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Journal of Bacteriology, August 2008, p. 5567-5575, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00577-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Fur-Dependent Detoxification of Organic Acids by rpoS Mutants during Prolonged Incubation under Aerobic, Phosphate Starvation Conditions{triangledown}

Mélanie L. Guillemet and Patrice L. Moreau*

Laboratoire de Chimie Bactérienne, Centre National de la Recherche Scientifique, 13009 Marseille, France

Received 25 April 2008/ Accepted 9 June 2008


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ABSTRACT
 
The activity of amino acid-dependent acid resistance systems allows Escherichia coli to survive during prolonged incubation under phosphate (Pi) starvation conditions. We show in this work that rpoS-null mutants incubated in the absence of any amino acid survived during prolonged incubation under aerobic, Pi starvation conditions. Whereas rpoS+ cells incubated with glutamate excreted high levels of acetate, rpoS mutants grew on acetic acid. The characteristic metabolism of rpoS mutants required the activity of Fur (ferric uptake regulator) in order to decrease the synthesis of the small RNA RyhB that might otherwise inhibit the synthesis of iron-rich proteins. We propose that RpoS ({sigma}S) and the small RNA RyhB contribute to decrease the synthesis of iron-rich proteins required for the activity of the tricarboxylic acid (TCA) cycle, which redirects the metabolic flux toward the production of acetic acid at the onset of stationary phase in rpoS+ cells. In contrast, Fur activity, which represses ryhB, and the lack of RpoS activity allow a substantial activity of the TCA cycle to continue in stationary phase in rpoS mutants, which decreases the production of acetic acid and, eventually, allows growth on acetic acid and Pi excreted into the medium. These data may help explain the fact that a high frequency of E. coli rpoS mutants is found in nature.


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INTRODUCTION
 
In the proximal part of the human colon, Escherichia coli may enter stationary phase as a result of phosphate (Pi) starvation. In fact, nutrients, including Pi and glucose, are efficiently absorbed in the small intestine. However, E. coli can acquire high levels of mucus-derived sugars in the colon (7, 25). Therefore, the elucidation of the mechanisms used by E. coli to survive under conditions of Pi starvation and excess carbon (C) could contribute to our understanding of the complex metabolic interactions between intestinal microbiota and hosts (45).

At the onset of Pi starvation under aerobic conditions, E. coli accumulates the {sigma}S (RpoS) factor, which enhances transcription by the {sigma}S-containing RNA polymerase (E{sigma}S) of ≥481 genes, including pdhR and poxB (42). PdhR represses expression of the genes encoding the pyruvate dehydrogenase complex (PDH), and poxB encodes pyruvate oxidase (pyruvate:Q reductase). The rerouting of the metabolic flux from PDH (pyruvate + coenzyme A + NAD+ -> acetyl coenzyme A + CO2 + NADH + H+) to PoxB (pyruvate + H2O + FAD -> acetate + CO2 + FADH2) decreases the production of NADH and thus the adventitious production of superoxide and hydrogen peroxide (H2O2) by NADH dehydrogenases (NDHs) in the aerobic respiratory chain (24, 26). However, the excretion of acetic acid (CH3COOH; pKa 4.76) into the medium may eventually threaten cell viability, unless the H+-consuming activity of the glutamate decarboxylase GadB is expressed (25). The gadB gene, which can be transcribed by E{sigma}S, is induced concomitantly with poxB at the onset of Pi starvation (25). Together, these data are consistent with the idea that genes of the RpoS regulon play a critical role in the viability of Pi-starved cells because of the protection afforded against oxidative and acid damage. This is in good agreement with the fact that RpoS levels increase strongly at the onset of Pi starvation, notably as a result of the accumulation of IraP, an inhibitor of RpoS degradation that is induced in response to Pi starvation (5, 18).

However, rpoS mutants starved for Pi can survive during prolonged incubation provided that they are shifted to anaerobiosis at the onset of stationary phase and that lysine is available in the medium, which triggers the activity of the H+-consuming lysine decarboxylase system CadBA (25). This may occur because the induction of the cadBA operon is strictly dependent upon anaerobic conditions and is totally independent of expression of the RpoS regulon (25). Interestingly, the process used by rpoS mutants to survive under anaerobic, Pi starvation conditions may be relevant to the survival of E. coli in the human gut, because many natural populations of E. coli contain a high frequency of rpoS mutants (16, 25). These results prompted us to determine whether rpoS mutants could also survive during prolonged incubation under aerobic, Pi starvation conditions. In this study, we demonstrated that rpoS mutants incubated in the absence of any amino acid survived during prolonged incubation because they could grow on and thus detoxify acetic acid.


