Department of Biology, North Central College, Naperville, IL 60540, USA
Correspondence
Jonathan E. Visick
jevisick{at}noctrl.edu
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ABSTRACT |
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Present address: Department of Molecular and Cell Biology, Brandeis University, Waltham, MA 02453, USA.
Present address: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
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INTRODUCTION |
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During long-term stationary phase, de novo protein synthesis is greatly reduced, and maintenance of existing proteins becomes crucial. To this end, the transition to stationary phase includes increased synthesis of chaperones, trehalose, catalase and other protective factors (Kolter et al., 1993). Long-term stationary-phase cells model many aspects of senescence in higher organisms, including gradual loss of viability of individual cells, decreased function of macromolecules and increased susceptibility to oxidative damage (Kolter et al., 1993
; Nyström, 2003
; Visick et al., 1998a
). Under these conditions, PCM is required for maximal stress resistance: strains carrying pcm deletions show reduced long-term survival when exposed to oxidative, osmotic or heat stress, or methanol (Visick et al., 1998a
). Based on these results, we have hypothesized that covalent damage (due to isoAsp formation) and conformational damage (due to destabilization of protein structure) synergize to result in impaired stress survival in ageing E. coli (Visick et al., 1998b
).
Surprisingly, ageing pcm mutants are deficient in stress resistance only when grown in rich media such as LuriaBertani (LB) broth. In glucose minimal medium, no difference in survival between wild-type and mutant strains has been found under conditions tested to date (Visick et al., 1998a). Growth of E. coli in rich media such as LB broth exposes the cells to elevated pH as organic acids are consumed (Stancik et al., 2002
), whereas glucose minimal medium would be acidified by acidic fermentation products (Böck & Sawers, 1996
), though such media are typically buffered to a near-neutral pH. Alkaline pH increases the rate of isoAsp formation in vitro (by sixfold for a model peptide at pH 9·0 compared to 7·5; Brennan & Clarke, 1994
), probably favouring succinimide formation (see Fig. 1
) by deprotonating the attacking peptide-bond nitrogen. Furthermore, exposure to alkaline pH in rich medium produces or intensifies other known long-term survival phenotypes (Farrell & Finkel, 2003
; Lazar et al., 1998
; Vulíc & Kolter, 2002
; Weiner & Model, 1994
). We therefore sought to determine (i) whether PCM is required for survival of pH stress during long-term stationary phase (as it is for other stresses that can affect protein conformation) and (ii) whether elevated pH in LB broth is sufficient to account for the difference in stress survival between rich and minimal media.
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METHODS |
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Growth media and pH buffers.
LB broth (Miller, 1972) was used as a rich growth medium for all experiments. Buffers (200 mM final concentration) were added to maintain a specific pH in LB broth as follows (Blankenhorn et al., 1999
): pH 5·5 or 6·0, MES; pH 7·0, MOPS; pH 8·0, TAPS; pH 9·0, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO). All buffers were adjusted to the desired pH with KOH. For growth in minimal medium, either MOPS-buffered minimal medium (pH 7·4; Neidhardt et al., 1974
) and an AMPSO-buffered derivative (pH 9·0) or M63 medium (pH 7·0; Miller, 1972
) and a derivative made by altering phosphate concentrations (pH 9·0; Farrell & Finkel, 2003
) were used. All cultures were grown at 37 °C under aerobic conditions.
Stationary-phase and stress-survival assays.
Long-term stationary-phase survival was measured as described previously (Visick et al., 1998a). Viable counts were performed to determine the number of viable cells after 24 h of aerobic growth at 37 °C (day 0) and daily for 10 days thereafter. When appropriate, methanol (0·5 % final concentration) or paraquat (0·25 mg ml1 final concentration) was added after the first 24 h growth. Results were normalized by expressing viable counts as percentages of the maximum number of viable cells for a particular culture; maximal density was reached on either day 0 or day 1. In all cases, the maximum number of viable cells ranged from 1x109 to 2·5x109 cells ml1, and no significant differences were observed between MC1000 and JV1068.
Growth curves.
