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Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K-12

¹ Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo-SP, Brazil
² School of Molecular and Microbial Biosciences, The University of Sydney, NSW 2006, Australia

Correspondence
Thomas Ferenci
tferenci{at}mail.usyd.edu.au

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Laboratory strains and natural isolates of Escherichia coli differ in their level of stress resistance due to strain variation in the level of the sigma factor ^S (or RpoS), the transcriptional master controller of the general stress response. We found that the high level of RpoS in one laboratory strain (MC4100) was partially dependent on an elevated basal level of ppGpp, an alarmone responding to stress and starvation. The elevated ppGpp was caused by two mutations in spoT, a gene associated with ppGpp synthesis and degradation. The nature of the spoT allele influenced the level of ppGpp in both MC4100 and another commonly used K-12 strain, MG1655. Introduction of the spoT mutation into MG1655 also resulted in an increased level of RpoS, but the amount of RpoS was lower in MG1655 than in MC4100 with either the wild-type or mutant spoT allele. In both MC4100 and MG1655, high ppGpp concentration increased RpoS levels, which in turn reduced growth with poor carbon sources like acetate. The growth inhibition resulting from elevated ppGpp was relieved by rpoS mutations. The extent of the growth inhibition by ppGpp, as well as the magnitude of the relief by rpoS mutations, differed between MG1655 and MC4100. These results together suggest that spoT mutations represent one of several polymorphisms influencing the strain variation of RpoS levels. Stress resistance was higher in strains with the spoT mutation, which is consistent with the conclusion that microevolution affecting either or both ppGpp and RpoS can reset the balance between self-protection and nutritional capability, the SPANC balance, in individual strains of E. coli.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Large numbers of influential studies in bacterial physiology, genetics and gene regulation have used laboratory strains of Escherichia coli K-12, but evidence is accumulating that stocks kept in various laboratories are not comparable. The MG1655 strain used in genomic studies differs in several respects between laboratories (Soupene et al., 2003). Another commonly used sequenced strain, W3110, also differs in important respects in various stocks (Jishage & Ishihama, 1997). There are also numerous physiological and regulatory differences between MG1655 and another commonly used K-12 lineage, MC4100 (King et al., 2004). Stored archival cultures of bacteria can be heterogeneous (Sutton et al., 2000), which is also likely to contribute to strain variation in stocks of K-12 strains. We are interested in the molecular basis of strain variation and in this study identify some of the factors important not only in strain differences but also in the settings for global gene regulation in bacteria.

One identified contributor to strain variation is the conditional advantage of the sigma factor RpoS (^S) in different environments. RpoS is responsible for the general stress response of E. coli (Weber et al., 2005) and strains with high levels of RpoS are more resistant to stress but grow more slowly under a variety of conditions (Ferenci, 2005). The balance between self-protection and nutritional capability, the SPANC balance, is frequently reset by mutation, resulting in many different combinations of stress resistance and nutritional capability in the species (King et al., 2004). Growth of bacteria on poor carbon sources or low levels of nutrients selects for reduced or abolished RpoS levels, whereas stressful environments require RpoS (King et al., 2006). Laboratory strains contain one of several different alleles of rpoS (Atlung et al., 2002). Here we investigate some of the factors that determine RpoS levels and its variation in strains of E. coli K-12.

The role of RpoS in E. coli is complementary to that of the alarmone ppGpp, whose concentration also responds to stress and shifts transcription away from vegetative growth towards stress resistance (Ferenci, 2005; Gentry et al., 1993; Jishage et al., 2002; Typas et al., 2007). ppGpp and RpoS co-operate both mechanistically and strategically, and expression of RpoS-controlled genes is partly dependent on ppGpp (Kvint et al., 2000; Nystrom, 2004). The level of ppGpp is also important because it helps to control the amount of RpoS in the cell, as ppGpp increases the cellular concentration of RpoS by several-fold during nutritional stress or upon entry into the stationary phase. The absence of ppGpp impairs or severely delays the accumulation of RpoS (Gentry et al., 1993). ppGpp also positively affects the efficiency of rpoS translation in stationary phase under stress conditions as well as rpoS basal expression under conditions of optimal growth (Brown et al., 2002; Hirsch & Elliott, 2002). The response to phosphate starvation additionally involves stabilization of RpoS protein mediated by ppGpp (Bougdour & Gottesman, 2007). At several levels then, ppGpp is intertwined with rpoS regulation and function; here we show ppGpp is also subject to strain variation.