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MATERIALS AND METHODS
 
Bacterial strains. The Escherichia coli K-12 strains used are listed in Table 1. The {Delta}ryhB1::cat mutation (19) was transferred by P1 transduction from strain JB90, obtained from J. Bos (Laboratoire de Chimie Bactérienne). The ColVK30 iucC::lacZ Tcr plasmid (3) was transferred by mating from strain ENZ1927—strain MS100 (39) transduced to argE::kan (2)—into strain ENZ1734, giving rise to strain ENZ1929 (Arg+ Tcr). Transductions and matings were performed as described by Miller (23).


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  		TABLE 1. Bacterial strains

Media and culture conditions. The minimal medium used for liquid cultures was essentially the MOPS (morpholinepropanesulfonic acid) medium described by Neidhardt et al. (27), which contained 40 mM MOPS (pH 7.4), 4 mM Tricine plus 10 µM FeSO4, 86 mM NaCl, 9.5 mM NH4Cl, 5 mM K2HPO4, and 20 mM glucose (final pH ~7.2) (25). The Pi-limiting MOPS medium contained 0.1 mM K2HPO4 plus 9.8 mM KCl and 40 mM glucose. When sodium glutamate was added, the final concentration of Na+ in the medium was adjusted to 86 mM with NaCl. 2-2'-dipyridyl was from Aldrich. Cultures (50 ml in 500-ml Erlenmeyer flasks) were agitated at 150 rpm in a covered water bath rotary shaker. All incubations were performed at 37°C. Culture optical densities were determined spectrophotometrically at 600 nm (OD600). The pHs of the media were determined at ~25°C (25).

Measurements of cell viability. To assess cell viability, serial dilutions were prepared in M9 buffer, and aliquots (20 µl) were spotted at least in triplicate onto LB medium plates, which were incubated under aerobic conditions (12, 23, 26). Beef liver catalase (2,000 U; Sigma) was spread on the plates in order to scavenge H2O2 adventitiously produced in LB medium (25).

Levels of glucose and of organic acids. The concentrations of pyruvate (Roche), glucose, D- and L-lactate, glutamate, and acetate (Scil Diagnostics) were determined by enzymatic tests performed according to the instructions of the manufacturers (25).

Measurement of β-galactosidase activity. β-Galactosidase levels were determined as described previously (11, 12).

Bioassay for Pi in culture media. The culture supernatants were adjusted to pH 7 with 1 M KOH, filter sterilized, distributed into 16- by 100-mm glass test tubes (1-ml aliquots), and supplemented with 31 µl nutrients (final concentrations of 20 mM glucose, 20 mM NH4Cl, and 1 mM KCl or 0.5 mM K2HPO4) (12, 26). Tester strain ENZ535 was grown for 24 h in Pi-limiting medium, washed in MOPS medium without K2HPO4, diluted 10-fold into the same medium, and used to inoculate (10 µl) the supplemented spent culture media. The cultures were incubated for 18 h at 150 rpm, and the number of viable cells was determined on LB medium plates. A calibration curve was determined with known concentrations of K2HPO4.


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RESULTS
 
The rpoS mutant strain survives prolonged incubation. When the wild-type (wt) strain ENZ535 (= MG1655) was incubated under aerobic conditions in MOPS minimal glucose medium containing a limiting concentration of Pi, the number of CFU dropped from ~109 to <101/ml between days 6 and 14 of incubation (Fig. 1A) (25). However, when 30 mM glutamate was added to the medium, the viability of the wt strain was barely affected because of the protection afforded by the Gad acid resistance system (Fig. 1A) (25). When rpoS-null mutant strains (ENZ985 rpoS::Tn10 and ENZ1698 rpoS::kan) were incubated without any amino acid, the number of CFU did not decrease below ~107/ml during an incubation period of 20 days (Fig. 1B). Therefore, the lack of RpoS activity, which prevents the induction of many defense genes (42), may somehow protect E. coli during prolonged incubation under aerobic, Pi starvation conditions.


Figure 1
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  		FIG. 1. rpoS mutants grow during prolonged incubation. Strains were inoculated 1:500 (time zero) into Pi-limiting medium (0.1 mM Pi plus 40 mM glucose) without glutamate or with 30 mM glutamate (GLU) and incubated further under aerobic conditions. Sodium ampicillin (Ap) or NaCl was added to a final concentration of 80 µg/ml every 2 days from day 4 of incubation to the end of the experiment. The numbers of CFU were determined on LB medium plates containing catalase. Parentheses indicate that no CFU were detected when 20-µl portions of the cultures were spotted directly or after washing in M9 buffer. The values are the means ± standard deviations for n independent cultures representative of several determinations. (A) ENZ535 (= MG1655) (wt; {diamond}; n = 3), ENZ535 + GLU ({square}; n = 2), ENZ535 + GLU + Ap ({blacksquare}; n = 2), ENZ535 + NaCl ({circ}; n = 2), and ENZ535 + Ap (•; n = 3). (B) ENZ985 (rpoS::Tn10; {diamond}; n = 6), ENZ1698 (rpoS::kan; {triangledown}; n = 2), ENZ985 (rpoS) + NaCl ({circ}; n = 2), ENZ985 (rpoS) + Ap (•; n = 4), and ENZ1222 (poxB; {triangleup}; n = 3).

