Ertan ; Ozyamak

Susan S Black

Morag J Maclean

Claire Anne Walker

Wendy Bartlett

Samantha Miller

Ian R Booth
email: i.r.booth@abdn.ac.uk

The critical role of S-lactoylglutathione formation

Keywords: methylglyoxal, potassium, glyoxalase, glutathione, detoxification

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Bacteria are subjected to frequent changes in the environment that can cause cell damage leading to death. Bacteria have evolved elaborate and complex stress management strategies to minimise damage and thus, to enhance their survival during environmental changes (Booth, 2002). Two major stratagems have been evolved by bacteria to cope with stress. Some systems are expressed essentially constitutively and the activity of the proteins is regulated in response to cytoplasmic or external cues arising from exposure to stress. Other systems are expressed at low levels during exponential growth and their transcription, translation and activity are increased as a response to environmental change (Hengge, 2009, Hengge-Aronis, 1999, Hengge-Aronis, 2002, Rosner & Storz, 1997, Wood, 2006). Metabolic activity in itself can create significant stress, for example the production of hydrogen peroxide and oxygen radicals is a consequence of aerobic growth and the resulting oxidative damage requires both intrinsic and adaptive enzyme activities (Korshunov & Imlay, 2010, Imlay, 2008).

However, pathogenic bacteria also frequently encounter oxidative stress as a component of the oxidative burst of the innate immune system. Similarly, bacteria encounter electrophiles both as a metabolic consequence and as an environmental challenge. Among the most frequently encountered electrophiles is methylglyoxal (MG), which is produced by bacteria from sugars and amino acids and is believed to have a role in macrophage-mediated killing (Eriksson et al., 2003, Eskra et al., 2001, Ficht, 2003).

MG is synthesised either from sugars by methylglyoxal synthase (MGS) (Totemeyer et al., 1998) or from threonine, serine and glycine by monoamine oxidase (Green & Lewis, 1968, Kim et al., 2004). In E. coli the dominant route appears to be from sugars and arises when there is an accumulation of phosphorylated glycolytic intermediates above the level of 1,3-diphosphoglycerate and a lowering of the pool of inorganic phosphate (Totemeyer et al., 1998, Ferguson et al., 1998). MGS activity is determined by the balance between inorganic phosphate, which is a strong inhibitor, and dihydroxyacetone phosphate (DHAP), the substrate, which exhibits strong homotropic activation ( , 1971). Thus, production of MG only occurs when there is simultaneous depletion of phosphate and extremely high concentrations of DHAP, conditions that arise when sugar metabolism is strongly stimulated leading to excess carbon flow into the upper end of glycolysis. Consistent with these observations, laboratory synthesis of MG has usually been associated with mutants that display defective regulation of gene expression for sugar transport and metabolism genes and/or loss of feedback inhibition of enzymes that regulate carbon flux (Ackerman et al., 1974, Freedberg et al., 1971, Kadner et al., 1992). For E. coli, accumulation of MG above ~0.3 mM in the medium results in growth inhibition and at levels above ~0.6 mM the survival of cells is affected. Damage to DNA and to proteins have been observed (Colanduoni & Villafranca, 1985, Ferguson et al., 2000) and both may contribute to the causes of cell death.

Protection against electrophiles is multifactorial with contributions from glutathione (GSH), detoxification enzymes, DNA repair enzymes, peptide export systems and regulated K + efflux systems (Ferguson & Booth, 1998, Ko et al., 2005, MacLean et al., 1998, Sukdeo & Honek, 2008, Xu et al., 2006). In E. coli, detoxification is primarily effected by the GSH-dependent glyoxalase system (GlxI and GlxII, products of the unlinked gloA and gloB genes) and their integration with the GSH adduct-gated KefGB K + export systems (Fig. 1). Other enzymatic systems, particularly a range of oxidoreductases (Murata et al., 1989, Xu et al., 2006, Ko et al., 2005), may also play a role in detoxification. In the GlxI-II pathway, the substrate for GlxI is created by the spontaneous reaction between MG and GSH forming hemithioacetal (HTA). GlxI isomerizes this to S-lactoylGSH (SLG), which is the substrate for GlxII, a hydrolase. The final products are the relatively non-toxic molecule D-lactate and GSH, which is recycled in the cytoplasm.

Although a GSH export system [START_REF] Pittman | A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm[END_REF], Owens & Hartman, 1986) has been identified there is no evidence for a role in MG detoxification.

Protection by the KefGB and KefFC systems is dependent on their role in modulation of the cytoplasmic pH (Ferguson et al., 2000, Ferguson & Booth, 1998, Ferguson et al., 1996a, Ferguson et al., 1995, MacLean et al., 1998). KefGB and KefFC are structurally-related K + efflux systems that are maintained inactive by the binding of GSH and are activated by binding of specific GSH adducts (Elmore et , 1990, Ferguson et al., 1993, Miller et al., 1997, Miller et al., 2000, Roosild et al., 2009). Activation leads to rapid K + efflux, which is quantitatively affected by several parameters: external K + concentration, the activity of K + uptake systems, the expression level of KefGB and KefFC and the intracellular concentration of the GSH adduct (Ferguson et al., 1996b, Ferguson et al., 1993, MacLean et al., 1998[START_REF] Mclaggan | Glutathionedependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process[END_REF]. K + efflux is accompanied by influx of H + and Na + (Bakker & Mangerich, 1982), but it is the lowering of the cytoplasmic pH that is critical for protection against MG (Ferguson et al., 2000, Ferguson et al., 1995). Lowering the cytoplasmic pH may slow the reaction of MG with guanine in DNA and with other macromolecules (Krymkiewicz, 1973).

We have previously shown that over-expression of GlxI causes increased rates of MG detoxification. However, 30-fold over-expression of GlxI only increased the rate of MG detoxification 2-to 3-fold (MacLean et al., 1998), indicating complex kinetics in which the formation of the substrate is potentially limited by the recycling of GSH by the second enzyme, GlxII. Here, we report the effects of both over-expression and depletion of GlxII. We show that the intracellular accumulation of SLG is critical for cell fate upon MG stress and that increased activity of the KefGB system can compensate for an impaired capacity to detoxify MG. The data are discussed in terms of the balance between GlxI, GlxII and the K + efflux systems in determining the fate of individual cells.

Results

Analysis of a strain over-expressing GlxII

The gloB gene, encoding for the GlxII enzyme, and its flanking regions were cloned (see Experimental procedures) into a moderate copy number vector (pHG165) to create pGlxII. Transformation into E. coli MJF274 (parent strain) led to an approximately 25-fold amplification of GlxII activity in extracts from mid-exponential grown cells (Table 1). This increase in GlxII activity reflects expression from the P gloB since the gloB gene was cloned with its own promoter F o r P e e r R e v i e w rather than under P lac which is separated from the gloB gene by ~1 kb of chromosomal DNA (see Experimental procedures). Cells expressing higher levels of GlxII grew at a similar rate as the parent strain in K 0.2 minimal medium (Fig. 2A).

Elevated synthesis of GlxII did not alter the rate of MG detoxification when cells were incubated with 0.7 mM MG (Fig. 2B). These data are consistent with the previous observation that the detoxification rate in parental cells is limited by the activity of GlxI, as indicated by the modest (2-to 3-fold) stimulation by overexpression of GlxI (MacLean et al., 1998). It can be inferred that GlxI activity is not restricted by the availability of GSH to form the substrate HTA, since one of the effects of increasing GlxII activity is to increase the rate of regeneration of GSH.

Thus, it is likely to be the intrinsic activity of GlxI that is limiting.

At least two linked components contribute to survival of exposure to MG -the rate of removal of MG and the activity of the protective KefGB system. Increased GlxII activity rendered cells more sensitive to MG despite the lack of change in the rate of detoxification (Fig. 2B,C). Elevated GlxII level is predicted to lower the pools of the substrate SLG. Since SLG is the strongest activator of KefGB, a reduced SLG pool will lower efflux activity (Fig. 2D) (MacLean et al., 1998). We have demonstrated a direct correlation between the level of KefGB activity, the degree of cytoplasmic acidification and survival (Ferguson et al., 1995, Ferguson et al., 1993, MacLean et al., 1998, Ness & Booth, 1999). Therefore, an explanation for the elevated sensitivity to MG in a strain expressing increased GlxII would be provided by the predicted lowering of the SLG pools (see below). Thus, we sought to test this hypothesis through the creation of a strain lacking GlxII, which would be unable to detoxify MG via the GlxI-GlxII pathway, but should accumulate SLG and thus maximise the activation of KefGB and to measure the consequent effects on SLG pools, KefGB activity, cytoplasmic pH and survival of MG stress.

Creation of a gloB null mutant

To assess the importance of the GlxII activity and the activation of KefGB for survival upon exposure to MG we inactivated gloB (see Experimental procedures). Previous attempts to inactivate the gloB gene by replacing the entire ORF with antibiotic resistance cassettes (kanamycin and spectinomycin) were unsuccessful (MacLean, 1998). We considered the possibility that replacement of the entire gloB gene might lead to polar effects with respect to the expression of the two genes on either side of gloB, namely mltD and yafS, which are separated by only 71 and 33 bp, respectively from the gloB ORF (Fig. 3A). The mltD gene encodes for a membrane-bound lytic murein transglycosylase, which plays a major role in peptidoglycan expansion and recycling (Scheurwater et al., 2008, Suvorov et al., 2008). The yafS gene is believed to encode an S-adenosyl-Lmethionine-dependent methyltransferase, but its physiological role remains unknown. From global array analysis under various growth conditions (GenExpDB, http://genexpdb.ou.edu), it is clear that both these genes are transcribed and thus either or both of these genes may be essential for cell function. Consequently we used a promoter prediction programme (see Supplementary information) for the design of the mutagenesis strategy. Based on this analysis, a 454 bp fragment (from 132 to 585) of the gloB structural gene was replaced (Experimental procedures) avoiding the putative promoter sequences for mltD and yafS. A short amino terminal sequence of the GlxII protein (residues 1 -43) may be expressed in the mutant strain MJF595 created in this study. However, from the crystal structure, this fragment is unlikely to form an enzymatically-active protein since the critical metal-and substrate-binding sites are located in other regions (Campos-Bermudez et al., 2007, Zang et al., 2001),.

The ΔgloB mutant grew at a similar rate as the parent in K 0.2 minimal medium (Fig. 3B) and exhibited no obvious phenotype. Thus GlxII is not an essential enzyme.

The residual activity corresponded to ~6% of the parental GlxII rate, but this low rate was close to the limit for detection. The residual activity was not due to GlxI, since increasing the expression of this enzyme in the ΔgloB mutant did not increase the rate of breakdown of SLG (Table 1). As expected the ΔgloB strain exhibited a reduced ability to detoxify MG, as previously noted for the ΔgloA strain (MacLean et al., 1998). Addition of 0.7 mM MG to cultures of both parent and mutant strains caused immediate growth inhibition without recovery over the course of the experiment (data not shown). MG disappeared from the medium in a linear fashion and, as expected, the rate was greatly reduced in cultures of for mutant and parent, respectively). We have previously observed a similar reduction in the capacity to detoxify MG in a ΔgloA null mutant (MacLean et al., 1998). However, cells retain the ability to breakdown MG, but at a much lower rate, which is consistent with the known presence of other enzymes that can metabolise MG (Misra et al., 1995, Misra et al., 1996).

Inactivation of gloB does not affect cell viability upon MG stress

Previously, we reported that a ΔgloA mutant, impaired in MG detoxification, exhibits increased sensitivity to MG, which can be explained by the persistence of the electrophile in the growth medium (MacLean et al., 1998). In both ΔgloA and ΔgloB mutants treated with 0.7 mM MG, the concentration remains above the lethal level (~0.6 mM MG) for ~6 h compared with ~2-3 h in the parent strain.

Thus, it was expected that the ΔgloB mutant would exhibit comparable sensitivity to MG as the ΔgloA mutant. However, a ΔgloB mutation did not increase the sensitivity of the cells to MG (Fig. 3D). Conversely, increased sensitivity to MG was only achieved by over-expression of GlxII from a plasmid (Fig. 2C &3D). Cell viability was also assessed upon exposure to higher MG concentrations (0.9 and 1.1 mM). Both the parent (MJF274) and ΔgloB (MJF595) strains exhibited reduced viability at the higher MG concentrations, but the death kinetics for the two strains were not significantly different in replicate experiments (data not shown).

FrmB and YeiG (EC 3.1.2.12), are major components of the formaldehyde detoxification pathway and have been reported to have low level hydrolytic activity against SLG (Gonzalez et al., 2006). The yeiG gene is transcribed constitutively whereas the frmB gene can be induced with formaldehyde (Gonzalez et al., 2006).