ppGpp directly and indirectly affects many important bacterial characteristics, from stress responses to pathogenesis to biofilm formation (Braeken et al., 2006; Cashel et al., 1996; Magnusson et al., 2005; Nakanishi et al., 2006). ppGpp accumulates in bacteria undergoing nutritional stress, such as amino acid, carbon or phosphate limitation (Bougdour & Gottesman, 2007; Cashel & Gallant, 1969; Lazzarini et al., 1971; Spira et al., 1995). Accumulation of ppGpp triggers the ‘stringent response’, in which a radical decrease in ribosome synthesis results in a general inhibition of protein synthesis and growth arrest (Cashel et al., 1996). The strategic overall role of ppGpp is the allocation of transcriptional resources so they are appropriate to the level of stress and growth rate of the bacteria. This, as with RpoS, necessarily involves a trade-off in the expression of genes related to stress resistance and vegetative growth. The presence of ppGpp may be beneficial in stressful environments but may be an impediment to vegetative growth in intermediate-stress situations. It is therefore interesting to analyse the evolutionary selection pressures imposed on the genes involved in ppGpp synthesis or degradation and how these shape bacterial behaviour.

ppGpp levels are controlled by two proteins, encoded by the genes relA and spoT (Cashel et al., 1996). Synthesis of ppGpp under intracellular amino acid starvation is dependent on relA. The RelA protein senses the presence of uncharged tRNAs at ribosomes, which triggers the RelA-dependent synthesis of ppGpp. relA mutants display a relaxed phenotype in response to amino acid limitation, characterized by a further decrease in ppGpp concentration and the continuous production of stable RNA (Cashel et al., 1996). SpoT is a bifunctional cytosolic enzyme capable of both ppGpp synthesis and degradation. Under nutritional stresses other than amino acid limitation, accumulation of ppGpp is largely relA-independent, as the rise in ppGpp concentration is mainly due to inhibition of SpoT hydrolytic activity (Murray & Bremer, 1996).

Because ppGpp is needed to obtain high levels of rpoS expression, it was expected that ppGpp would also affect the equilibrium between metabolic capacity essential during vegetative growth and stress resistance, the so-called SPANC balance (Ferenci, 2005). ppGpp may have a direct effect on bacterial nutritional ability, as it binds to RNA polymerase and affects the expression of many genes either positively or negatively (Cashel et al., 1996; Magnusson et al., 2005; Reddy et al., 1995). Additionally, ppGpp may affect cellular properties indirectly through RpoS (Cashel et al., 1996); we tested this prediction in this study. We also showed that ppGpp levels are remarkably strain-specific and that spoT is particularly important in microevolution, fixing the basal level of ppGpp in response to selective pressures on stress resistance and nutrition. Finally, we demonstrate that strain variation in ppGpp contributes to the difference in the level of RpoS in E. coli and discuss the consequences of polymorphisms at the core of bacterial regulation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Strains and media.
The strains used in this study are described in Table 1. Bacteria were cultured in T-salts, a Tris-buffered minimal medium supplemented with different concentrations of glucose and KH₂PO₄ (Spira et al., 1995), and minimal medium A (MMA; as described by Miller, 1972). For growth on acetate and L-agar plates (Miller, 1972), overnight cultures were harvested, washed twice and resuspended in MMA to a density of 2x10⁸ cells ml^–1. Aliquots of suspensions (5 µl) were spotted on MMA-acetate or L-agar plates and incubated at 37 °C.

Transductional transfer of spoT alleles and rpoS : : Km.
The different spoT alleles were transferred by P1 transduction (Miller, 1972). Strains CF12269, CF12271 and CF12273 served as lysate donors for the spoT1655, spoT202 and spoT203 alleles, respectively, and MC4100TF served as the recipient strain. The proximity of the spoT locus to zib563 : : Tn10 (80 % co-transduction) was used to select the transductants for tetracycline resistance; thes transductants were later tested for spoT-dependent phenotypes, such as ppGpp level, iodine staining and growth on acetate. For the transduction of the spoT4100 allele into MG1655, the zib563 : : Tn10 marker was first transduced into MC4100 and then a lysate of MC4100 zib563 : : Tn10 was used to transduce MG1655. The rpoS : : Km mutation was transduced into other strains using strain ZK1000 as a donor.