Cultures of the rpoS mutant strain do not contain acetic acid. The catabolism of glucose into acetic acid is the primary cause of death of rpoS+ cells incubated under aerobic, Pi starvation conditions (25). To determine whether the survival of the rpoS mutant strain might be related to acetic acid metabolism, the concentrations of glucose and of acetate and the pHs of the media were determined in cultures of the rpoS mutant strain and in cultures of the wt strain incubated without and with glutamate. In cultures of the wt strain, the addition of 30 mM glutamate (Table 2) increased the pH of the medium, the concentration of glucose consumed and the concentration of acetate excreted. These data support the idea that the survival of rpoS+ cells primarily results from proton consumption, which neutralizes acetic acid. In cultures of the rpoS mutant strain (Table 2), the pH of the medium was as high as 6.8 and glucose was catabolized in its totality, but no acetate was obtained.


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  		TABLE 2. Metabolic patternsa

rpoS mutants grow during prolonged incubation. The lack of acetic acid in cultures of the rpoS mutant strain might be accounted for by two different processes: (i) the rerouting of glucose metabolism toward products such as glycogen, which would divert the metabolic flux away from the production of acetic acid, and/or (ii) the consumption of acetic acid that was produced. Excess glucose may be converted into glycogen in nongrowing cells. However, the synthesis of glycogen depends upon the expression of the RpoS regulon (14). Therefore, it is unlikely that rpoS mutants could convert glucose into glycogen during prolonged incubation under aerobic, Pi starvation conditions. Alternatively, rpoS mutants could catabolize glucose into acetic acid and consume acetic acid. It is known that exponentially growing cells (incubated in rich LB medium or in minimal medium containing a limiting concentration of glucose) first excrete acetate and then grow on acetate; the acetate switch, which occurs at the approach of the stationary phase, allows growth to continue for a while (diauxic growth) when the preferred carbon source(s) is exhausted (33, 43). In this light, an intriguing possibility is that rpoS mutants could grow during prolonged incubation, thereby consuming toxic products such as acetic acid that might be produced through glucose metabolism.

To determine whether rpoS359-null mutant cells grew during prolonged incubation, ampicillin was added in the medium. Ampicillin kills cells that are dividing during prolonged incubation (41). When 80 µg/ml ampicillin was added every 2 days from day 4 of incubation, the viability of the wt strain, incubated without and with glutamate, was not significantly affected (Fig. 1A). In stark contrast, the viability of the rpoS mutant strain changed dramatically between days 6 and 14 of incubation whether ampicillin was added or not: the viable counts dropped ≥106-fold in the presence of ampicillin, whereas they increased ~10-fold in the absence of ampicillin (Fig. 1B). Therefore, it appears that most rpoS mutant cells divide after day 6 of incubation, whereas most wt cells, not protected or protected by the Gad acid resistance system, remain nondividing during prolonged incubation.

rpoS mutant cells detoxify organic acids. The presence of ampicillin did not change the metabolic pattern of the wt strain: ~30 mM glucose was catabolized primarily into ~30 mM acetate in cultures incubated for 14 days without and with ampicillin (Table 2). In stark contrast, the metabolic pattern of the rpoS mutant strain changed dramatically following the addition of ampicillin: whereas no organic acids were obtained in the absence of ampicillin (Table 2), significant levels of acetate (9.2 mM), pyruvate (3.9 mM), and D-lactate (1.8 mM) were obtained in 14-day-old cultures incubated with ampicillin (Table 2), which is reminiscent of the metabolic profile of poxB mutants (Table 2) (25).

Next we performed a time course experiment in the absence of ampicillin in order to determine whether cultures of rpoS mutants could fleetingly accumulate organic acids. In cultures of the wt strain, the levels of acetic acid steadily increased and reached a plateau (~30 mM acetate at pH 4.8) on day 8 of incubation (Fig. 2G and J), when cell viability dramatically decreased (Fig. 2A). In stark contrast, in cultures of the rpoS mutant strain, the levels of organic acids reached a peak on day 8 of incubation (~7 mM acetate, ~1.5 mM pyruvate, ~0.4 mM lactate, and <0.02 mM glutamate) and then decreased to 0 by day 12 of incubation (Fig. 2J and data not shown). Likewise the viability of the rpoS mutants and the pH of the medium were at their lowest levels on day 8 of incubation (3 x 107 CFU/ml at pH 6.2) and then increased again to reach their maximal levels on day 12 of incubation (2 x 108 CFU/ml at pH 6.8) (Fig. 2A and G). Therefore, the consumption of the organic acids that were excreted into the medium occurs concomitantly with growth of rpoS mutants (Fig. 1B).