LB broth buffered to either pH 7·0 or pH 9·0 was inoculated with MC1000 or JV1068, using a 1 : 100 dilution of a broth culture or a 1 : 100 dilution of a single resuspended colony from a plate. Inocula consisted of either cells grown overnight at 37 °C in LB broth or on LB plates or aged cells incubated at 37 °C for five additional days. Viable counts were used to verify initial c.f.u. ml1; there was no significant difference in the number of viable cells in the inocula under either condition. Cultures were grown aerobically at 37 °C and growth was monitored spectrophotometrically (Milton-Roy Spectronic 20D spectrophotometer using cuvettes with 1 cm path length) by OD600. Length of lag phase and generation time were calculated for each growth curve according to the Baranyi growth model (Baranyi & Roberts, 1994), using the MicroFit program (www.ifr.ac.uk/microfit). Statistical significance of differences in averaged lag and generation times was evaluated using Student's t test.
Measurement of isoAsp damage.
IsoAsp content of proteins from crude E. coli extracts was measured essentially as described previously (Visick et al., 1998a). Cytosolic extracts were prepared from cells maintained for 24 h, 5 or 10 days after onset of stationary phase in LB broth adjusted to the desired pH. Cells were washed and resuspended in buffer containing 50 mM Tris/HCl (pH 8·0) and 300 mM NaCl, lysed by sonication and centrifuged at 20 000 g to pellet debris. Protein concentration was estimated by the Lowry method (Lowry et al., 1951
), following precipitation with trichloroacetic acid. Base-labile methyl esters produced by transfer of the methyl group from S-adenosyl-L-[methyl-14C]methionine to isoAsp residues by purified recombinant human PCM were measured as described by Li & Clarke (1992)
. IsoAsp content was then calculated as picomoles of methyl groups transferred per milligram total protein in the extract.
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RESULTS |
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While these experiments demonstrate the importance of pH, they do not establish whether the inability to detect survival phenotypes for the pcm mutant in minimal medium (Visick et al., 1998a) can be accounted for entirely by the difference in pH. To address this question directly, we compared cells maintained in M63 medium (buffered with potassium phosphate to pH 7·0; Miller, 1972
) with those maintained in M63 at pH 9·0 (adjusted by manipulating phosphate concentrations; Farrell & Finkel, 2003
). The pcm mutant strain JV1068 survived exposure to 0·5 % methanol to the same extent as MC1000 at pH 7·0 (open symbols in Fig. 4
), but at pH 9, the mutant (closed triangles) exhibited a survival defect relative to its parent (closed circles) comparable to that observed in rich medium (Fig. 3c
). The actual pH remained at the target level in the pH 7·0 cultures (dashed line in Fig. 4
) but initially decreased somewhat in those buffered to pH 9·0 (dotted line), probably due to the effect of acidic products that resulted from growth on glucose. The appearance of differences between the strains coincided with the point at which the pH reached 8·5. Methanol appeared to have a somewhat stronger deleterious effect on both strains in this medium than in LB broth (compare Figs 3 and 4
). Similar results were obtained (data not shown) when the strains were exposed to oxidative stress in minimal medium or when stress survival in MOPS-buffered minimal medium (pH 7·4) was compared to AMPSO-buffered minimal medium (pH 9·0).
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PCM accelerates recovery from long-term stationary phase
In the course of performing the survival assays described above, we noticed that for the pcm mutant, the number of c.f.u. ml1 after 24 h growth at pH 9·0 (day 0) was often lower than after an additional 24 h incubation in stationary phase (day 1), particularly if the culture was started from a colony stored for a few days on a plate, rather than directly from frozen stock. In contrast, wild-type cultures (as well as any culture grown at pH 7·0 or in unbuffered broth) were almost always at their maximum c.f.u. ml1 on day 0. This phenomenon can be observed, for example, in Fig. 3(c) (compare JV1068 with MC1000 on day 0 and day 1). We therefore investigated whether PCM might be involved in recovery from long-term stationary phase, perhaps affecting either the growth rate (i.e. doubling time) of recovering cells or their lag time before exponential growth begins.
We monitored growth of fresh (grown in unbuffered LB broth overnight) and aged (maintained in LB broth for 5 days) MC1000 and JV1068 after dilution 1 : 100 into fresh LB buffered to pH 7·0 or pH 9·0. Viable counts (data not shown) were used to verify that the initial number of c.f.u. ml1 was essentially the same for both strains, whether fresh or aged. At pH 7·0, wild-type and pcm mutant cells displayed the same growth characteristics regardless of age (Table 1), although aged cells of both types required a slightly (
20 min) longer lag period before resuming exponential growth. Generation time was not affected by age or genotype.