ppGpp assay.
ppGpp was extracted from cells growing at 37 °C in T-salts minimal medium containing 100 µCi (3.7 MBq) KH₂³²PO₄ ml^–1. To measure basal levels of ppGpp, cells growing exponentially in T-salts were resuspended in T-salts supplemented with 0.2 % glucose and 0.2 mM KH₂³²PO₄ at OD₆₀₀ 0.1. Samples were harvested after 90, 100, 110 or 120 min. ppGpp was extracted from amino acid-starved cells using the same method except that amino acid starvation was triggered by adding 1 mg serine hydroxamate ml^–1 to the cultures.

The labelled samples were mixed immediately with 0.5 vols of cold formic acid and stored overnight at –20 °C. The extracts were centrifuged for 5 min at 10 000 r.p.m. to precipitate cell debris; 3–5 µl was applied to PEI-cellulose TLC plates. The labelled nucleotides were resolved by one-dimensional TLC using 1.5 M KH₂PO₄ as solvent. The amounts of ppGpp on the chromatograms were estimated by measuring the radioactivity of the spots in a Phosphor-Imager (Molecular Dynamics) and calculating the level of ppGpp relative to that of GTP+ppGpp. The densitometric analysis was performed with the help of the Image J software (available at http://rsb.info.nih.gov/ij/).

Determination of growth rate.
The growth rate of K-12 strains was measured in cultures growing in unlimited T-salts minimal medium. Overnight cultures were diluted to OD₆₀₀ 0.02 and grown at 37 °C for 4 h. Samples were taken every hour and OD₆₀₀ was determined. The growth rate (µ, h^–1) was calculated using the formula µ=ln(N/N₀)/t, where N₀ is the initial and N is the final cell concentration, and t is the time course of the growth curve.

Steady-state growth conditions in chemostats.
T-salts supplemented with 0.02 % glucose and 1.0 mM KH₂PO₄ was used to set up an 80 ml chemostat culture as described by Notley-McRobb et al. (2002). The dilution rate was set to 0.1 h^–1. Daily samples were taken to monitor the rpoS status of members of the population. The rpoS status was determined by iodine staining (see below).

Detection of rpoS status by iodine staining.
The RpoS level was qualitatively assessed by staining glycogen with an iodine solution as described by Hengge-Aronis & Fischer (1992). Patches of bacteria or diluted chemostat samples were grown overnight on L-agar plates, stored at 4 °C for 24 h and then flooded with iodine. The intensity of the brown colour varies according to glycogen concentration in the cell and indirectly measures the level of RpoS (King et al., 2004; Spira & Ferenci, 2008). rpoS⁺ strains stain brown to dark brown.

Stress resistance assays.
Bacteria from overnight cultures grown in L-broth were washed twice in 0.9 % (w/v) NaCl and diluted in 0.9 % NaCl to a density of 4x10³ cells ml^–1. For oxidative stress, freshly diluted H₂O₂ (final concentration 1.5 mM) was added to 1 ml culture and held at room temperature. Samples (100 µl) were taken at 5, 10, 15 and 20 min, spread on L-agar plates and incubated overnight at 37 °C. For osmolarity, suspensions of 4x10³ cells ml^–1 were incubated in 1.5 M NaCl at room temperature. Samples (100 µl) were withdrawn at 1, 3 and 4 h and plated onto L-agar. For cold stress, bacteria resuspended in 0.9 % NaCl were held at 4 °C and survival was assessed at 2, 4, 6 and 8 days.