Figure 2
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  		FIG. 2. Effects of fur and ryhB mutations on viability and metabolic patterns of rpoS+ and rpoS mutant cells. Strains were inoculated 1:500 (time zero) into Pi-limiting medium (0.1 mM Pi plus 40 mM glucose) and incubated further under aerobic conditions. The pHs of the culture supernatants were determined and adjusted to pH 7, and the concentrations of glucose and acetate were determined. The values are the means ± standard deviations for n independent cultures representative of several determinations. (A, D, G, and J) ENZ535 (wt; {circ}; n = 3), ENZ985 (rpoS; {blacksquare}; n = 2), ENZ1782 (fur; {triangleup}; n = 2), ENZ1709 (rpoS fur; {blacktriangledown}; n ≥ 2), and ENZ1787 (rpoS fur; {blacktriangleup}; n ≥ 2). (B, E, H, and K) ENZ1786 (rpoS ryhB; {blacksquare}; n = 2) and ENZ1906 (rpoS fur ryhB; {blacklozenge}; n ≥ 3). (C, F, I, and L) ENZ1781 (ryhB; {triangledown}; n = 2), ENZ1921 (fur ryhB; {diamond}; n = 3), and ENZ535 (wt; {circ}) and ENZ1782 (fur; {triangleup}) as in A, D, G and J.

Pi excreted into the medium allows rpoS mutants to grow on acetic acid. Growth of rpoS mutants (Fig. 1B) implies that a source of Pi was obtained. A bioassay was used to measure the concentrations of Pi equivalent in the spent culture media. The calibration curve (Fig. 3A) shows that the yield of tester cells was directly proportional to the concentration of K2HPO4 in the range of 500 to ~4 µM. The apparent overestimate in the growth yield (CFU/ml) that occurs at growth-limiting concentrations of K2HPO4 (<4 µM) (17) may be accounted for, at least in part, by the small size of the cells (data not shown).


Figure 3
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  		FIG. 3. Bioassay for Pi equivalent in culture media. (A) The calibration curve was determined by measuring the yield of tester cells after overnight incubation in MOPS medium containing increasing concentrations of K2HPO4 ([1.3 ± 0.1] x 106 CFU/ml with 0 µM K2HPO4). The values are from three independent experiments. Open and closed arrowheads indicate the yield of tester cells obtained using 24-day-old spent culture media (see below) supplemented with nutrients containing 0.5 mM K2HPO4 (controls for growth capacity) and 1 mM KCl, respectively, for the rpoS+ (n = 4) and rpoS mutant strains (n = 3) (standard deviations ≤ 10%). (B and C) Strains ENZ535 (rpoS+) and ENZ985 (rpoS) were inoculated 1:500 (time zero) into Pi-limiting medium (0.1 mM Pi plus 40 mM glucose) and incubated for 24 days under aerobic conditions. The OD600s of the cultures (rpoS+, {circ}; rpoS, {square}) and the concentrations in the culture supernatants (adjusted to pH 7) of Pi equivalent (rpoS+, {blacktriangleup}; rpoS, {blacktriangledown}) were determined between 2 and 10 h of incubation (B) and between 1 and 24 days of incubation (C). The values are the means for two independent cultures (standard deviations ≤ 30%) representative of several determinations.

The concentration of Pi in the spent media decreased sharply from 100 to ~1 µM when the rpoS+ and rpoS mutant strains approached the stationary phase (Fig. 3B). Then, the concentration of Pi decreased further to ≤0.3 µM when the strains progressively entered the stationary phase (Fig. 3B and C). However, the levels of Pi equivalent eventually went into reverse during prolonged incubation. In cultures of the rpoS+ strain, Pi levels increased from ≤0.3 to ~25 µM between days 3 and 13 of incubation (Fig. 3C), which indicates that rpoS+ cells eventually excrete significant amounts of Pi. In cultures of the rpoS mutant strain, Pi levels slightly increased after 4 days of incubation, remained steady at ~1 µM between days 6 and 10, and increased sharply to ~15 µM between days 10 and 13 of incubation (Fig. 3C). Therefore, the consumption of Pi that is excreted into the medium may occur concomitantly with growth of rpoS mutants.

Fur activity is required for the survival of the rpoS mutant strain. Because rpoS mutants were exposed to 5 to 7 mM acetate at slightly acidic pH for several days before they resumed growth (Fig. 2A, G, and J), we wondered whether some mechanisms, such as the synthesis of acid shock proteins (ASPs), could help cells to resume growth. It has been shown that the addition of 8 mM acetate in medium at pH 6 decreases the growth rate of E. coli twofold (31). The global regulators PhoB and Fur are good candidates for a role in the protection of rpoS mutants. PhoB (regulator of the phosphate starvation regulon) (17) induces the synthesis of the ASP Asr, which scavenges protons in the periplasm and thus protect cells against acid damage (34, 35). Fur (ferric uptake regulator) is required for the synthesis of ASPs in Salmonella and Shigella (13, 28); the synthesis of Fur increases at the onset of Pi starvation in E. coli, but the physiological role, if any, of Fur in Pi-starved cells is not known (38).