Strains lacking

K + efflux systems are hyperactive in a gloB null mutant

We have previously established that KefGB and KefFC are activated by electrophiles through the formation of GSH adducts [START_REF] Elmore | Activation of potassium efflux from Escherichia coli by glutathione metabolites[END_REF], Ferguson et al., 1993, Ferguson et al., 1995, Ferguson et al., 1997). From the study of a ΔgloA mutant we inferred that SLG was the metabolite activating KefGB during exposure to MG, since the HTA formed by a reversible reaction with GSH in such a mutant was insufficient to activate the K + efflux system (MacLean et al., 1998).

We predicted that a ΔgloB mutant, lacking GlxII activity, would accumulate SLG as a result of GlxI activity and thus, KefGB activity should be enhanced. Analysis of K + efflux patterns in the parent strain MJF274 and the mutant MJF595 (ΔgloB), using a range of MG concentrations, supported this model. K + efflux experiments are performed with high cell densities (OD 650nm ~0.8). Consequently in the parent strain the MG concentration is continuously declining due to the high rate of detoxification in such conditions (0.7 mM MG falls to ~0.3 mM in 30 min). Thus efflux assays were performed with 0.7 -3 mM MG over a time course of 20 min such that depletion of MG is minimised during the assay period (Fig. 4A,B). The rate and extent of K + efflux from the parent strain was dependent on the concentration of MG. The initial K + loss upon treatment with 0.7 mM MG was followed by steady recovery of the K + pool due to rapid MG detoxification. No such recovery was observed in the ΔgloB strain and the rate and extent of K + loss was faster than observed in the parent strain (Fig. 4B). This effect was most marked at the lower MG concentrations and thus we investigated the concentration dependence of efflux in both strains (Fig. 4C). First order rate constants for the initial K + efflux were measured over the first 3 min. In the parent strain, the rate of efflux observed at very low MG concentrations (<200 µM) was not significantly different from the rate of K + loss in the absence of MG. For higher concentrations the rate constant increased and approached a maximum for concentrations ≥3 mM MG (Fig. 4C). In contrast, the ΔgloB mutant exhibited rapid K + loss even at concentrations as low as 25 µM MG and the rate was not further stimulated by limited by the number of KefGB proteins since it has previously been shown that the levels of KefGB can be rate-limiting for K + efflux and that higher rate constants are achieved when the channels are over-expressed (Ness & Booth, 1999).

The data from the mutant strain followed a hyperbolic function, saturated above approximately 0.2 mM MG and a mathematical fitting matched well with experimental data points (adjusted R 2 = 0.96) (Fig. 4C). Data points for the parent strain followed the same trend but could not be fitted as well (adjusted R 2 = 0.65), which can be attributed to the difficulty in determining the rate constants at lower MG concentrations. However, the maximum rate of K + efflux observed with the parent strain is always lower, which is consistent with the influence of GlxII on SLG pools (see below). These data show that KefGB activity is critically dependent both on the activity of the GlxII enzyme and on the MG concentration.

Intracellular accumulation of SLG upon MG stress

The data above were consistent with KefGB activation being effected by the SLG concentration reaching a certain threshold. In the absence of GlxII, SLG can accumulate to this threshold concentration even at very low MG concentrations.

We developed an LC-MS/MS assay to quantify intracellular GSH and SLG pools to obtain insight into the in vivo dynamics of SLG formation in both the gloB null mutant and in the parent strain. In addition we measured SLG formation in a strain over-expressing GlxI. Cells were grown in K 0.2 minimal medium and GSH and SLG were extracted from cells with formic acid using a silicone oil centrifugation technique (see Experimental procedures). Pools of SLG and GSH were measured at intervals (10 s, 2.5 min and 5 min) after addition of MG (0 -0.7 mM) using conditions identical to those for the K + efflux assays. GSH pools prior to MG addition were identical in the parent and ΔgloB strains within experimental error and no SLG could be detected prior to addition of MG. MG addition caused a rapid increase in SLG in both the mutant and parent in a concentration dependent manner (Fig. 5A); in the parent SLG pools were below the detection limit of the analytical technique at concentrations less than 0.2 mM 5B) but caused detectable K + efflux (data not shown) and a drop in cytoplasmic pH through activation of KefGB (see below). The SLG pool rose rapidly and, in the parent, was at its maximum value 10 s after addition of MG (Fig. 5B). Pools of SLG in the ΔgloB strain increased rapidly, even with only 25 µM MG (Fig. 5C), achieving ~50% of the maximum value in 10 s, then climbed slowly over the next 2.5 min and remained essentially constant for the assay period (Fig. 5A).

The steady state was achieved more rapidly at higher concentrations of MG (Fig. 5C). Approximately 80% of the GSH pool was converted to SLG when ΔgloB cells were incubated with 0.1 mM MG (Fig. 5C). The SLG pools in the parent never reached the values observed for the ΔgloB mutant, even when an almost 4-fold higher MG concentration was used. In the parent, the presence of GlxII maintains SLG pools at a low level. Higher SLG concentrations can only be achieved by over-expression of the GlxI enzyme. Accumulation was rapid in strain MJF274/pMJM1 (GlxI) in which the highest SLG level was generally observed at 10 s and decreased thereafter (Fig. 6A). SLG levels were significantly higher than in the parent strain (~2.5-fold at 10 s, Fig. 6B). However, these did not reach the levels seen in the ΔgloB mutant, reflecting the continuing breakdown. Cells expressing high levels of GlxII did not accumulate significant SLG when incubated with MG (0.1 -0.7 mM MG; data not shown).

These data are consistent with observed K + efflux rates, since the fastest rates of K + efflux correlate with the accumulation of high pools of SLG in the ΔgloB mutant and in strains expressing high levels of . KefGB activity exhibited an initial rapid rate for ~3 min followed by a slow decline; this change in kinetics appears to be an intrinsic property of the efflux system since the SLG pools remain high during the slow phase of efflux and the cytoplasmic K + pool remains substantial. To get a better insight into the relationship between SLG accumulation and KefGB activity we measured the initial rate constant for K + efflux and compared this with the SLG pool (Fig. 7A). The precise relationship between KefGB activity and SLG pools is expected to be complex since the formation of SLG is accompanied by removal of the KefGB inhibitor, GSH. The rate constant for efflux shows a non-linear dependence on SLG concentration with a tendency for the rate constant towards ~0.13 min -1 when SLG has reached the maximum concentration (Fig. 7A). By this analysis KefGB appears to achieve 50% of its (Roosild et al., 2010) bind to the same pocket on the KTN domain of the KefGB protein complex with affinities in the high µM range (C. Pliotas, S. Miller, I.R. Booth & T. Rasmussen, unpublished data). KefGB activity would thus be determined by the rate at which GSH is released and replaced by SLG, which would explain the apparent low affinity for the activating ligand.

Greater cytoplasmic acidification is achieved in a ΔgloB mutant

The activity of the KefGB efflux system ultimately results in acidification of the cytoplasm, which limits the toxicity of MG (Ferguson et al., 1995, Ness & Booth, 1999). We investigated whether the increased activity of KefGB in the ΔgloB cells affected the cytoplasmic pH (pH i ) upon MG stress. Experiments were conducted under the same conditions as for K + efflux and SLG pool analysis. Cytoplasmic pH fell to a steady state level within 60 s of addition of MG and the change was dependent upon the presence of both KefGB and KefFC. Thus the pH change had different kinetics from K + efflux, which continued for at least 15 min, albeit at progressively slower rates. The discrepancy between the change in pH and K + loss is explained by previous studies that demonstrated that K + efflux is accompanied by entry of both H + and Na + (Bakker & Mangerich, 1982).

Cytoplasmic pH changed both as a function of the MG concentration and the strain (a representative dataset showing the kinetics of the pH changes is presented in Fig. S2; Supplementary information) (Fig. 7B). There was no consistent difference in the cytoplasmic pH between the parent and the ΔgloB strain prior to addition of MG. After MG addition the cytoplasmic pH always fell to a lower value in the mutant, which is the predicted consequence of increased activity of KefGB (Fig. 7B). Over-expression of GlxII (strain MJF595 pGlxII) led to a small drop (~0.1 pH units). Overall, a simple correlation was found between the initial rate of loss of viability and the change in the steady state pH i (Fig. 7C). (MacLean et al., 1998), which suggested that the failure to observe increased sensitivity to MG in a ΔgloB strain (Fig. 3) might be accounted for by the observed increased activation of KefGB and sustained lowering of the cytoplasmic pH (Figs. 4 & 7). We therefore constructed a strain lacking both KefGB (and KefFC) and GlxII (MJF596; kefB, kefC::Tn10, ΔgloB). The triple mutant grew at the same rate as the parent strain MJF274 in K 0.2 minimal medium (data not shown) and was similarly impaired with regard to MG detoxification as the gloB null mutant (0.463 ± 0.063 μM MG • min -1 ; n = 3). Cells were very sensitive to MG (Fig. 8), despite their similar rate of detoxification to the strain lacking only GlxII. No viable cells were observed in the triple mutant after 2 h incubation with 0.7 mM MG. A mutant lacking both K + efflux systems (MJF276; ΔkefB, ΔkefC) that retains detoxification, was less sensitive to MG than the triple mutant, but cells were killed more rapidly than either the parent strain or the single ΔgloB mutant (Fig. 8). These data emphasize the importance of the efflux systems for survival during exposure to MG.

The role of KefGB in protection against MG

Discussion

MG is produced by cells when metabolism is unbalanced, in particular when carbon flow is either out of balance with growth potential or when regulatory mechanisms are sub-optimal. Most descriptions of MG production have arisen from genetic studies of regulation of metabolism (Ackerman et al., 1974, Freedberg et al., 1971, Kadner et al., 1992) or from chemostat studies of growth physiology (Burke & Tempest, 1990, Tempest et al., 1983, Russell, 1993, Russell, 1998). The activity of the GlxI & II system provides an intrinsic advantage to cells when they are exposed to MG by catalysing the GSH-dependent conversion of MG to D-lactate, which can either be excreted or further metabolised. Mutants lacking GlxI exhibit a high sensitivity to MG; both growth inhibition at low MG concentrations (0.1 mM) and cell death at higher concentration (0.7 mM) are increased ~2-fold (MacLean et al., 1998). In most detoxification pathways one would not necessarily look further than the loss of the capacity to remove the toxic molecule for an explanation of the phenotype. However, rapid cell death takes place in a time frame during which the MG concentration remains relatively constant. This is further supported by the ΔgloB mutant studied here, which does not share the increased sensitivity to MG seen in a ΔgloA strain despite similar continued exposure to high MG concentrations (Fig. 1). The observation that null mutants for each enzyme exhibit opposite phenotypes is essentially a property of their linkage to the KefGB efflux system that modulates cytoplasmic pH.

We have previously established that KefGB, but not KefFC, is strongly activated by metabolites derived from MG and GSH. Based on an analysis of the ΔgloA mutant and of strains possessing either KefGB or KefFC we proposed that KefGB was activated by the product of the GlxI, SLG. The survival properties of the mutants become explicable by this linkage and establish that the dominant factor determining survival is not detoxification but activation of the protecting K + efflux system. Thus, ΔgloA (GlxI) and ΔgloB (GlxII) mutants exhibit almost identical rates of MG detoxification that are approximately 3-fold lower than observed in the parent (MacLean et al., 1998) (Fig. 3 ). However, in a GlxI mutant the only adduct formed is HTA, whereas in a GlxII mutant, SLG accumulates to high levels and the GSH pool is severely depleted. GSH is an inhibitor of KefGB and HTA is only a weak activator, which would result in only limited activation of KefGB in a ΔgloA strain. In contrast, the enhanced pools of SLG and depletion of GSH in a GlxII mutant, create optimal conditions for activation of KefGB and result in a corresponding greater acidification of the cytoplasmic pH. Support for this model comes from our studies on the effect of the over-expression of GlxI or GlxII. The former stimulates detoxification, which suggests that this enzyme is limiting for the pathway. However, the stimulation is not proportional to the increase in enzyme activity, which is consistent with a fairly small gap between the relative activities of GlxI and GlxII, such that as GlxI activity is increased, GlxII becomes the limiting factor. Under these circumstances SLG will accumulate, with the effect of stimulating KefGB activity, which explains the dramatically increased protection against MG by over-expression of GlxI (MacLean et al., 1998). In contrast, over-expression of GlxII does not modify the detoxification rate, but sensitises cells to MG (Fig. 2 ). An increase in GlxII, in the context of a low but constant level of GlxI, depletes the SLG pool resulting in limited activation of KefGB and hence only The relationship between the magnitude of the SLG pool, KefGB activity and pH i is complex, but can be understood from the properties of the systems and the cell itself. The buffering capacity of the cytoplasm is at its lowest value around cytoplasmic pH values in the range pH 7 -8 (Booth, 1985). Thus the cell is simultaneously at its most sensitive to perturbation of the environment due to limited buffering capacity, but also has the greatest potential to modulate cytoplasmic pH through changes in the balance of proton entry and efflux pathways. Previous work established that K + efflux from E. coli is compensated by the entry of both H + and Na + (Bakker & Mangerich, 1982) in a ratio of approximately 1K + = 0.3H + and 0.7Na + . The extremely rapid, but limited, acidification of the cytoplasm upon activation of KefGB can be explained by initial rapid K + -linked H + movements that are subsequently compensated by the reversibility of the Na + /H + antiports (Padan et al., 2001) leading to exchange of cytoplasmic H + for external Na + . Thus, the pH would be stabilised at a value set by the intrinsic properties of the KefGB system and the Na + /H + antiports (NhaB and NhaA) (Dover & Padan, 2001, Padan et al., 2001). Our recent work (Roosild et al., 2010) shows that a single binding site on KefGB is shared by GSH and SLG. Activation of KefGB by MG requires displacement of GSH and binding of SLG. The cytoplasmic pool of GSH is suggested to be ~20 mM (Bennett et al., 2009, Fahey, 2001[START_REF] Mclaggan | Glutathionedependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process[END_REF] . We observed that conversion to SLG is maximally ~80% (in a ΔgloB strain) but more often SLG is at a much lower concentration than GSH (Fig. 5B). Consequently, the activation of KefGB is not maximal at the normal levels of abundance of GlxI and GlxII. Thus, the change in cytoplasmic pH is constrained by the relative abundance of GlxI and GlxII activities and the concentration of MG.