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DISCUSSION |
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Stress survival, including both pH stress (reviewed by Slonczewski & Foster, 1996) and protein-denaturing conditions such as heat and oxidative stress (reviewed by Storz & Zheng, 2000
; Yura et al., 2000
), has been extensively studied in E. coli. Most studies, however, have focused on stress resistance either during exponential growth or shortly after the transition to stationary phase, and little is known about possible combinatorial effects of multiple stresses. Like our pcm phenotypes, other long-term effects, including the growth advantage in stationary phase (GASP) phenotype (Farrell & Finkel, 2003
), surA phenotypes (Lazar et al., 1998
), psp phenotypes (Weiner & Model, 1994
) and viability loss preventable by ethanol (Vulíc & Kolter, 2002
), also become apparent or more pronounced at alkaline pH, suggesting that pH is a major factor in the ability of ageing E. coli to survive stress. It is significant in this regard that even for wild-type, repair-proficient cells, the effect of oxidative stress or methanol on survival was much greater under alkaline conditions than at neutral pH (Figs 3 and 4
). The mechanisms by which alkaline pH potentiates loss of viability under stress merit further study: while growing and early stationary cells effectively buffer cytoplasmic pH, it may be that internal pH is less effectively maintained during long-term stationary phase.
Our data suggest that PCM-mediated repair might be more important at high pH due to increased isoAsp damage in vivo: damaged proteins accumulated to higher levels sooner under alkaline conditions than at neutral pH (Fig. 5), mirroring previous in vitro measurement of isoAsp formation rates (Brennan & Clarke, 1994
). Assuming the molecular mass of a typical protein to be 40 000 Da, the amount of methylatable isoAsp we observed at pH 9·0 would represent about one damage site per 17 individual protein molecules after 5 days, versus one damage site per 70 molecules at pH 7·0. This suggests the potential for the reduced activity of a much larger number of cellular proteins under alkaline conditions, perhaps more rapidly surpassing some threshold of damage beyond which survival is impaired. However, isoAsp content began to plateau between 5 and 10 days of ageing at pH 9·0 (Fig. 5
), at a concentration roughly the same as the maximum accumulation observed in PCM-deficient mice (Lowenson et al., 2001
). Perhaps most of the susceptible residues have isomerized by this point, but we cannot exclude the possibility of an as-yet-unknown additional physiological mechanism that limits the amount of isoAsp damage. Our previous finding (Visick et al., 1998b
) that isoAsp accumulation increases in cells deficient in both PCM and SurE an acid phosphatase (Zhang et al., 2001
) of unknown substrate specificity co-transcribed with pcm favours the second hypothesis.
Effect of PCM on recovery: a model for PCM function in vivo
Two of our results seem incongruous with the simple model in which PCM is required to repair isoAsp damage that accumulates during long-term stationary phase due to reduced metabolism and protein synthesis. First, isoAsp levels after 10 days were no higher in the pcm mutant than in wild-type cells (Fig. 5), consistent with previous results for unbuffered LB (Visick et al., 1998b
). Second, it seems surprising that PCM would play a role during recovery from stationary phase (Table 1
), in the presence of plentiful nutrients. Taken together, however, these results may suggest a new model for isoAsp protein repair in vivo. While PCM is active in exponential and early stationary phases (Li et al., 1997
), its activity may be limited in long-term stationary phase, due to poor expression, instability, limiting concentrations of the methyl donor (S-adenosylmethionine) and/or excess of product (S-adenosylhomocysteine). If so, then even a repair-proficient cell would be unable to prevent accumulation of isoAsp damage during nutrient limitation, but it may be able to respond more rapidly to the return of favourable conditions when PCM is present to enable repair of existing proteins in addition to de novo synthesis.
This hypothesis can also account for the stress-survival phenotypes of pcm mutants during long-term stationary phase. It is increasingly clear that stationary phase is really a dynamic state in which some cells (particularly those that have or acquire mutations increasing fitness for current culture conditions) reproduce at the expense of others (Finkel & Kolter, 1999; Vulíc & Kolter, 2001
). PCM may thus function primarily to repair damaged proteins in those cells that are able to divide: those that undergo a recovery period even in the absence of added nutrients. Our previous finding (Visick et al., 1998a
) that aged pcm mutants fail to outcompete younger cells the GASP phenotype (Finkel & Kolter, 1999
), which requires the ability to divide during long-term stationary phase supports this idea. Future experiments will test this new hypothesis directly.
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ACKNOWLEDGEMENTS |
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Received 16 December 2004;
revised 2 March 2005;
accepted 24 March 2005.
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