PhoB might play no significant role in the viability of the rpoS mutant strain because rpoS {Delta}phoBR mutants behaved like rpoS mutants during prolonged incubation under aerobic, Pi starvation conditions (data not shown). In contrast, Fur appeared to play a key role in the survival of the rpoS mutant strain, since the viability of rpoS {Delta}fur double mutants (ENZ1709 and ENZ1787) steadily decreased between days 3 and 25 of incubation, in such a way that no viable counts were obtained on day 25 of incubation when 20-µl aliquots of cultures (OD600 ~ 0.65) were spotted directly on LB medium plates (Fig. 2A). Since the introduction of the fur mutation had no effect on the viability of the rpoS+ strain (Fig. 2A), Fur activity may be specifically required for the viability of rpoS mutants during prolonged incubation under aerobic, Pi starvation conditions.

Fur activity is required for the detoxification of acetic acid. The levels of acetic acid remained steady between days 8 and 12 of incubation in cultures of the rpoS fur double mutant strain (~15 mM acetate at pH 5.5), whereas they dropped in cultures of the rpoS strain (~0 mM acetate at pH 6.9 on day 12 of incubation) (Fig. 2G and J), which provides evidence that the detoxification of acetic acid by rpoS mutants requires Fur activity. Furthermore, close examination of the metabolic patterns reveals that during the first 4 days of incubation, the rpoS fur strain consumed less glucose (–5 mM) but excreted more acetate (+7 mM) than the rpoS strain did (Fig. 2D and J). Since the incubation media of the rpoS mutant strain contained 5 mM acetate together with 12 mM glucose on day 4 of incubation (Fig. 2D and J), a concentration of glucose that inhibits acetate catabolism in growing cells (6), it is unlikely that the low levels of acetate in cultures of rpoS mutants could be accounted for merely by a coassimilation of acetate and glucose. Therefore, Fur activity may help the rpoS mutant strain to combat acetic acid toxicity both by decreasing the production and by increasing the consumption of acetic acid.

Fur activity is required primarily to inhibit RyhB synthesis. Notably, the Fur(Fe2+) active complex represses fur, iron-uptake genes, and ryhB (9, 19, 22). The small antisense RNA RyhB triggers a rapid degradation of target mRNAs (20), thereby inhibiting the synthesis of Fur (40) and of iron-rich proteins (e.g., proteins containing hemes and Fe-S clusters) collectively known as Fe proteins (19-22). The Fe proteins belong notably to the tricarboxylic acid (TCA) cycle (e.g., aconitases AcnA and -B, fumarase FumA, and the succinate dehydrogenase [SDH] SdhCDAB) and to the aerobic respiratory chain (e.g., the NDH-I NuoA-N and the cytochrome oxidase CydAB) (19-22). Therefore, the control of iron homeostasis by the Fur-RyhB system may indirectly affect aerobic metabolism. It has been suggested that the RyhB-mediated degradation of the SdhCDAB mRNA might account for the inability of fur mutants to grow on succinate as the sole carbon source (19). Interestingly, the same process might account for the inability of the rpoS fur mutant strain to consume acetate, since acetate metabolism requires the full activity of the TCA cycle, including SDH (8, 29). However, the fur mutation might also decrease the metabolism of acetate in rpoS mutants independently of RyhB activity. During growth on acetate, the glyoxylate shunt (isocitrate -> succinate + malate) is required to replenish the TCA cycle (8). The enzymes of the glyoxylate shunt are encoded by the aceBAK operon, which is apparently activated by Fur; the binding of the Fur(Fe2+) complex in the promoter region of aceBAK may relieve the inhibitory effect of another regulator(s), such as IclR (21, 44).

To determine whether the release of repression of ryhB could help explain the complex metabolic changes caused by the fur mutation, the ryhB::cat mutation (19) was introduced into the rpoS fur strain. The defects obtained in viability and in the metabolism of glucose and of acetic acid in the rpoS fur strain (Fig. 2A, D, G, and J) were totally reversed in the rpoS fur ryhB strain after 24 days of incubation (Fig. 2B, E, H, and K). In contrast, the introduction of the ryhB mutation into the rpoS strain had no significant effect (Fig. 2B, E, H, and K). Therefore, a crucial role of Fur in the rpoS strain may be to repress the expression of ryhB, which otherwise would inhibit the synthesis of Fe proteins required for the metabolism of glucose and of acetic acid.