The data presented here point to an important relationship between the intrinsic activity of the different enzymes and transporters. The effects of GlxI over-expression indicate that although this enzyme is, on a population-basis, limiting for the rate of detoxification, the two enzymes are relatively closely matched. A 30-fold increase in GlxI activity produces only a 2-fold enhancement Given that most detoxification enzymes are expressed at low levels in the cell there is considerable scope for cell-to-cell variation in enzyme level. GlxI and GlxII are encoded by separate unlinked genes and there is no coordination of their expression. Consequently, in each cell their abundance should vary independently. We have calculated the abundance (in cells grown to mid-exponential phase in minimal medium) to be ~130 GlxI and ~1500 GlxII molecules per cell (see Supplementary information). Stochastic variation of protein abundance will have a greater impact on GlxI and thus some cells will have a balance that favours SLG accumulation (high GlxI: GlxI > GlxII) whereas others (low GlxI: GlxII > GlxI) will have lower cytoplasmic pools of this adduct. Thus, on an individual cell basis, some cells will experience greater protection than others.

The independence of the expression of KefGB from the detoxification enzymes creates a further dimension of variability that is superimposed on the modulation of SLG pools. This means that some cells may gain further protection; the optimal solution would be simultaneous enhanced levels of GlxI and KefGB, coupled with low activity of GlxII. In this way the cell will experience maximum protection coupled with detoxification.

In this study we have defined the relationship between sensitivity to MG and the relative activities of the major detoxification pathway, GlxI-II, and the KefGB K + efflux system. The study demonstrates the benefits to the cell of linking an intermediate in detoxification to the activation of the protective K + efflux system. Small changes in detoxification enzymes determine the maximum activity of KefGB. It is perhaps counterintuitive that cells possess protective capacity that is not fully utilised. However, stochastic distribution of the proteins between cells in a population ensures that a few cells are highly protected and survive exposure to MG. In addition, it seems plausible that avoiding maximum stimulation of KefGB in all cells prevents excessive acidification that might be itself detrimental. The paradox is that in wild type populations the majority (99.9%) of cells are terminally-damaged (i.e. unable to form colonies after dilution and plating on fresh medium) but continue to contribute to MG removal. Thus, the survivors are aided by the dead and dying cells.

Experimental procedures

Strains and plasmids

All experiments were performed with E. coli K-12 derivative strains and are listed in Table 2. See further below for details of strains and plasmids created in this study.

Table 2. List of strains and plasmids

Strain

Genotype Source / Reference MJF274 F - (Ferguson et al., 1993) , thi, rha, lacZ, kdpABC5, lacI,

Growth media

For physiological assays cells were grown either in K 0.2 minimal medium containing ~0.2 mM K + or K 115 minimal medium containing ~115 mM K + (Epstein & Kim, 1971) depending on the experimental design. Both media were supplemented with 0.2% (w/v) glucose, 0.0001% (w/v) thiamine, 0.4 mM MgSO 4 and 6 μM (NH 4 ) 2 SO 4 •FeSO 4 . LK complex medium (Rowland et al., 1985) was used for cell growth for DNA manipulations. In the case of strains carrying an antibiotic resistance marker, no antibiotics were used in physiological assays since the routinely used resistance determinants were stable and were not lost in response to the absence of antibiotics from the growth media. Solid media contained 14 g/L agar. To prepare solid K 0.2 medium the agar was washed with a 1 M NaCl solution to displace trace amounts of K + and then washed several times with distilled water before use.

Cloning of the gloB gene

The gloB gene was PCR-amplified using whole cells of MJF274 as a template.

Primers GloBI (5'-CAACCAGCGTCGACTGTAC) and GloBII (5'-GGTATCACCCAGTGGATCC ) were designed 1.1 kb downstream and 1kb upstream of gloB respectively. The primers contained HincII and BamHI restriction sites, respectively (underlined). The amplified 2.8 kb product was cleaned and digested with HincII and BamHI restriction enzymes. The cloning vector pHG165 was digested with HindIII, followed by treatment with Klenow enzyme to fill the HindIII site. Subsequently, the vector was digested with BamHI.

The PCR product was ligated into the linear plasmid to create plasmid pGlxII. The gloB gene was disrupted with a chloramphenicol resistance cassette (Cm R ) by homologous recombination mediated by λ-phage functions (recombineering).

Construction of gloB null mutants

Recombineering primers gloB KO-A (5'-TGCCGCTCATTTTTCAGAATTACGGGTAGTGTTATTTGATTTTTTGCCCGACCA GCAATAGACATAAGCG) and gloB KO-B (5'-GATCCCGGAGACGCAGAGCCAGTATTAAACGCCATTGCCGCCAATAACTGAT AAATAAATCCTGGTGTCCC) contained homologous sequences to the 3' and 5' of the gloB gene, respectively. The incorporation of the Cm R was predicted to create a fused ORF with the remaining 5' gloB sequence. Therefore, a double stop codon was incorporated into primer gloB KO-B (underlined) to prevent any possible expression of the fusion protein driven by a gloB promoter. A 454 bp fragment (from 132 to 585) of the structural gene, where 1 is the first bp of the start codon, was replaced by the Cm R cassette. Hence a short sequence of the GlxII protein may be expressed. The Cm R cassette was amplified by colony-PCR from a strain carrying the resistance marker (MG1655 zwf<cm>; kindly provided by Michelle Brewster, SAIC, Frederick, USA) using the recombineering primers above. DNA was purified using a QIAGEN PCR product purification kit and quantified. Recombineering was performed using strain DY330 (Yu et al., 2000) and cells were plated on nitrocellulose filters placed onto LK solid medium containing 10 µg/ml chloramphenicol (LK-Cm). Putative gloB integrants showed normal colony morphology and were purified twice on LK-Cm plates to check for the stability of the resistance marker. Recombination at the correct locus was investigated by PCR analysis using a primer pair flanking the gloB gene (gloB KO check F: 5'-CGCTTCGCAATTGATTTC; gloB KO check R: 5'-GGCGAGTAATATCGCTTT), showing the two distinctly different sized PCR products before and after recombination. The mutation was further confirmed by DNA sequencing of the PCR product across the borders of the site of recombination using primers gloB KO check F & R and Cm Seq 1

(5'-CTTCATTATGGTGAAAGTTGG) and Cm Seq 3

(5'-GATAATAAGCGGATGAATGG) located within the Cm R cassette thus verifying successful creation of strain DY330 gloB<>cm (<> indicates replacement by recombineering approach (Yu et al., 2000)). The mutation was transduced into strain MJF274 by P1 transduction creating strain MJF595. An overnight culture of strain DY330 gloB<>cm was grown in LK medium at 32°C (shaking at 250 rpm). The culture was diluted 100-fold into fresh LK medium also containing 0.2% glucose, 10 mM MgCl 2 and 5 mM CaCl 2 . Cells were grown at 32˚C with shaking at 250 rpm until minor growth was apparent (after 30 -40 min) when P1 phage lysate was added. The cultures were incubated further until cell lysis was evident.

Lysates were transferred to a 50 ml centrifuge tube containing chloroform (50 µl per 10 ml lysate) and incubated at 37˚C for 30 min without shaking. derivative strain grew at a similar rate as the parent strain and had no obvious phenotype (data not shown) possibly indicating the acquisition of a secondary mutation allowing cells to recover normal growth.

K + efflux assays

K + efflux from cells was measured as described by previously [START_REF] Elmore | Activation of potassium efflux from Escherichia coli by glutathione metabolites[END_REF], Ferguson et al., 1993). Briefly, cells were grown to late logarithmic phase (OD 650nm ~0.8) in K 115 medium, collected by filtration onto a cellulose acetate membrane (0.45 µm), washed with K 0 buffer containing ~5 mM (K 5 buffer) and suspended in K 0 buffer. Cells were rapidly transferred into thermally insulated glass pots and kept at 37˚ C under continuous stirring. Samples (1 ml) were taken at various time points, cells pelleted in a microcentrifuge at 14000 rpm for 30 s, the supernatant quickly aspirated and the K + content of the cells determined by flame photometry after lysis by boiling in distilled water. MG (Sigma, M0252) was added from stock solutions to the test suspension 2 min after suspending cells in K 0 buffer. The first order rate constants for K + efflux (k) were calculated by transforming the values of K + levels to the natural logarithm and by determining the slope of the decline in the linear range after addition of MG. For illustration purposes the K + levels were normalised to the value at t 0 min , defined as 100%.

Cell viability and MG detoxification assays

Overnight cultures were grown in K 0.2 medium and diluted into fresh, pre-warmed K 0.2 medium to an OD 650nm of ~0.05. Cells were grown to mid-exponential phase (OD 650nm ~0.4) and diluted 10-fold into pre-warmed K 0.2 medium also containing MG. Samples were taken at various time points and cell viability and MG detoxification assays performed as described previously (Ferguson et al., 1993, Totemeyer et al., 1996). Viable cells were recovered on solid K 0.2 media plates.

Preparation of cytoplasmic cell extracts and GlxII enzyme assays

Cells were grown in K 0.2 medium to mid-exponential phase (OD 650nm ~0.4) exactly as for the assays above, however, they were harvested at this point by centrifugation at 4300 x g. Cells were washed, suspended in 50 mM potassium phosphate buffer (pH 6.6) and disrupted by two passages through a French press at 18000 Psi. Bulk cell debris and membrane fractions were removed by centrifugation at 4300 x g and subsequent centrifugation at 110000 x g.

Cytoplasmic cell extracts were stored at -20°C until enzyme assays were performed. Protein quantification was performed using the Lowry assay (Lowry et al., 1951). GlxII activities were measured as a modification of a previously described method (Racker, 1951, Oray & Norton, 1982), in which SLG hydrolysis is measured by the decrease in A 240nm . Enzyme assays were performed in 50 mM potassium phosphate buffer (pH 6.6) at 37°C using a Shimadzu UV 2101PC

spectrophotometer. The reaction mixture contained 1 mM SLG (Sigma, L7140) in a total volume of 0.4 ml, using a 0.1 cm path length spectrophotometer cuvette.

Enzyme activity was expressed as units per cytoplasmic cell protein (U•mg -1 ) using a molar extinction coefficient of 3060 M -1 cm -1 (Racker, 1951), where 1 unit is defined as the amount of enzyme catalysing the formation of 1 µmol • min -1 SLG.

Enzyme activities were determined using two different enzyme concentrations to ensure that the enzyme was rate-limiting.

Determination of intracellular GSH and SLG levels by LC-MS/MS

The choice of the experimental design to extract the metabolites from the cells was guided by the need to correlate SLG levels directly with K + efflux. Therefore, cells were grown and treated as in K + efflux experiments as described above except that cells were grown in K 0.2 medium. After suspension of cells in K 0 buffer 1 ml samples were taken at various time points before and after exposure to MG and transferred into previously prepared microcentrifuge tubes. Microcentrifuge ( Kroll & Booth, 1981)). Thus, the total cytoplasmic volume in the assay is ~0.64 µl (8x10 8 cells), and a concentration of 100 µM in the extraction volume equates to an intracellular concentration of ~6.35 mM.