Quantification of RpoS.
Bacteria from overnight cultures were resuspended in L-broth and grown at 37 °C up to OD₆₀₀ 2.0, then 2x10⁹ cells were centrifuged, resuspended in 200 µl application buffer (0.5 M Tris/HCl, 2 % SDS, 5 % 2-mercaptoethanol, 10 %, v/v, glycerol and 0.01 % bromophenol blue) and boiled for 5 min. Proteins were resolved in a 12.5 % denaturing polyacrylamide gel and transferred to a nitrocelullose membrane (GE HealthCare) by capillary action. Following blocking with 5 % skim milk, the membrane was incubated with 2000-fold diluted monoclonal anti-RpoS antibodies (Neoclone) and 20 000-fold diluted peroxidase-conjugated anti-mouse IgG (Pierce). The Super Signal West Pico kit (Pierce) was used to detect the RpoS bands as recommended by the manufacturer. The membrane was exposed to X-ray films for various periods of time and the signal intensities were scanned and computed using the Image J software.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Level of ppGpp and nature of the relA/spoT genes in a high-RpoS strain
The MC4100 strain from the Ferenci lab (subsequently called MC4100TF) maintains higher levels of RpoS than other K-12 strains, including MG1655 (King et al., 2004). As ppGpp is an important factor in controlling RpoS synthesis and function (Gentry et al., 1993; Kvint et al., 2000), the level of ppGpp in MC4100 was compared to that of MG1655. As shown in Table 2, the basal level of ppGpp was twofold greater in MC4100TF than in MG1655 under steady-state growth conditions at similar growth rates. The higher ppGpp in MC4100 was unexpected, given the known genotypes (relA⁺ in MG1655 and relA1 in MC4100) and the wild-type spoT genes in both. Amino acid starvation induced by an amino acid analogue resulted in a relaxed response in MC4100TF consistent with the presence of the relA1 defect (data not shown), which was closer to the expected result. To test if undetected genetic differences were present, both the relA and spoT genes associated with ppGpp synthesis were sequenced in MC4100TF. The relA1 insertion mutation in MC4100 was indeed found in MC4100TF (data not shown). Surprisingly, the spoT sequence in MC4100TF differed from that in MG1655 and included a substitution (H255Y) and a two-amino acid insertion between residues 82 and 83 (+QD) of the SpoT protein. The gene with this pair of changes is subsequently called spoT4100.

A different MC4100 stock maintained in another laboratory, MC4100BS, was also sequenced for spoT and contained the same pair of changes as MC4100TF. The spoT4100 from MC4100BS also elevated ppGpp in the MG1655 background to similar basal levels as found in the MC4100TF transductant. The mutational differences in spoT and ppGpp between MG1655 and MC4100 indicate the variability of these features of E. coli laboratory strains.

Differences in RpoS levels between MG1655 and MC4100 lineages
The effect of different endogenous ppGpp concentrations on the level of RpoS protein were tested. As shown in Fig. 1, using anti-RpoS antibodies and three to six replicate immunoblots of each strain, different levels of RpoS were found both as a function of strain background and as a result of different spoT alleles. The difference between MG1655 and MC4100TF (Fig. 1) is fully consistent with an earlier study (King et al., 2004). More surprisingly, in spoT1655 strains, RpoS levels were also lower in MG1655 than in MC4100TF. Since both MC4100TF transduced with the spoT1655 allele and MG1655 showed similar ppGpp levels (Table 2), it is necessary to invoke other differences in RpoS regulation to explain the protein levels. The increased RpoS levels found in spoT4100 strains relative to spoT1655 strains were consistently higher in either MC4100TF or MG1655 backgrounds. Likewise, ppGpp was approximately twofold higher in the spoT4100 strain. With the spoT202/203 mutations and even higher ppGpp, RpoS did not increase substantially above the spoT4100 level, suggesting saturation of rpoS regulation by ppGpp. The trends in protein amount in Fig. 1 between spoT1655 and spoT mutants are consistent with the effect of ppGpp levels (Table 2) on rpoS expression (Gentry et al., 1993).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Quantification of RpoS protein. Bacteria were harvested from early stationary phase cultures of strains with various spoT alleles; samples were lysed and proteins were resolved by SDS-PAGE. The blots were incubated with anti-RpoS monoclonal antibodies. The bands were scanned and quantified. Values represent the mean±SEM of at least three independent experiments. A mutant lacking RpoS is also shown.

Effect of ppGpp and spoT differences on growth rates, nutrition and the SPANC balance
The influence of spoT alleles on RpoS levels suggested that ppGpp would also affect downstream RpoS-dependent phenotypes. A simple test of RpoS function is through testing for glycogen accumulation, which is RpoS-dependent and proportional to its protein level (Spira & Ferenci, 2008). As shown in Fig. 2(c), staining with iodine showed that glycogen accumulation is very much in parallel with the independently determined RpoS estimations. The strong staining in the high-ppGpp, high-RpoS strains (MC4100TF spoT202, MC4100TF spoT203) contrasts with the poor staining in MG1655 and MC4100BS. It is also obvious that there is an increase in staining in spoT4100 compared with spoT1655. These data are entirely consistent with those in Fig. 1.