The inactivation of both fur and ryhB improves the survival of rpoS+ cells. The inactivation of both fur and ryhB increased the viability of rpoS+ cells during the first 8 days of incubation (Fig. 2C). The higher viability of the fur ryhB double mutants was correlated with lower levels of acetic acid in the incubation medium: cultures of the fur ryhB strain contained only 13 mM acetate at pH 6.3 on day 4 of incubation (Fig. 2I and L), whereas the cultures of the wt, fur, and ryhB strains contained up to 21 mM acetate at pH ~5.1 (Fig. 2I and L). The lower levels of acetic acid in the cultures of the fur ryhB strain did not result merely from a defect in glucose metabolism, since fur ryhB mutants metabolized glucose as efficiently as wt cells and ryhB mutants and more efficiently than fur mutants during the first 8 days of incubation (Fig. 2F). Eventually, the fur ryhB strain metabolized more glucose (~34 mM) than the wt and ryhB strains (~30 mM) and much more than the fur strain (~24 mM) during a 24-day incubation period (Fig. 2F). Therefore, the lack of both Fur and RyhB may improve the aerobic metabolism of glucose in such a way that the excretion of toxic acetic acid is significantly delayed.

Fur(Fe) activity does not change at the onset of Pi starvation in rpoS+ cells. A simple hypothesis that could account for the above results is that rpoS+ cells might contain low levels of Fur(Fe2+) repressor, which in turn might lessen the repression of ryhB and favor the production of acetic acid, whereas rpoS mutants might contain high levels of Fur(Fe2+) repressor, which in turn might repress ryhB and favor the detoxification of acetic acid. A putative decrease in Fur(Fe2+) repressor activity in rpoS+ cells might account for the increase in Fur levels that occurs at the onset of Pi starvation (38).

The activity of the Fur repressor was tested by monitoring the expression of the iucC::lacZ fusion, which is strictly controlled by the Fur(Fe2+) complex (3, 39). As shown in Fig. 4, the levels of β-galactosidase remained quite stable (~70 U) when the strain ENZ1929 (rpoS+/iucC::lacZ) entered stationary phase. The fusion was expressed at a slightly higher level (~110 U) when the rpoS mutant strain ENZ1930 entered stationary phase. Similarly, the fusion was induced to a higher level (~1,530 U) in rpoS mutants than in rpoS+ cells (~635 U) when the strains were incubated between 10 and 12 h in the presence of dipyridyl (Fig. 4), a cell-permeable iron chelator that removes the metal from Fur (19, 39). Therefore, the activity of the Fur(Fe2+) repressor may remain stable in rpoS+ cells, whereas it may somewhat decrease at the onset of stationary phase in rpoS mutants.


Figure 4
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  		FIG. 4. Expression of the Fur regulon. Strains ENZ1929 (rpoS+/iucC::lacZ) and ENZ1930 (rpoS/iucC::lacZ) were inoculated 1:500 (time zero) into Pi-limiting medium (0.1 mM Pi plus 40 mM glucose) and incubated under aerobic conditions. The OD600 of the cultures (rpoS+, solid line; rpoS, dashed line) and the specific activity of β-galactosidase were determined (rpoS+, •; rpoS, {blacksquare}). At 6 and 10 h of incubation, samples were withdrawn, supplemented with 250 µM dipyridyl (+ DP), and further incubated for 2 h, and the levels of β-galactosidase were determined (rpoS+, {circ}; rpoS, {square}). The values are the means ± standard deviations for three (rpoS+) and two (rpoS) independent cultures representative of several determinations.


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DISCUSSION
 
The data presented here demonstrate that rpoS mutants survived during prolonged incubation in MOPS minimal medium that initially contained 40 mM glucose and a limiting concentration of Pi (0.1 mM), whereas rpoS+ cells died under the same incubation conditions. The behavior of the rpoS mutant strain may be accounted for by the fact that most rpoS mutants grow after 6 days of incubation, which eventually allows rpoS mutants to consume in their totality the organic acids, mainly acetic acid, that are excreted during glucose metabolism. In fact, rpoS mutants may resume growth on day 3 of incubation, when Pi is excreted by the bulk of the population. Then, rpoS mutants may consume Pi together with glucose and eventually with organic acids. Finally, by day 13 of incubation, when no C sources remain, growth of rpoS mutants may stop and Pi accumulates in the medium.

The detoxification of organic acids by rpoS mutants requires the activity of the global regulator Fur, whose primary role is to inhibit the synthesis of the small RNA RyhB. Fur activity is required at two different steps: (i) during the first days of incubation, to increase the catabolism of glucose and to decrease the production of acetic acid, and (ii) between days 8 and 12 of incubation, to allow the consumption of organic acids. In contrast, rpoS+ cells excrete high levels of acetic acid during the first days of incubation and cannot consume acetic acid during prolonged incubation, even though rpoS+ and rpoS mutant cells eventually excrete similar levels of Pi during prolonged incubation.

