5, and although there is no direct evidence of FNR binding to the katG promoter, transcript profiling suggests that FNR activates katG expression (Constantinidou et al., 2006). If the katG FNR site(s) are functional, then although the katG promoter architecture is different to that of yhjA, where the FNR and OxyR sites are very close together, the seemingly incompatible combination of FNR and OxyR might be used to control expression of at least two genes associated with the peroxide stress response in E. coli.
In conclusion, it has been shown that the YhjA protein is likely to be a CCP that contributes to the survival of E. coli upon exposure to peroxide stress, and that expression of the yhjA gene is driven from an FNR-dependent class II promoter that also requires OxyR for activity. This unusual use of FNR and OxyR to activate expression suggests that yhjA plays a specific role in countering oxidative stress in E. coli, possibly in particular micro-aerobic environments. Further extensive in vitro analysis is required to determine the exact mechanism by which two regulators that respond to such disparate signals combine to activate yhjA expression.
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ACKNOWLEDGEMENTS |
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Edited by: S. J. W. Busby
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 1 December 2006;
revised 19 January 2007;
accepted 22 January 2007.
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) and the isogenic yhjA mutant (
) were grown to OD600