Measurement of intracellular pH

The magnitude of the pH gradient was estimated from the distribution of a weak acid, and pH i (internal pH) calculated from knowledge of pH o (external pH). Cells were grown in K 0.2 minimal medium until they reached an OD 650nm ~0.8. The cell suspension was transferred into two thermally insulated glass pots (37°C) and 14 C benzoic acid (4.5 μM final concentration; specific activity 0.1 μCi•ml -1 ) and 3 H-water (specific activity 1 μCi•ml -1 ), as marker of extracellular water, were added to the cultures (Kroll & Booth, 1981). After 5 min incubation, 1 ml samples were taken at timed intervals and the cells and supernatant separated by centrifugation (14000 rpm for 20 s). For each sample, 100 μl of the supernatant was transferred to a scintillation vial containing 200 μl of cell suspension that had not been treated

with radioactivity; the remaining supernatant was aspirated from the cell pellet and discarded. Cell pellets were suspended in 200 μl K 0.2 buffer containing 0.2% glucose and the suspension transferred into a scintillation vial containing 100 μl of the same buffer. Samples of supernatant and pellet were counted for radioactivity on a preset 3 H/ 14 C program of a Tri-Carb 2100 TR liquid scintillation analyzer. The pH gradient and subsequently the pH i were calculated as described previously (Booth et al., 1979). Enzyme activities were performed on three independent cytoplasmic cell extracts from each strain. For each extract the GlxII activity was measured using two different protein concentrations to ensure that the enzyme was rate-limiting.

Activities increased proportionally with protein concentration and were averaged.

The mean activity and standard deviation in independent extracts is shown.

Strains: MJF274 (parent strain), MJF595 (ΔgloB), MJF274 pGlxII, MJF274 pMJM1

(control, parent strain over-expressing GlxI).

Fig. S2. The relationship between intracellular pH and MG

The intracellular pH was determined for MJF274 (A) and MJF595 (B) cells treated with a range of MG concentrations (0 -0.8 mM) in K 0.2 . * MG was added at t 15 min thus the t 15 min time point has been added as a duplicate of t 14 min to illustrate the rapid kinetics of cytoplasm acidification. The data are mean ± s.e.m.

Supplementary information

Promoter predictions for mltD and yafS

To guide our experimental approach in creating a gloB null mutant, an assessment of promoter elements for the respective genes was undertaken. In the first instance, promoter predictions were accessed on RegulonDB, a curated database containing information on the transcriptional regulatory network of E. coli K-12 (Gama-Castro et al., 2008). At the time of preparation of this paper RegulonDB stated four potential σ 70 promoters for mltD with the furthest predicted -35 element being 184 bp upstream of the start codon (Table 3.) and thereby within the gloB gene. No promoter predictions were stated for yafS on the RegulonDB database. This analysis was complemented using the web-based tool BPROM that can predict σ 70 promoters (www.softberry.com). The upstream sequences (500 bp from protein encoding sequence) of mltD and yafS were analysed with BPROM using the default settings and potential -10 and -35 promoter elements identified.

BPROM predicted one promoter region for mltD, located up to 180 bp upstream (between positions 234109 and 234135 on the chromosome, Table 4) thereby overlapping with the furthest promoter prediction stated in RegulonDB. Two putative promoter regions were predicted for yafS by BPROM ( 1) The length of presented sequence.

2) LDF threshold (default).

3) The number of predicted promoters.

4) The positions of predicted promoters and their scores with 'weights' of two conserved promoter boxes. Promoter position is assigned to the first nucleotide of the transcript (transcription start site position).

See the following web address for more details:

http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup= gfindb

Estimation of the number of GlxI & GlxII enzymes per cell

The number of GlxI and GlxII molecules in a single cell can be estimated from the knowledge of the enzyme activity of the purified proteins, the activity in cell extracts and the weight of total cellular protein.

The purified GlxI enzyme exhibits a maximal activity of ~676 µmol • min -1 • mg -1 protein. The dimeric protein has molecular mass of ~30 kDa (Clugston et al., 1998), thus 1 mol of dimeric GlxI can convert ~20280 mol of substrate per minute.

The specific GlxI activity in E. coli extracts is ~0.016 µmol • min -1 • mg -1 total cell protein (MacLean et al., 1998). Given the activity of the pure protein, this equates to ~7.89 x 10 -13 mol GlxI per mg of total cell protein. We can approximate that 1 mg total cell protein is equivalent to ~3.6 x 10 9 cells based the following assumptions: OD 650nm of 1 = 1 x 10 9 cells/ml, OD 650nm of 1 = 0.5 mg cell dry weight/ml [START_REF] Elmore | Activation of potassium efflux from Escherichia coli by glutathione metabolites[END_REF], protein content of cell dry weight: 55 % (Neidhardt & Umbarger, 1996)(. Therefore a single cell will have 2.17 x 10 -22 mol of GlxI, which after consideration of the Avogadro constant (6.022 x 10 23 ) equates to an estimated number of GlxI molecules of ~130 dimers per cell. The same calculations can be performed for the GlxII enzyme. The maximal activity of the purified enzyme is ~112 µmol • min -1 • mg -1 protein and the molecular mass is ~ 28.4 kDa [START_REF] O'young | Escherichia coli glyoxalase II is a binuclear zinc-dependent metalloenzyme[END_REF]. Thus 1 mol of pure protein can hydrolase ~3180 mol of substrate per minute. An approximation of the number of GlxII molecules from the activity in cell extracts requires further considerations.

GlxII is a metallo-enzyme and it has been reported that the active site of the purified E. coli enzyme is loaded with zinc ions [START_REF] O'young | Escherichia coli glyoxalase II is a binuclear zinc-dependent metalloenzyme[END_REF]

Introduction

Bacteria have evolved elaborate and complex stress management strategies to minimise damage and thus, to enhance their survival during environmental changes (Booth, 2002). In addition, metabolic activity in itself can create significant stress, for example the production of hydrogen peroxide and oxygen radicals is a consequence of aerobic growth and the resulting oxidative damage requires both intrinsic and adaptive enzyme activities (Korshunov & Imlay, 2010, Imlay, 2008). Similarly, bacteria encounter electrophiles both as a metabolic consequence and as an environmental challenge. Among the most frequently encountered electrophiles is methylglyoxal (MG), which is produced by bacteria from sugars and amino acids and is believed to have a role in macrophage-mediated killing (Eriksson et al., 2003, Eskra et al., 2001, Ficht, 2003). MG is synthesised either from sugars by methylglyoxal synthase (MGS) (Totemeyer et al., 1998) or from threonine, serine and glycine by monoamine oxidase (Green & Lewis, 1968, Kim et al., 2004). In E. coli the dominant route appears to be from sugars and arises when there is an accumulation of phosphorylated glycolytic intermediates above the level of 1,3-diphosphoglycerate and a lowering of the pool of inorganic phosphate (Hopper & Cooper, 1971;Totemeyer et al., 1998, Ferguson et al., 1998). MGS activity is determined by the balance between inorganic phosphate, which is a strong inhibitor, and dihydroxyacetone phosphate (DHAP), the substrate, which exhibits strong homotropic activation (Hopper & Cooper, 1971). Thus, production of MG only occurs when there is simultaneous depletion of phosphate and extremely high concentrations of DHAP, conditions that arise when sugar metabolism is strongly stimulated leading to excess carbon flow into the upper end of glycolysis. For E. coli, accumulation of MG above ~0.3 mM in the medium results in growth inhibition and at levels above ~0.6 mM the survival of cells is affected. Damage to DNA and to proteins has been observed (Krymkiewicz, 1973;Colanduoni & Villafranca, 1985, Ferguson et al., 2000) and both may contribute to cell death.

Protection against electrophiles is multifactorial with contributions from glutathione (GSH), detoxification enzymes, DNA repair enzymes, peptide export systems and (Ferguson & Booth, 1998, Ko et al., 2005, MacLean et al., 1998, Sukdeo & Honek, 2008, Xu et al., 2006). In E. coli, detoxification is primarily effected by the GSH-dependent glyoxalase system (GlxI and GlxII, products of the unlinked gloA and gloB genes) and their integration with the GSH adduct-gated KefGB K + export systems (Fig. 1). Other enzymatic systems, particularly a range of oxidoreductases (Murata et al., 1989, Xu et al., 2006, Ko et al., 2005), may also play a role in detoxification. In the GlxI-II pathway, the substrate for GlxI is created by the spontaneous reaction between MG and GSH forming hemithioacetal (HTA). GlxI isomerizes this to S-lactoylGSH (SLG), which is the substrate for GlxII, a hydrolase. The final products are the relatively non-toxic molecule D-lactate and GSH, which is recycled in the cytoplasm.

Although a GSH export system [START_REF] Pittman | A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm[END_REF], Owens & Hartman, 1986) has been identified, there is no evidence for a role in MG detoxification.

Protection by the KefGB and KefFC systems is dependent on their role in modulation of the cytoplasmic pH (Ferguson et al., 2000, Ferguson & Booth, 1998, Ferguson et al., 1996a, Ferguson et al., 1995, MacLean et al., 1998). KefGB and KefFC are structurally-related K + efflux systems that are maintained inactive by the binding of GSH and are activated by binding of specific GSH adducts [START_REF] Elmore | Activation of potassium efflux from Escherichia coli by glutathione metabolites[END_REF], Ferguson et al., 1993, Miller et al., 1997, Miller et al., 2000, Roosild et al., 2009). Activation leads to rapid K + efflux, which is quantitatively affected by several parameters: external K + concentration, the activity of K + uptake systems, the expression level of KefGB and KefFC and the intracellular concentration of the GSH adduct (Ferguson et al., 1996b, Ferguson et al., 1993, MacLean et al., 1998[START_REF] Mclaggan | Glutathionedependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process[END_REF]. K + efflux is accompanied by influx of H + and Na + (Bakker & Mangerich, 1982), but it is the lowering of the cytoplasmic pH that is critical for protection against MG (Ferguson et al., 2000, Ferguson et al., 1995). Lowering the cytoplasmic pH may slow the reaction of MG with guanine in DNA and with other macromolecules (Krymkiewicz, 1973).

We have previously proposed a model in which GlxI plays the major role in both determining the rate of MG detoxification and in modulation of KefGB activity by the intermediate SLG (Fig. 1) (MacLean et al., 1998). However, the original model could not be fully tested since mutants lacking GlxII could not be created and

Results

Modulation of GlxII activity

To assess the importance of the GlxII activity and the activation of KefGB for survival upon exposure to MG we inactivated gloB (see Experimental procedures).

Previous attempts to replace the gloB gene with antibiotic resistance cassettes (kanamycin and spectinomycin) were unsuccessful. We considered the possibility that replacement of the entire gloB gene might lead to polar effects with respect to the expression of the two genes on either side of gloB, namely mltD and yafS, which are separated by only 71 and 33 bp, respectively from the gloB ORF (Fig. 2A). The mltD gene encodes for a membrane-bound lytic murein transglycosylase, which plays a major role in peptidoglycan expansion and recycling (Scheurwater et al., 2008, Suvorov et al., 2008). The yafS gene is believed to encode an S-adenosyl-L-methionine-dependent methyltransferase, but its physiological role remains unknown. From global array analysis under various growth conditions (GenExpDB, http://genexpdb.ou.edu), it is clear that both these genes are transcribed and thus either or both of these genes may be essential for cell function. ) may be expressed in the mutant strain MJF595 created in this study. However, from the crystal structure, this fragment is unlikely to form an enzymatically-active protein since the critical metal-and substrate-binding sites are located in other regions (Campos-Bermudez et al., 2007, Zang et al., 2001).

The ∆gloB mutant grew at a similar rate to the parent in K 0.2 minimal medium (Fig. S1A) and exhibited no obvious growth phenotype. Thus GlxII is not an essential enzyme during normal exponential growth. Some residual activity was detectable (corresponding to ~6% of the parental GlxII activity), but was close to the analytical limit of the assay. The residual activity was not due to GlxI, since increasing the expression of this enzyme in the ∆gloB mutant did not increase the rate of breakdown of SLG (Table 1). Addition of 0.7 mM MG to both parent and mutant strains, in early exponential phase (OD 650 = 0.05), caused immediate growth inhibition without recovery over the course of the experiment (Fig. S1A). MG disappeared from the medium in a linear fashion and, as expected, the rate was greatly reduced in ∆gloB cultures (Fig. 2B; 0.444 ± 0.015 µM MG • min -1 and 1.155 ± 0.21 µM MG • min -1 , for mutant and parent, respectively). We have previously observed a similar reduction in the capacity to detoxify MG in a ∆gloA null mutant (MacLean et al., 1998). However, cells retain the ability to breakdown MG, but at a much lower rate, which is consistent with the known presence of other enzymes that can metabolise MG (Misra et al., 1995, Misra et al., 1996).

The gloB gene, encoding for the GlxII enzyme, and its flanking regions were cloned into a moderate copy number vector to create pGlxII (see Experimental procedures). Transformation into E. coli MJF274 (parent strain) led to an approximately 25-fold amplification of GlxII activity in extracts from mid-exponential grown cells (Table 1). Cells expressing higher levels of GlxII grew at a similar rate to the parent strain in K 0.2 minimal medium (Fig. S1B). Elevated synthesis of GlxII did not alter the rate of MG detoxification when cells were incubated with 0.7 mM MG (Fig. 2B). These data are consistent with previous observations that GlxI activity limits the rate of MG detoxification in parental cells (Maclean et al., 1998).