However, we found that during the first days of incubation, fur ryhB mutants behave phenotypically like rpoS mutants, as both strains produce low levels of acetic acid during glucose catabolism. How do fur and ryhB mutations allow rpoS+ cells to excrete less acetic acid? The ryhB mutation, which prevents the small-RNA-RyhB-mediated degradation of target mRNAs, may enhance the synthesis of Fur and of Fe proteins that belong notably to the TCA cycle, namely, AcnA and -B, FumA, and SDH, and to the aerobic respiratory chain, namely, NuoA-N (NDH-I) and CydAB (8, 19-22, 40, 44). The fur mutation, which prevents the synthesis of Fur and relieves the repression of iron transport genes, may increase the intracellular pool of iron and prevent Fur from competing for iron with Fe proteins (15, 36, 40). Overall, an increase in the activity of the Fe proteins Acn, FumA, and SDH may directly increase the activity of the TCA cycle, and an increase in the activities of NDH-I and CydAB may indirectly increase the flux of glucose toward the TCA cycle; an efficient regeneration of NADH into NAD+ by the aerobic respiratory chain (NDH-I-Q-CydAB) may enhance the activity of glucose 6-phosphate dehydrogenase in glycolysis, of PDH, and of {alpha}-ketoglutarate dehydrogenase and malate dehydrogenase (MDH) in the TCA cycle (8). Therefore, the behavior of fur ryhB mutants may be accounted for by an increase in the flux of glucose toward the TCA cycle, which may consequently decrease the flux through the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway, which produces acetic acid (6) (Fig. 5). In this view of the role of Fe proteins in Pi-starved cells, the low rate of excretion of acetic acid that occurs during the first days of incubation of fur ryhB and of rpoS mutants (provided that Fur represses ryhB) may primarily result from the activity of RyhB-controlled Fe proteins in the TCA cycle and in the aerobic respiratory chain. In contrast, in rpoS+ cells, low levels of Fe proteins might inhibit the activity of the TCA cycle and of the aerobic respiratory chain and direct the metabolic flux mainly toward the Pta-AckA pathway, which leads to the excretion of acetic acid (1, 6, 8).


Figure 5
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  		FIG. 5. Diagram showing the proposed metabolic flux distribution in rpoS+ (pathways 1 and 2) and rpoS mutant strains (pathways 3 and 4) during prolonged incubation in Pi-limiting medium. Abbreviations: AceCoA, acetyl coenzyme A; CIT, citrate; CYT, cytochrome quinol oxidase; FAD, flavin adenine dinucleotide; FUM, fumarate; G6P, glucose 6-phosphate; GLX, glyoxylate; ICT, isocitrate; KG, {alpha}-ketoglutarate; MAL, malate; OA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; Q, ubiquinone; SUC, succinate; SucCoA, succinyl coenzyme A.

How might the RpoS activity decrease the activity of Fe proteins? Because the synthesis of Fe proteins is normally decreased posttranscriptionally by RyhB when iron is scarce (15), a possibility would be that the induction of the RpoS regulon might decrease the intracellular pool of iron; the sequestration of iron by RpoS-dependent proteins, such as the Dps and Bfr ferritin-like proteins, might remove the metal from Fur, which would release the repression of ryhB. To test this hypothesis, we monitored the level of expression of the iucC gene, which is strictly controlled by the Fur(Fe2+) repressor (3, 39). On the basis of these measurements, we estimate that the activity of the Fur(Fe2+) repressor does not change significantly at the onset of Pi starvation in rpoS+ cells. We cannot exclude the possibility that the binding of the Fur(Fe2+) repressor might be weaker in the operator region of ryhB than in the operator region of iucC; a limited decrease in Fur(Fe2+) activity could allow some induction of ryhB, while iucC would remain fully repressed. However, the levels of expression of iucC were somewhat lower in rpoS+ than in rpoS mutant cells at the onset of stationary phase, which would imply that RyhB levels should be lower, rather than higher, in rpoS+ cells than in rpoS mutant cells.

Since the behavior of Pi-starved cells cannot be explained merely by changes in RyhB levels, an alternative hypothesis is that the accumulation of E{sigma}S at the onset of stationary phase might decrease the transcription of genes encoding RyhB-controlled Fe proteins that are required for the activity of the TCA cycle. This hypothesis is supported by recent microarray analyses, which show that the accumulation of RpoS reduces the expression of genes involved in the TCA cycle about twofold (8, 30, 42).

Since RpoS and RyhB activities may increase independently the production of acetic acid in rpoS+ cells during the first days of incubation, we propose that the combined action of high levels of RpoS and basal levels of RyhB decrease, at the transcriptional and translational levels, respectively, the synthesis of RyhB-controlled Fe proteins required for the activity of the TCA cycle. In contrast, the TCA cycle may remain substantially functional in rpoS mutants despite the presence of low levels of RyhB; metabolism through the TCA cycle and growth are blocked in rpoS mutants only if ryhB is fully induced as a result of the introduction of a fur mutation.