Inactivation of gloB does not affect cell viability upon MG stress

Previously, we reported that a ∆gloA mutant, impaired in MG detoxification, exhibits increased sensitivity to MG, which can be explained by the persistence of the electrophile in the growth medium (MacLean et al., 1998). When mutant cultures (either ∆gloA or ∆gloB) were treated with 0.7 mM MG, the concentration remains above the lethal level (~0.6 mM MG) for ~6 h due to the slow rate of detoxification in the absence of the Glx pathway. In contrast, the parent strain detoxifies MG to non-lethal levels within ~2-3 h (at low cell density, OD 650 ~ 0.05).

Thus, it was expected that the ∆gloB mutant would exhibit a similar sensitivity to MG as the ∆gloA mutant. However, survival of the ∆gloB mutant during MG exposure was not impaired (Fig. 2C). Surprisingly, over-expression of GlxII increased sensitivity to MG despite having no effect on the rate of detoxification (Fig. 2B,C).

We further addressed if the viability phenotype of the ∆gloB mutant is a reflection of compensatory enzyme activities in vivo. FrmB and YeiG (EC 3.1.2.12), are major components of the formaldehyde detoxification pathway and have been reported to have low level hydrolytic activity against SLG (Gonzalez et al., 2006).

The yeiG gene is transcribed constitutively whereas the frmB gene can be induced with formaldehyde (Gonzalez et al., 2006). Strains lacking GlxII and lacking either YeiG (MJF595 ∆yeiG) or FrmB (MJF595 ∆frmB) were created (Experimental procedures) and cell viability determined during exposure to 0.7 mM MG. The level of survival of both double mutants (∆gloB-∆yeiG or ∆gloB-∆frmB) was indistinguishable from the ∆gloB mutant (Fig. S2), indicating that these systems do not have a physiologically significant role in MG detoxification.

K + efflux systems are hyperactive in a gloB null mutant

We have previously established that KefGB and KefFC are activated by electrophiles through the formation of GSH adducts [START_REF] Elmore | Activation of potassium efflux from Escherichia coli by glutathione metabolites[END_REF], Ferguson et al., 1993, Ferguson et al., 1995, Ferguson et al., 1997). From the study of a ∆gloA mutant we inferred that SLG was the metabolite activating KefGB during exposure to MG, since the HTA formed by a reversible reaction with GSH in such a mutant was insufficient to activate the K + efflux system (MacLean et al., 1998).

We predicted that a ∆gloB mutant, lacking GlxII activity, would accumulate SLG and thus, KefGB activity should be enhanced; conversely we predicted that overexpression of GlxII should diminish SLG pools and thus lower the rate of K + efflux.

Analysis of K + efflux patterns in the parent strain MJF274 and the ∆gloB mutant MJF595, using a range of MG concentrations, supports this model. Accurate measurements of K + efflux require cells to be incubated at higher cell densities than are used for growth and viability measurements (OD 650 ~0.8 for efflux assays compared with ~0.05 for growth and viability). Consequently, in the parent strain, the MG concentration is continuously declining due to the high rate of detoxification in such conditions (0.7 mM MG falls to ~0.3 mM in 30 min). Control experiments for cell viability and MG detoxification were carried out at high cell density under conditions identical to the measurements of cytoplasmic pH and K + efflux (Fig. S3A,B). The rate constant for efflux was measured over the first 3 min, a period in which there was minimal lowering of the MG concentration (Fig. S3B).

The rate and extent of K + loss was faster in the ∆gloB strain than in the parent (Fig. 3). This effect was most marked at the lower MG concentrations and thus first order rate constants for the initial K + efflux were measured at a range of concentrations (Fig. 3C). In the parent strain, the rate of efflux observed at very low MG concentrations (<200 µM) was not significantly different from the rate of K + loss in the absence of MG. For higher concentrations the rate constant increased and approached a maximum for concentrations ≥3 mM MG (Fig. 3C). In contrast, the ∆gloB mutant exhibited rapid K + loss even at concentrations as low as 25 µM MG and the rate was not further stimulated by treatment with ≥ 200 µM MG (Fig. 3B,C). In cells over-expressing GlxII MG addition did not stimulate significant K + efflux (Fig 3D).

Intracellular accumulation of SLG upon MG stress

We developed an LC-MS/MS assay to quantify intracellular GSH and SLG pools to obtain insight into the in vivo dynamics of SLG formation. Cells were grown in K 0.2 minimal medium and GSH and SLG were extracted from cells with formic acid using a silicone oil centrifugation technique (see Experimental procedures). Pools of GSH and SLG were measured over the time period used to measure the rate constant for KefGB and using experimental conditions identical to those for the K + efflux assays. GSH pools prior to MG addition were identical in the parent and ∆gloB strains within experimental error and no SLG could be detected prior to addition of MG (Table S1). Addition of MG (0.2 mM) caused a rapid increase in SLG, coincident with depletion of GSH, in both ∆gloB and parent; the increase was ~20-fold greater in the mutant than the parent (Fig. 4A). In the parent, SLG pools were below the detection limit of the analytical technique at concentrations <0.2 mM MG (Fig. 4B). At higher MG concentrations the SLG pool rose rapidly then declined slowly over the period of the assay (Fig. 4A). Pools of SLG in the ∆gloB strain also increased rapidly, even with only 25 µM MG, achieving ~50% of the maximum value in 10 s, then climbed slowly over the next 2.5 min (Fig. 4C).

Despite the absence of GlxII, complete conversion of the GSH pool to SLG was never observed, free GSH was always measurable ~34±4 µM (Table S1).

High SLG pools were also observed in strains over-expressing GlxI in which the highest SLG level was generally observed at 10 s and decreased thereafter (Table S1), consistent with the higher rates of detoxification observed in this strain (Maclean et al., 1998). SLG levels were significantly higher than in the parent strain (~2.5-fold at 10 s, Fig. 4B andD). However these did not reach the levels seen in the ∆gloB mutant, reflecting the continuing breakdown. Cells expressing high levels of GlxII did not accumulate significant SLG when incubated with MG (0.1 -0.7 mM MG; Table S1).

Relationship between SLG pool and KefGB activity

Measurements of the SLG pools and rate constant for K + efflux under similar conditions allowed the determination of the adduct dependence of KefGB activity.

The very slow MG breakdown in the ∆gloB strain allowed both the SLG pool and KefGB activity to be measured across a wide range of MG concentrations. Parallel measurements were made with the parent (Fig. 5A). The rate constant for efflux shows a non-linear dependence on SLG concentration (Fig. 5A). Data from the parent strain was consistent with this relationship (Fig. 5A). When excess MG was supplied (3 mM), which maximally activates KefGB, the rate constant was 0.1 ± 0.02 min -1 and 0.13 ± 0.01 min -1 for the parent and ∆gloB respectively (Fig. 3A,B). Remarkably, strong KefGB activation (to ~25% maximum activity) was observed in the ∆gloB strain even with only 25 µM MG (Fig. 3C), which corresponded to a six-fold excess of GSH over SLG (Table S1). The precise relationship between KefGB activity and SLG pools is expected to be complex since the formation of SLG is accompanied by removal of the KefGB inhibitor, GSH.

Greater cytoplasmic acidification is achieved in a ∆gloB mutant

The activity of the KefGB efflux system ultimately results in acidification of the cytoplasm, which limits the toxicity of MG (Ferguson et al., 1995, Ness & Booth, 1999). We investigated whether the increased activity of KefGB in the ∆gloB cells affected the cytoplasmic pH (pH i ) upon MG stress. Experiments were conducted under the same conditions as for K + efflux and SLG pool analysis. Cytoplasmic pH fell to a steady state level within 60 s of addition of MG and the change was dependent upon the presence of both KefGB and KefFC. Thus the pH change had different kinetics from K + efflux, which continued for at least 15 min, albeit at progressively slower rates. The discrepancy between the change in pH and K + loss is explained by previous studies that demonstrated that K + efflux is accompanied by entry of both H + and Na + (Bakker & Mangerich, 1982).

Cytoplasmic pH changed both as a function of the MG concentration and the strain (Fig. 5B) (a representative dataset showing the kinetics of the pH changes is presented in Fig. S4). There was no consistent difference in the cytoplasmic pH between the parent and the ∆gloB strain prior to addition of MG. After MG addition the cytoplasmic pH always fell to a lower value in the mutant, which is the predicted consequence of increased activity of KefGB (Fig. 5B). Over-expression of GlxII (strain MJF595 pGlxII) led to a small drop (~0.1 pH units, data not shown). Overall, a simple correlation was found between the initial rate of loss of viability and the change in the steady state pH i (Fig. 5C).

The role of KefGB in protection against MG

Our failure to observe increased sensitivity to MG in a ∆gloB strain (Fig. 2C) might be accounted for by the increased activation of KefGB and sustained lowering of the cytoplasmic pH (Figs. 3C &5C). We therefore constructed a strain lacking both KefGB (and KefFC) and GlxII (MJF596; kefB, kefC::Tn10, ∆gloB). The triple mutant grew at the same rate as the parent strain MJF274 in K 0.2 minimal medium (data not shown) and was similarly impaired in MG detoxification as the gloB null mutant (0.463 ± 0.063 µM MG • min -1 ; n = 3). Cells were very sensitive to MG (Fig. 6), despite their similar rate of detoxification to the strain lacking only GlxII.

No viable cells were observed in the triple mutant after 2 h incubation with 0.7 mM MG. A mutant lacking both K + efflux systems (MJF276; ∆kefB, ∆kefC) that retains detoxification, was less sensitive to MG than the triple mutant, but cells were killed more rapidly than either the parent strain or the single ∆gloB mutant (Fig. 6).

These data suggest that the efflux systems for K + are more critical for survival than the detoxification pathway at low cell density.

Discussion

The glyoxalase pathway provides an intrinsic advantage to cells when they are exposed to MG by catalysing the GSH-dependent conversion of MG to D-lactate, which can either be excreted or further metabolised. Mutants lacking GlxI exhibit a high sensitivity to MG; both growth inhibition at low MG concentrations (0.1 mM)

and cell death at higher concentration (0.7 mM) are increased ~2-fold (MacLean et al., 1998). In most detoxification pathways one would not necessarily look further than the loss of the capacity to remove the toxic molecule for an explanation of the phenotype. However, rapid cell death takes place in a time frame during which the MG concentration remains relatively constant. This is further supported by the 12 ∆gloB mutant studied here, which does not share the increased sensitivity to MG seen in a ∆gloA strain despite similar continued exposure to high MG concentrations (Fig. 2). We have previously established that KefGB, but not KefFC, is strongly activated by metabolites derived from MG and GSH. Based on an analysis of the ∆gloA mutant and of strains possessing either KefGB or KefFC we proposed that KefGB was activated by the product of GlxI, SLG. The survival properties of the mutants studied here become explicable by this linkage and establish that the dominant factor determining survival is not detoxification but activation of the protecting K + efflux system. Thus, ∆gloA (GlxI) and ∆gloB (GlxII)

mutants exhibit almost identical rates of MG detoxification that are approximately 3-fold lower than observed in the parent (Fig. 2B, MacLean et al., 1998).

However, in a GlxI mutant the only adduct formed is HTA, whereas in a GlxII mutant, SLG accumulates to high levels and the GSH pool is severely depleted (Fig. 4A; Table S1). GSH is an inhibitor of KefGB and HTA is only a weak activator, which would result in only limited activation of KefGB in a ∆gloA strain.

In contrast, the enhanced pools of SLG and depletion of GSH in a GlxII mutant create optimal conditions for activation of KefGB (Fig. 3B,C) and result in a corresponding greater acidification of the cytoplasmic pH (Fig. 5B). Support for this model comes from our studies on the effect of the over-expression of GlxI or GlxII. The former stimulates detoxification, which suggests that this enzyme is limiting for the pathway. However, the stimulation is not proportional to the increase in enzyme activity, which is consistent with a fairly small gap between the relative activities of GlxI and GlxII, such that as GlxI activity is increased, GlxII becomes the limiting factor. Under these circumstances SLG accumulates (Table S1), with the effect of stimulating KefGB activity, which explains the dramatically increased protection against MG by over-expression of GlxI (MacLean et al., 1998). In contrast, over-expression of GlxII does not modify the detoxification rate, but sensitises cells to MG (Fig. 2C). An increase in GlxII, in the context of a low but constant level of GlxI, depletes the SLG pool (Table S1) resulting in limited activation of KefGB and hence only moderate protection. Finally, the combination of mutations that eliminate KefGB, KefFC and GlxII renders cells as sensitive to MG as if they had a ∆gloA mutation (Fig. 6). The relationship between the magnitude of the SLG pool, KefGB activity and pH i is complex, but can be understood from the properties of the systems and the cell itself. The buffering capacity of the cytoplasm is at its lowest value around cytoplasmic pH values in the range pH 7 -8 (Booth, 1985). Thus the cell is simultaneously at its most sensitive to perturbation of the environment due to limited buffering capacity, but also has the greatest potential to modulate cytoplasmic pH through changes in the balance of proton entry and efflux pathways. Previous work established that K + efflux from E. coli is compensated by the entry of both H + and Na + in a ratio of approximately 1K + ~ 0.4H + and 0.6Na + (Bakker & Mangerich, 1982). The extremely rapid acidification of the cytoplasm upon activation of KefGB can be explained by initial rapid K + -linked H + movements that are subsequently compensated by the reversibility of the Na + /H + antiports (Padan et al., 2001) leading to exchange of cytoplasmic H + for external Na + . Thus, the pH would be stabilised at a value set by the intrinsic properties of the KefGB system and the Na + /H + antiports (NhaB and NhaA) (Dover & Padan, 2001, Padan et al., 2001). Our recent work (Roosild et al., 2010) shows that a single binding site on KefGB is shared by GSH and glutathione adducts. Activation of KefGB by MG requires displacement of GSH and binding of SLG. The cytoplasmic pool of GSH is suggested to be ~20 mM (Bennett et al., 2009, Fahey, 2001[START_REF] Mclaggan | Glutathionedependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process[END_REF]. We observed that activation of KefGB in a ∆gloB strain requires only low concentrations of SLG, even in the presence of a six-fold excess of GSH (Fig.