Taken together, the results provide compelling evidence for the regulation of metabolism by the Fur-RyhB system during prolonged incubation of rpoS mutants. Moreover, our results are consistent with the idea that Fur by itself, independently of its control of RyhB, may improve metabolic activities in rpoS mutants. In fact, the rpoS fur ryhB triple mutant strain consumed glucose and acetate, and it increased the pH of the medium more slowly than the rpoS and rpoS ryhB mutant strains did. Fur may act at two different levels. First, the activity of the Fur(Fe2+) repressor, which represses iron uptake genes (22), may decrease the intracellular levels of free Fe2+ and thus decrease oxidative damage (through Fenton chemistry) in metabolic enzymes (15). This idea is supported by the fact that the viability of the rpoS fur ryhB strain decreased more strongly than that of the rpoS and rpoS ryhB strains between days 3 and 8 of incubation. In addition, the activity of the Fur(Fe2+) repressor may increase the expression of the aceBAK operon (21, 44); the AceBAK enzymes of the glyoxylate shunt may be required for growth on acetate after day 8 of incubation (8, 29, 43).

These results lead us to propose the following model to explain the different strategies used by rpoS+ and rpoS mutant cells to survive during prolonged incubation under aerobic, Pi starvation conditions (Fig. 5). At the entry into stationary phase, rpoS+ cells accumulate RpoS, which increases the E{sigma}S-mediated transcription of at least 481 genes (gadABC, poxB, katE, dps, bfr, pdhR, etc.). The accumulation of RpoS as well as other factors, such as Rsd, 6S RNA, and ppGpp, contribute to decrease the E{sigma}70-mediated transcription of genes encoding proteins required for the activity of the TCA cycle, such as Acn, FumA, and SDH (8, 30, 37, 42). Moreover, RyhB targets and triggers the degradation of the mRNA encoding the same iron-rich proteins Acn, FumA, and SDH (19-22, 44). Interestingly, RyhB may somehow increase the expression of Pta and AckA (21). Overall, the combined action of RpoS and RyhB may redirect the metabolic flux from the TCA cycle toward the Pta-AckA pathway, which leads to production of acetic acid (Fig. 5, pathway 1). Then, the increase in PoxB synthesis and the decrease in PDH synthesis, triggered, respectively, by RpoS and PdhR, lead to conversion of pyruvate directly into acetic acid (24) (Fig. 5, pathway 2). We showed previously that Pi-starved cells do not use fermentative enzymes such as pyruvate-formate lyase PflB (25). Therefore, all the changes triggered in rpoS+ cells by RpoS and RyhB may help to protect stationary-phase cells against oxidative damage that otherwise would be generated through the activity of NDHs in the aerobic respiratory chain (24). Moreover, acetic acid can be neutralized through the induction of the H+-consuming activity of the glutamate-dependent Gad system (25). However, if rpoS+ cells survive in the presence of glutamate, they cannot resume growth on acetate and Pi that are excreted into the medium, probably because high levels of acetate at modestly acidic pH increase the activity of E{sigma}S and specifically decrease the E{sigma}70-mediated transcription of the rRNA operons (31, 32).

In contrast, when rpoS mutants enter stationary phase, the combined action of Fur, which, notably, represses ryhB, and E{sigma}70 allows the cells to synthesize RyhB-controlled Fe proteins, which in turn helps to maintain, directly and indirectly, the activities of glucose 6-phosphate dehydrogenase, PDH, Acn, malate dehydrogenase, FumA, and SDH of the TCA cycle (Fig. 5, pathway 3) (8). Such metabolic activities increase oxidative damage, through a high rate of production of NADH, but allow rpoS mutants to consume high levels of glucose and to excrete low levels of acetic acid, which prevents acetic acid from dramatically perturbing cellular homeostasis (31, 32). Then, surviving rpoS mutants can resume growth when Pi is excreted into the medium by the bulk of the population, which enables cells to consume the organic acids that were previously excreted (Fig. 5, pathway 4) (8, 29, 43).

Could the different strategies used by rpoS+ and rpoS mutant cells to survive during prolonged incubation in media initially limited in Pi help explain the behavior of E. coli in nature? We have suggested that E. coli cells that grow in the proximal part of the human colon may enter the stationary phase as a result of Pi starvation, when high levels of C may be obtained from the degradation of mucus (25). Our results support the hypothesis that both rpoS+ and rpoS mutant cells could survive in the aerobic part of the gut by using different strategies: rpoS+ cells could tolerate high levels of organic acids provided that glutamate is available, whereas rpoS mutants could survive in the absence of glutamate through the detoxification of organic acids. These data may help explain the fact that a high frequency of E. coli rpoS mutants is found in nature (16).


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ACKNOWLEDGMENTS
 
We acknowledge F. Mansour and S. Benomar for help with some of the experiments. We thank J. Bos, S. Gottesman, J. Imlay, R. Kolter, and D. Touati for generous gifts of strains.


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FOOTNOTES
 
* Corresponding author. Mailing address: CNRS-LCB, 31 chemin Joseph Aiguier, 13009 Marseille, France. Phone: (33) 4 91 16 43 53. Fax: (33) 4 91 71 89 14. E-mail: moreau{at}ibsm.cnrs-mrs.fr Back

{triangledown} Published ahead of print on 13 June 2008. Back


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Journal of Bacteriology, August 2008, p. 5567-5575, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00577-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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