4A, 5A). Even in the parental strain, SLG is always at a lower concentration than GSH and consequently, the activation of KefGB is not maximal at the normal levels of abundance of GlxI and GlxII. Thus, the change in cytoplasmic pH is constrained by the relative abundance of GlxI and GlxII activities and the concentration of MG.

The data presented here point to an important relationship between the intrinsic activity of GlxI, GlxII and KefGB. The effects of GlxI over-expression indicate that although this enzyme is, on a population-basis, limiting for the rate of detoxification. A 30-fold increase in GlxI activity produces only a 2-fold enhancement of the detoxification rate (MacLean et al.,1998) (see also Fig. S3B).

From this we infer that GlxI and GlxII activities in cells are similar, such that large increases in GlxI cannot be manifested as increased detoxification rates due to the limitation that is imposed by GlxII activity. Given that most detoxification enzymes are expressed at low levels in the cell there is considerable scope for cell-to-cell variation in enzyme level. GlxI and GlxII are encoded by separate unlinked genes and there is no coordination of their expression. Consequently, in each cell their abundance should vary independently. We have calculated the abundance (in cells grown to mid-exponential phase in minimal medium) to be ~130 GlxI and ~1500 GlxII molecules per cell (see Supplementary information). Stochastic variation of protein abundance will have a greater impact on GlxI and thus some cells will have a balance that favours SLG accumulation (high GlxI: GlxI > GlxII)

whereas others (low GlxI: GlxII > GlxI) will have lower cytoplasmic pools of this adduct. Thus, on an individual cell basis, some cells will experience greater protection than others. The independence of the expression of KefGB from the detoxification enzymes creates a further dimension of variability that is superimposed on the modulation of SLG pools. This means that some cells may gain further protection; the optimal solution would be simultaneous enhanced levels of GlxI and KefGB, coupled with low activity of GlxII. In this way the cell will experience maximum protection coupled with detoxification.

In this study we have defined the relationship between sensitivity to MG and the relative activities of the major detoxification pathway, GlxI-II, and the KefGB K + efflux system. The study demonstrates the benefits to the cell of linking an intermediate in detoxification to the activation of the protective K + efflux system.

Small changes in detoxification enzymes determine the maximum activity of KefGB. It is perhaps counterintuitive that cells possess protective capacity that is not always fully utilised. At low cell density detoxification capacity is secondary to ion channel activity in determining single cell fate. Conversely at high cell densities detoxification capacity is the primary determinant of cell survival.

Stochastic distribution of the proteins between cells in a population ensures that a few cells are highly protected and survive exposure to MG. In addition, it seems plausible that avoiding maximum stimulation of KefGB in all cells prevents excessive acidification that might be itself detrimental. The paradox is that in wild type populations the majority (99.9%) of cells are terminally-damaged (i.e. unable to form colonies after dilution and plating on fresh medium) but continue to

Experimental procedures

Strains and plasmids

All experiments were performed with E. coli K-12 derivative strains and are listed in Table 2. See further below for details of strains and plasmids created in this study.

Growth media

For physiological assays cells were grown either in K 0.2 minimal medium containing ~0.2 mM K + or K 115 minimal medium containing ~115 mM K + (Epstein & Kim, 1971) depending on the experimental design. Both media were supplemented with 0.2% (w/v) glucose, 0.0001% (w/v) thiamine, 0.4 mM MgSO 4 and 6 µM (NH 4 ) 2 SO 4 •FeSO 4 . LK complex medium (Rowland et al., 1985) was used for cell growth for DNA manipulations. In the case of strains carrying an antibiotic resistance marker, no antibiotics were used in physiological assays since the routinely used resistance determinants were stable and were not lost in the absence of antibiotics. Solid media contained 14 g/L agar. To prepare solid K 0.2 medium the agar was washed with a 1 M NaCl solution to displace trace amounts of K + and then washed several times with distilled water before use.

Cloning of the gloB gene

The gloB gene was PCR-amplified using whole cells of MJF274 as a template.

Primers GloBI (5'-CAACCAGCGTCGACTGTAC) and GloBII (5'-GGTATCACCCAGTGGATCC) were designed 1.1 kb downstream and 1kb

upstream of gloB respectively. The primers contained HincII and BamHI restriction sites, respectively (underlined). The amplified 2.8 kb product was digested with HincII and BamHI restriction enzymes. The cloning vector pHG165 was digested with HindIII, followed by treatment with Klenow enzyme to fill the HindIII site. Subsequently, the vector was digested with BamHI. The PCR product was ligated into the vector to create plasmid pGlxII from which the gloB gene is expressed from its native promoter.

Construction of gloB null mutants

The gloB gene was disrupted with a chloramphenicol resistance cassette (Cm R ) by homologous recombination mediated by λ-phage functions (recombineering). Michelle Brewster, SAIC, Frederick, USA) using the recombineering primers above. Recombineering was performed using strain DY330 and essentially as described by Yu et al., (Yu et al., 2000). Putative gloB integrants showed normal colony morphology and were purified twice on LK solid media containing 10 µg/ml chloramphenicol to check for the stability of the resistance marker. Recombination at the correct locus was investigated by PCR analysis using a primer pair flanking the gloB gene (gloB KO check F: 5'-CGCTTCGCAATTGATTTC; gloB KO check R: 5'-GGCGAGTAATATCGCTTT). The mutation was further confirmed by DNA sequencing of the PCR product across the borders of the site of recombination using primers gloB KO check F & R and Cm Seq 1

Recombineering primers gloB KO

(5'-CTTCATTATGGTGAAAGTTGG) and Cm Seq 3

(5'-GATAATAAGCGGATGAATGG) located within the Cm R cassette thus verifying successful creation of strain DY330 gloB<>cm (<> indicates replacement by recombineering approach (Yu et al., 2000)). The mutation was transduced into strain MJF274 by P1 transduction creating strain MJF595. An overnight culture of strain DY330 gloB<>cm was grown in LK medium at 32°C (shaking at 250 rpm).

The culture was diluted 100-fold into fresh LK medium also containing 0.2% [START_REF] Elmore | Activation of potassium efflux from Escherichia coli by glutathione metabolites[END_REF], Ferguson et al., 1993). Briefly, cells were grown to late logarithmic phase (OD 650nm ~0.8) in K 115 medium, collected by filtration onto a cellulose acetate membrane (0.45 µm), washed with K 0 buffer containing ~5 mM (K 5 buffer) and suspended in K 0 buffer. Cells were rapidly transferred into thermally insulated glass pots and kept at 37˚ C under continuous stirring. Samples (1 ml) were taken at various time points, cells pelleted in a microcentrifuge at 14000 rpm for 30 s, the supernatant quickly aspirated and the K + content of the cells determined by flame photometry after lysis by boiling in distilled water. MG (Sigma, M0252) was added from stock solutions to the test suspension 2 min after suspending cells in K 0 buffer. The first order rate constants for K + efflux (k) were calculated by transforming the values of K + levels to the natural logarithm and by determining the slope of the decline in the linear range after addition of MG. For illustration purposes the K + levels were normalised to the value at t 0 min , defined as 100%.

Cell viability and MG detoxification assays

Overnight cultures were grown in K 0.2 medium and diluted into fresh, pre-warmed K 0.2 medium to an OD 650nm of ~0.05. Cells were grown to mid-exponential phase (OD 650nm ~0.4) and diluted 10-fold into pre-warmed K 0.2 medium also containing MG. Samples were taken at various time points and cell viability and MG detoxification assays performed as described previously (Ferguson et al., 1993, Totemeyer et al., 1996). Viable cells were recovered on solid K 0.2 media plates.

Preparation of cytoplasmic cell extracts and GlxII enzyme assays

Cells were grown in K 0.2 medium to mid-exponential phase (OD 650nm ~0.4) exactly as for the assays above, however, they were harvested at this point by centrifugation at 4300 x g. Cells were washed, suspended in 50 mM potassium phosphate buffer (pH 6.6) and disrupted by two passages through a French press at 18000 Psi. Bulk cell debris and membrane fractions were removed by Cytoplasmic cell extracts were stored at -20°C until enzyme assays were performed. Protein quantification was performed using the Lowry assay (Lowry et al., 1951). GlxII activities were measured as a modification of a previously described method (Racker, 1951, Oray & Norton, 1982), in which SLG hydrolysis is measured by the decrease in A 240nm . Enzyme assays were performed in 50 mM potassium phosphate buffer (pH 6.6) at 37°C using a Shimadzu UV 2101PC

spectrophotometer. The reaction mixture contained 1 mM SLG (Sigma, L7140) in a total volume of 0.4 ml, using a 0.1 cm path length spectrophotometer cuvette.

Enzyme activity was expressed as units per cytoplasmic cell protein (U•mg -1 ) using a molar extinction coefficient of 3060 M -1 cm -1 (Racker, 1951), where 1 unit is defined as the amount of enzyme catalysing the formation of 1 µmol • min -1 SLG.

Enzyme activities were determined using two different enzyme concentrations to ensure that the enzyme was rate-limiting.

Determination of intracellular GSH and SLG levels by LC-MS/MS

The choice of the experimental design to extract the metabolites from the cells was guided by the need to correlate SLG levels directly with K + efflux. Therefore, cells were grown and treated as in K + efflux experiments as described above except that cells were grown in K 0.2 medium. After suspension of cells in K 0 buffer 1 ml samples were taken at various time points before and after exposure to MG and transferred into previously prepared microcentrifuge tubes. Microcentrifuge tubes contained 40 µl 2.5 M formic acid (Sigma, 251364) with 50 µM Glu-Glu (Sigma, G3640, as an internal standard for LC-MS/MS analysis), overlayed with 500 µl silicone oil mixture prepared from silicone oils of different densities (AR20: Fluka, 10836; AP100: Fluka, 10838; proportion of 3:2). The sample tubes were centrifuged at 14000 rpm for 30 s. Cells that passed through the silicone into the formic acid were permeabilised and thus all cell reactions ceased immediately.

Medium and silicone oil were removed by vacuum aspiration and the formic acid, with the cell debris, was transferred into fresh microcentrifuge tubes. The samples were centrifuged at 14000 rpm for 15 min at 4° C, the supernatants removed to separate tubes and stored at -20°C. Subsequently, samples were analysed by LC-MS/MS using quantification based on standard curves for GSH (Sigma,

Measurement of intracellular pH

The magnitude of the pH gradient was estimated from the distribution of a weak acid, and pH i (internal pH) calculated from knowledge of pH o (external pH). Cells were grown in K 0.2 minimal medium until they reached an OD 650nm ~0.8. The cell suspension was transferred into two thermally insulated glass pots (37°C) and 14 C benzoic acid (4.5 µM final concentration; specific activity 0.1 µCi•ml -1 ) and 3 H-water (specific activity 1 µCi•ml -1 ), as marker of extracellular water, were added to the cultures (Kroll & Booth, 1981). After 5 min incubation, 1 ml samples were taken at timed intervals and the cells and supernatant separated by centrifugation (14000 rpm for 20 s). For each sample, 100 µl of the supernatant was transferred to a scintillation vial containing 200 µl of cell suspension that had not been treated

with radioactivity; the remaining supernatant was aspirated from the cell pellet and discarded. Cell pellets were suspended in 200 µl K 0.2 buffer containing 0.2% glucose and the suspension transferred into a scintillation vial containing 100 µl of the same buffer. Samples of supernatant and pellet were counted for radioactivity on a preset 3 H/ 14 C program of a Tri-Carb 2100 TR liquid scintillation analyzer. The pH gradient and subsequently the pH i were calculated as described previously (Booth et al., 1979). Table 1. GlxII activity.

Figure legends

Enzyme activities were performed on three independent cytoplasmic cell extracts from each strain. For each extract the GlxII activity was measured using two different protein concentrations to ensure that the enzyme was rate-limiting.

Activities increased proportionally with protein concentration and were averaged.

The mean activity and standard deviation from independent extracts are shown.

Strains: MJF274 (parent strain), MJF595 (∆gloB), MJF274 pGlxII, MJF274 pGlxI (pMJM1). C. 1 st order rate constants (k) for K + efflux over a range of MG concentrations. K + efflux from the parent ( ) and ∆gloB ( ) were measured using different MG concentrations (0.025 to 3 mM). Rate constants were determined over a period of 3 min after the addition of MG (t 2 to t 5 ; see also Experimental Growth of the gloB null mutant (A) and strain over-expressing gloB (pGlxII, B) are not affected. Cells from the parent and the ∆gloB mutant or over-expressing strains were grown overnight in K 0.2 minimal media, diluted into fresh media and cultured to OD 650nm of ~0.4. Cells were then diluted 10-fold into fresh media in the absence (controls) or presence of 0.7 mM MG. The data are representative of three independent replicates.

Table S1. SLG and GSH pools.

The SLG (A) and GSH (B) pools were measured for the parent strain, ∆gloB, pGlxI and pGlxII at 0, 10, 150 and 300 s intervals following treatment with a range of MG

  . 3C; 0.444 ± 0.015 μM MG • min -1 and 1.155 ± 0.21 μM MG • min -1 ,

  GlxII and lacking either YeiG (MJF595 ΔyeiG) or FrmB (MJF595 ΔfrmB) were created (Experimental procedures) and cell viability determined during exposure to 0.7 mM MG. The level of survival of both double mutants (ΔgloB-ΔyeiG or ΔgloB-ΔfrmB) was indistinguishable from the gloB null mutant in experiments (Fig.S1), indicating that these systems do not have a physiologically significant role in detoxification of MG.

  ≥ 200 µM MG. Higher rates of K + efflux in the mutant were probably

  that SLG is the activator molecule for KefGB

  Finally, the combination of mutations that eliminate KefGB, KefFC and GlxII renders cells as sensitive to MG as if they had a ΔgloA mutation.

  rate, indicating that the two activities are in a similar range.

  Cell debris was removed by centrifugation at 4300 x g for 15 min and the supernatant passed through a filter (Whatman PURADISC TM filter, 0.25 µm) into a sterile tube containing 20 µl chloroform. Lysates were either used immediately to infect a recipient strain (see below) or kept at 4°C for long-term storage. To create strain MJF595 (gloB<>cm) an overnight culture of MJF274 was grown in LK medium at 37°C (shaking at 250 rpm). 200 µl aliquots were transferred into microcentrifuge tubes and cells harvested at 12000 rpm for 30 s. Cells were suspended in 100 µl P1 salts solution (100 mM MgCl 2 and 5 mM CaCl 2 ) using a Gilson pipette and a range of volumes of P1 donor lysate (see above) were added (5 -50 µl). Tubes were incubated at 37˚ C for 30 min. Subsequently cells were centrifuged at 12000 rpm for 30 s and suspended in 0.5 ml LK containing 20 mM sodium citrate (LK-NaCitrate). Cells were pelleted again as above and suspended in 1 ml LK-NaCitrate and incubated at 37°C for 1 h. Cells were harvested as described above and suspended in 100 µl LK-NaCitrate and plated onto selective LK-NaCitrate plates containing chloramphenicol (10 µg/ml). The plates were incubated overnight at 37˚C and the obtained transductants were purified twice in succession on selective LK-NaCitrate plates. The gloB<>cm mutation was also transduced into strain MJF276 by P1 transduction to create strain MJF596 (ΔkefB, ΔkefC, ΔgloB<>cm ). Strains MJF625 and MJF626 were made by transducing MJF595 with P1 donor lysates prepared from strains JW2141 and JW0346 respectively thus creating strains that lack known enzymes with minor S-lactoylglutathione hydrolase activity in addition to GlxII. Interestingly, we observed some differences in the transfer of the gloB null mutation into different E. coli strains by P1 transduction. The gloB<>cm mutation, initially created in strain DY330, could be transduced into strain MJF274, as described above, and transductants were obtained after overnight incubation (~16 h). In contrast, upon transfer of the mutation into strain MG1655, evident after prolonged incubation (~40 h) despite similar growth rates of both parent strains. Notably, upon purification, the MG1655

  µl 2.5 M formic acid (Sigma, 251364) with 50 µM Glu-Glu (Sigma, G3640, as an internal standard for LC-MS/MS analysis), overlayed with 500 µl silicone oil mixture prepared from silicone oils of different densities (AR20: Fluka, 10836; AP100: Fluka, 10838; proportion of 3:2). The sample tubes were centrifuged at 14000 rpm for 30 s. Cells that passed through the silicone into the formic acid were permeabilised and thus all cell reactions ceased immediately.Medium and silicone oil were removed by vacuum aspiration and the formic acid, with the cell debris, was transferred into fresh microcentrifuge tubes. The samples were centrifuged at 14000 rpm for 15 min at 4° C, the supernatants removed to separate tubes and stored at -20°C. Subsequently, samples were analysed by LC-MS/MS using quantification based on standard curves for GSH (Sigma, G6529) and SLG (Sigma, L7140) prepared on the same day as the cellular samples by adding appropriate volumes from frozen stocks to the formic acid/Glu-Glu extraction solution. The LC-MS/MS was performed using a Thermo Surveyor-TSQ Quantum system with electrospray ionisation (ESI) in the positive ion mode.A Stability BSC 17 (5 µ) column (150 mm x 2 mm) was used and the analytes eluted with a mobile phase comprising, 50% 7.5 mM ammonium formate, pH 2.6 (formic acid) and 50% acetonitrile at a flow rate of 0.2 ml • min -1 . The column was maintained at 45°C. ESI conditions were as follows: spray voltage 4 kV, sheath gas pressure 60, auxiliary gas 0 and capillary temperature 375° C. Detection was carried out in SRM mode at a collision pressure of 1.4 and a collision energy of 13V using the following SRM transitions: GSH m/z 308 -m/z 179, SLG m/z 380 -m/z 233 and Glu-Glu (internal standard) m/z 277 -241. Quantification was performed using Xcalibur software. All samples and standards were diluted 1:100 with water prior to injection (1 µl) and were maintained at 4°C in the autosampler.It was not possible to resolve SLG and GSH chromatographically, however, parent masses and fragment masses were suitably different to allow discrimination by the subsequent MS. Control experiments with a gloA null mutant verified that the LC-MS/MS assay did not detect the isomer of SLG, namely HTA. HTA is the product of the chemical equilibrium between MG and GSH (Fig.1). The equilibrium of this reaction is in favour of HTA which is then converted to SLG by the action of the GlxI enzyme. Note that, in this assay, measured GSH levels do not necessarily reflect in vivo GSH levels during the MG detoxification process because of the chemical equilibrium with HTA. Measured GSH levels will be a of actual GSH levels and GSH that was conjugated as HTA at the time of cytoplasmic extraction. The HTA molecule is unstable upon disturbance of the equilibrium with GSH + MG, i.e. upon dilution of the cytoplasmic cell volume in formic acid the equilibrium is driven back to GSH and MG. Furthermore, metabolite concentrations presented in this study are the levels as quantified in the extraction volume (40 μl formic acid). An approximation of intracellular concentrations can be derived from the relationship between OD 650nm and cytoplasmic volume (1 ml cell culture at OD 650nm 2 = ~1.6 μl cytoplasmic volume;

Fig. 1 .

 1 Figure legends

Fig. 2 .

 2 Fig. 2. Over-expression of GlxII sensitises E. coli to MG.A. Over-expression of GlxII does not affect growth. Cells of the parent strain MJF274 (circles) and MJF274 bearing plasmid pGlxII (diamonds) were grown overnight in K 0.2 minimal media, diluted into fresh media and cultured to OD 650nm

Fig. 3 .

 3 Fig. 3. Mutant strain lacking GlxII exhibits a reduced capacity to detoxify MG, but is not more sensitive to the electrophile. A. Genomic context of the gloB gene in E. coli. The gloB gene (756 bp) encodes for the GlxII enzyme (EC 3.1.2.6, hydroxyacylglutathione hydrolase). The flanking genes mltD and yafS are transcribed divergently from gloB. Arrows indicate gene boundaries and transcriptional orientation. Genome coordinates are shown above the arrows. Bar and dashed lines indicate the deleted genomic region in strain MJF595 (ΔgloB). Arrows on the scale of the genome coordinates indicate promoter elements (-35 elements) for mltD and yafS as predicted by BPROM (see Supplementary information).B. Growth of gloB null mutant is not affected. Cells of strain MJF274 (circles) and the mutant strain MJF595 (ΔgloB, diamonds) were grown overnight in K 0.2 minimal media, diluted into fresh media and cultured to OD 650nm

Fig. 4 .Fig. 6 .

 46 Fig. 4. K + efflux systems are hyperactive in a gloB null mutant.A and B. K + efflux from the parent strain MJF274 (A) and MJF595 (ΔgloB; B) upon exposure to different MG concentrations. Cells were grown to an OD 650nm of ~0.8 in K + -rich minimal medium (K 115 ), harvested and suspended in K + -free buffer. K + efflux was measured in the absence (control; ▲) and in the presence of 0.7 mM () and 3 mM MG (). MG was added 2 min (indicated by arrow) after resuspension of cells in K + -free buffer. Control data were averaged for illustration.Data shown are representative of three independent replicates. At time zero MJF274 contained ~494 ± 8 µmol K + and MJF595 contained 485 ± 18 µmol K +

Fig. 7 .

 7 Fig. 7. Complex relationships exist between SLG levels, activation of K + efflux systems and modulation of pH iA. 1st order rate constants for K . + efflux (k) in strain MJF595 (ΔgloB) (derived from fitting in Fig.4C) were plotted against mean SLG levels at t 10 s after addition of selected MG concentrations. The data point in gray illustrates the lack of K +

Fig. 8 .

 8 Fig. 8. Survival of gloB null mutant upon MG stress depends on the activity of K + efflux systems.Cells that lack the K + efflux systems KefGB and KefFC, in addition to GlxII, are highly sensitive to MG exposure. Cells of strains MJF274 (), MJF595 (ΔgloB; ), MJF276 (kefB, kefC::Tn10; ) and MJF596 (ΔgloB, kefB, kefC::Tn10; ) were grown in K 0.2 minimal media, exposed to 0.7 mM MG and viable cells enumerated exactly as for experiments presented in Fig.3. The mean and standard deviation of three independent experiments are shown.

Fig. S1 .

 S1 Fig. S1. Survival of gloB null mutant upon MG stress is not dependent on enzymes with minor SLG hydrolase activity.Cells that lack either YeiG or FrmB in addition to GlxII are not more sensitive to MG stress than the single mutant lacking GlxII. Cells of strains MJF274 (), MJF595 (ΔgloB; ), MJF625 (ΔgloB, ΔyeiG;,) and MJF626 (ΔgloB, ΔfrmB; ) were grown in K 0.2 minimal media, exposed to 0.7 mM MG and viable cells

  Fig.

  had not been developed. Thus the model rested on assumptions and essential elements were untested. Here, we report construction and characterisation of GlxII mutants (∆gloB). We integrate measurements of the SLG pools with assays of other parameters involved in MG detoxification and cell survival. In addition to supporting the original model, this comprehensive dataset demonstrate unequivocally the importance for survival of activation of KefGB and the consequent lowering of cytoplasmic pH. In particular we demonstrate that increased activity of the KefGB system can compensate for an impaired capacity to detoxify MG. The data are discussed in terms of the balance between GlxI, GlxII and the K + efflux systems in determining the fate of individual cells.

  removal. Thus, the survivors are aided by the dead and dying cells.

  homologous sequences to the 3' and 5' of the gloB gene, respectively. The incorporation of the Cm R was predicted to create a fused ORF with the remaining 5' gloB sequence. Therefore, a double stop codon was incorporated into primer gloB KO-B (underlined) to prevent possible expression of a fusion protein driven by a gloB promoter. A 454 bp fragment (from 132 to 585) of the structural gene, where 1 is the first bp of the start codon, was replaced by the Cm R cassette. Hence a short sequence of the GlxII protein may be expressed. The Cm R cassette was amplified by colony-PCR from a strain carrying the resistance marker (MG1655 zwf<cm>; kindly provided by

  x g and subsequent centrifugation at 110000 x g.

  SLG (Sigma, L7140) prepared on the same day as the cellular samples by adding appropriate volumes from frozen stocks to the formic acid/Glu-Glu extraction solution. The LC-MS/MS was performed using a Thermo Surveyor-TSQ Quantum system with electrospray ionisation (ESI) in the positive ion mode.A Stability BSC 17 (5 µ) column (150 mm x 2 mm) was used and the analytes eluted with a mobile phase comprising, 50% 7.5 mM ammonium formate, pH 2.6 (formic acid) and 50% acetonitrile at a flow rate of 0.2 ml • min -1 . The column was maintained at 45°C. ESI conditions were as follows: spray voltage 4 kV, sheath gas pressure 60, auxiliary gas 0 and capillary temperature 375° C. Detection was carried out in SRM mode at a collision pressure of 1.4 and a collision energy of 13V using the following SRM transitions: GSH m/z 308 -m/z 179, SLG m/z 380 -m/z 233 and Glu-Glu (internal standard) m/z 277 -241. Quantification was performed using Xcalibur software. All samples and standards were diluted 1:100 with water prior to injection (1 µl) and were maintained at 4°C in the autosampler.It was not possible to resolve SLG and GSH chromatographically, however, parent masses and fragment masses were suitably different to allow discrimination by the subsequent MS. Control experiments with a gloA null mutant verified that the LC-MS/MS assay did not detect the isomer of SLG, namely HTA. HTA is the product of the chemical equilibrium between MG and GSH (Fig.1). The equilibrium of this reaction is in favour of HTA which is then converted to SLG by the action of the GlxI enzyme. Note that, in this assay, measured GSH levels do not necessarily reflect in vivo GSH levels during the MG detoxification process because of the chemical equilibrium with HTA. Measured GSH levels will be a composite of actual GSH levels and GSH that was conjugated as HTA at the time of cytoplasmic extraction. The HTA molecule is unstable upon disturbance of the equilibrium with GSH + MG, i.e. upon dilution of the cytoplasmic cell volume in formic acid the equilibrium is driven back to GSH and MG. Furthermore, metabolite concentrations presented in this study are the levels as quantified in the extraction volume (40 µl formic acid). An approximation of intracellular concentrations can be derived from the relationship between OD 650nm and cytoplasmic volume (1 ml cell culture at OD 650nm 2 = ~1.6 µl cytoplasmic volume;(Kroll & Booth, 1981)). Thus, the total cytoplasmic volume in the assay is ~0.64 µl (8x10 8 cells), and a concentration of 100 µM in the extraction volume equates to an intracellular concentration of ~6.35 mM.

Fig. 1 .

 1 Fig. 1. Detoxification of MG in enteric bacteria. Schematic representation of the major pathways of MG synthesis and detoxification, including the link to KefGB. Abbreviations: MG, methylglyoxal;

Fig. 2 .

 2 Fig. 2. Mutant strain lacking GlxII exhibits a reduced capacity to detoxify MG, but is not more sensitive to the electrophile. A. Genomic context of the gloB gene in E. coli. The gloB gene (756 bp) encodes for the GlxII enzyme (EC 3.1.2.6, hydroxyacylglutathione hydrolase). The flanking genes mltD and yafS are transcribed divergently from gloB. Arrows indicate gene boundaries and transcriptional orientation. Genome coordinates are shown above the arrows. Bar and dashed lines indicate the deleted genomic region in the ∆gloB strain (MJF595). Arrows on the scale of the genome coordinates indicate promoter elements (-35 elements) for mltD and yafS as predicted by BPROM (see Supplementary information). B. The gloB null mutant has impaired MG detoxification. Rate of MG detoxification does not change when GlxII is over-expressed. Cells from the parent (MJF274, ), ∆gloB (MJF595, ) and pGlxII ( ) were grown to OD 650nm of ~0.4 in K 0.2 minimal media and diluted 10-fold into fresh media containing 0.7 mM MG. At intervals the medium was assayed for the disappearance of MG. The data are representative of three independent replicates. C. The gloB null mutant exhibits similar death kinetics to the parent strain upon MG stress. Cells over-expressing GlxII are more sensitive to MG. Cells from the parent (MJF274, ), ∆gloB (MJF595, ) and pGlxII ( ) were grown exactly as in B and diluted into media containing 0.7 mM MG. Cell samples were taken at

Fig. 3 .

 3 Fig. 3. K + efflux systems are hyperactive in a gloB null mutant. A and B. K + efflux from the parent (MJF274, A) and ∆gloB (MJF595, B) upon exposure to different MG concentrations. Cells were grown to an OD 650nm of ~0.8 in K + -rich minimal medium (K 115 ), harvested and suspended in K + -free buffer. K + efflux was measured in the absence (control; , ) and in the presence of 0.7 mM (, ) and 3 mM MG ( , ). MG was added 2 min (indicated by arrow) after resuspension of cells in K + -free buffer. Control data were averaged for illustration. Data shown are representative of three independent replicates. At time zero MJF274 contained ~494 ± 8 µmol K + and MJF595 contained 485 ± 18 µmol K + per g dry cell weight.

  procedures) and multiplied by -1 for illustration purposes. Data represent the mean of three independent replicates (standard deviations are shown). Data sets for both strains were fitted using an exponential association function in the Origin 8.0 software [Equation: y = y0 + A1*(1 -exp(-x/t1)) + A2*(1 -exp(-x/t2))] and the output for each data set is shown as a dashed line. D. 1 st order rate constants (k) for K + efflux of the parent, ∆gloB and pGlxII when treated with 0.7 mM MG. Rate constants were determined as for 3C. * Not significantly different to the rate of spontaneous K + loss from cells untreated with MG.

Fig. 4 .

 4 Fig. 4. SLG accumulates rapidly to high levels in a gloB null mutant.A. Changes in GSH and SLG levels upon MG exposure were quantified in both the parent strain and the gloB null mutant. Cells were grown in K 0.2 minimal medium to an OD 650nm of ~0.8, handled as in K + efflux assays and cells sampled at various time points. MG (0.2 mM) was added immediately after suspending cells in K 0 buffer (t 0 s ). GSH (open symbols) and SLG levels (closed symbols) from the parent (, ) and ∆gloB (MJF595, , ) were quantified by LC-MS/MS. Data

Fig. 5 .Fig. 6 .

 56 Fig. 5. Complex relationships exist between SLG levels, activation of K + efflux systems and modulation of pH i .A. 1st order rate constants for K + efflux (k) from the ∆gloB mutant (derived from fitting in Fig.3C), and the parent were plotted against mean SLG levels at t 10 s after addition of selected MG concentrations. The data point in gray illustrates the lack of K + efflux in the absence of SLG. B. The change in intracellular pH (∆pH i ) in the parent ( ) and ∆gloB ( ) as a function of the concentration of exogenously applied MG. The ∆pH i was calculated as (t 10-14 min ) -(t 15-18 min ) where MG was added at t 15 min for each MG concentration. The data shown are means ± s.e.m. Data sets for both strains were fitted using an exponential association function in the Origin 8.0 software [Equation: y = A1* exp(-x/t1)+y0] and the output for each data set is shown as a dashed line. The adjusted R 2 values for the parent and ∆gloB were 0.76 and 0.96, respectively.C. 1 st order rate constants (k) for survival of the parent ( ), ∆gloB ( ), pGlxII ( ) and ∆gloB; ∆kefC; ∆kefB (MJF596, ) upon MG exposure were derived from viable cell counts over the first 60 min after addition of 0.7 mM MG. These data were plotted against the ∆pH i calculated as described in 5B. Data plotted are means ± s.e.m. Data points were fitted using an exponential association function in the Origin 8.0 software [Equation: y = A1* exp(-x/t1)+y0]. The output is shown as a dashed line and the adjusted R 2 = 0.89. * As a consequence of the method used to determine pH i and subsequently derive ∆pH i , the absence of a drop in pH i upon addition of MG, as is the case for MJF596, can lead to a negative value for

Fig. S1 .

 S1 Fig. S1. Growth of strains deleted for gloB and over-expressing gloB are similar to the parent strain.

  concentrations. The mean and standard deviations are shown. The values given for SLG and GSH concentrations are the final concentrations after extraction from ~ 0.8 x10 9 cells in 40 µl formic acid. An estimation of intracellular SLG and GSH concentrations can be derived from the knowledge of the relationship between OD and cytoplasmic volume determined as 1ml cells at OD 650 = 2 has a cytoplasmic volume of 1.6 µl (Ahmed and Booth 1981).

Fig. S2 .

 S2 Fig. S2. Survival of gloB null mutant upon MG stress is not dependent on enzymes with minor SLG hydrolase activity.

Table 1 . GlxII activity.

 1

	Schematic representation of the major pathways of MG synthesis and
	detoxification, including the link to KefGB. Abbreviations: MG, methylglyoxal;
	GSH, glutathione; HTA, hemithioacetal; SLG, S-lactoylGSH; D-Lac, D-Lactate;
	GlxI, glyoxalase I; GlxII, glyoxalase II; GlxIII, glyoxalase III; MGS, methylglyoxal
	synthase; DHAP, dihydroxyacetone phosphate; P i , inorganic phosphate.

Table 5 )

 5 . The -35 element of the first region was located 68 bp (end of -35 box) upstream from the 31 start codon and thereby also within the gloB gene. A second promoter was predicted further away (~450 bp from start codon), however, this was not considered in our strategy to inactivate gloB since this may have resulted in the expression of considerable GlxII fragment.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 3 . Computational σ 70 promoter predictions for mltD as stated on RegulonDB.

 3

	Promoter	Position	Box -35	Spacer	Box -10	Score
	name	+1		between -10
				& -35 box
	mltDp1	234025 GTTTTGCAT 17	GATAGGTT 5.07
	mltDp2	234041 AACCTGAAG 13	GTTAAGGT 7.33
	mltDp3	234074 TAATTAATG	16	ATTATTGC	2.54
	mltDp4	234100 TTTTTTTAA	16	TTTAATTA	3.72

See the following web address for more details: http://regulondb.ccg.unam.mx/

Table 4 . σ 70 promoters for mltD as predicted by BPROM

 4

	1) Length of sequence:	500
	2) Threshold for promoters:	0.20
	3) Number of predicted promoters:	1
	4) Promoter Position:	354		LDF: 7.12
	-10 box at position	339	TGATTTAAT Score 44
	-35 box at position	321	TTTAAG	Score 35

Table 4 . σ 70 promoters for yafS as predicted by BPROM

 4

	1) Length of sequence:	500
	2) Threshold for promoters:	0.20
	3) Number of predicted promoters:	2
	4) Promoter Position:	144		LDF: 5.21
	-10 box at position	129	TCCTAAAGT Score
	-35 box at position	108	TTTACT	Score
	Promoter Position:	470		LDF: 3.82
	-10 box at position	455	TGTTAAGAT Score
	-35 box at position	433	TTGTCA	Score

Table 2 . List of strains and plasmids

 2

	Strain	Genotype	Source / Reference
	MJF274	F -, thi, rha, lacZ, kdpABC5, lacI, trkD1	(Ferguson et al., 1993)
	MJF276	MJF274, kefB, kefC::Tn10	(Ferguson et al., 1993)
	MJF595	MJF274 ∆gloB<>cm	This study
	MJF596	MJF276 ∆gloB<>cm	This study
	MJF625	MJF595 ∆yeiG::kan	This study
	MJF626	MJF595 ∆frmB::kan	This study
	DY330	W3110 ∆lacU169gal490 pgl∆8 λcI857 ∆(cro-bioA)	(Yu et al., 2000)
	JW2141	BW25113 ∆yeiG::kan	(Baba et al., 2006)
	JW0346	BW25113 ∆frmB::kan	(Baba et al., 2006)
	Plasmid	Description	Source / Reference
	pHG165	pBR322 copy number derivative of pUC8 (Stewart et al., 1986)
		A pHG165 derivative into which gloA was
		cloned and is transcribed from its own
	pMJM1	promoter.	(MacLean et al., 1998)
		A pHG165 derivative into which gloB was
		cloned and is transcribed from its own
	pGlxII	promoter.	This study

Molecular Microbiology

Acknowledgements

This work was funded by the Medical Research Council UK (EO), by The University of Aberdeen, by the Wellcome Trust (GR040174 & 086903) and by the BBSRC SysMO (BB/F003455/1).

Cells that lack either YeiG or FrmB in addition to GlxII are not more sensitive to MG stress than the single mutant lacking GlxII. Cells from the parent ( ), ∆gloB ( ), MJF625 (∆gloB, ∆yeiG; ) and MJF626 (∆gloB, ∆frmB; ) were grown in K 0.2 minimal media, exposed to 0.7 mM MG and viable cells enumerated exactly as for experiments presented in Fig. 2. The mean and standard deviation of three independent experiments are shown.

Fig. S3. High cell density survival and MG detoxification

A. The ∆gloB strain exhibits similar death kinetics to the parent strain upon exposure of high cell density cultures (OD 650nm ~0.8) to 0.8 mM MG.

B. MG detoxification ability of cells from the parent ( ), ∆gloB mutant ( ), pGlxI ( ) and pGlxII ( ) assayed at high cell density (OD 650nm ~0.8).

Fig. S4. The relationship between intracellular pH and MG

The intracellular pH was determined for the parent (A) and ∆gloB (B) cells treated with a range of MG concentrations (0 -0.8 mM) in K 0.2 . The intracellular pH of parent cells was measured over 15 min after addition of 0.8 mM MG (C). * MG was added at t 15 min thus the t 15 min time point has been added as a duplicate of t 14 min to illustrate the rapid kinetics of cytoplasm acidification. The data are mean ± s.e.m.