View larger version (82K): [in this window] [in a new window]   Fig. 2. Growth of bacterial patches on different carbon sources. Washed cultures (5 µl) containing equal amounts of cells were grown on minimal agar plates containing 0.4 % acetate as the sole carbon source (a); the faster-growing colonies in some of the slow-growing patches are rpoS mutants selected after 2 days on plates (King et al., 2004). Bacterial patches were grown on L-agar as described above and growth was recorded after 12 h at 37 °C (b); these patches were stained with iodine to detect the level of glycogen in the cell (c). The intensity of staining is a function of glycogen content and indirectly of RpoS protein concentration (Spira & Ferenci, 2008).

The difference in growth rates with the set of strains in Table 2 was evident, even with good carbon sources such as glucose. Furthermore, growth rate differences can be seen even in rich Luria–Bertani medium (Fig. 2b). The slower growth of high-ppGpp strains was at least partially due to RpoS, as rpoS mutants were able to grow faster even with rich nutrients. Even with the very high-ppGpp strains containing the spoT202/203 alleles, an rpoS mutation relieves most of the growth inhibition. Hence, selection for improved growth rates with any carbon source can be a potential cause of polymorphisms affecting RpoS and ppGpp.

Effect of ppGpp differences on stress resistance
The spoT alleles affected the nutritional side of the SPANC balance, so twofold variation in basal ppGpp levels, such as between MC4100TF and MC4100TF spoT1655, should also be reflected in differences in the general stress responses of the bacteria. The effects of H₂O₂, high osmolarity and cold stress were tested on these strains (Fig. 3). In each stress condition, the strain with the spoT4100 allele performed better. Particularly interesting is the disparity upon cold storage. Given the advantage of the spoT4100 allele in stressful situations (as may occur in prolonged laboratory storage at 4 °C), the difference in stress survival may explain the selection pressure for mutations in spoT towards higher ppGpp levels in the MC4100 lineage or any E. coli strain stored for long periods in a stressful environment. The above results suggest that an increase in basal ppGpp levels can improve resistance and tilt the SPANC balance away from effective nutrition; lower ppGpp improves growth on different carbon sources, but reduces stress survival.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Effect of the spoT background on stress resistance. Cultures of strains MC4100TF (), MC4100TF spoT1655 () and MC4100BS () were challenged for resistance to 1.5 mM H2O2, high osmolarity (1.5 M NaCl) and cold (4 °C). Viable bacteria were determined from plate counts.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Loss of RpoS in bacteria with different ppGpp backgrounds during steady-state growth. Cultures of strains MC4100TF (), MC4100TF spoT1655 (), MC4100TF spoT202 (), MG1655 () and MG1655 spoT4100 () were grown separately for 4 days in glucose-limited chemostats at a 0.1 h–1 dilution rate. Samples were harvested daily, diluted and plated on L-agar. Changes in rpoS status were followed by iodine staining. The proportion of RpoS+ cells is shown; the value for each strain is representative of at least two experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The structure–function relationships in the bi-functional SpoT protein are only partly understood (Gentry & Cashel, 1996), so the spoT mutations found in this study can only be tentatively interpreted. The insertion at amino acid residues 82/84 probably compromises the ppGppase activity of the protein. Interestingly, one of the high-ppGpp mutations in spoT (spoT202) is nearby, affecting residue 78 (T78I, M. Cashel, personal communication). The spoT203 mutation affecting residue 140 (R140C, M. Cashel, personal communication) and the H255Y substitution in the spoT4100 strain are more likely to affect synthesis in a positive way. Further protein–function studies are needed to fully resolve these questions, as current speculations are based on the structure of a hybrid protein related to SpoT (Hogg et al., 2004).

In many respects, spoT4100 resembles the previously described spoT1 mutation that had elevated basal levels of ppGpp (Lagosky & Chang, 1980; Sarubbi et al., 1989). Strain variation in spoT has already been associated with the presence of a spoT1 allele in a subset of K-12 lineages (Laffler & Gallant, 1974). Indeed, sequencing of the archival spoT1 gene revealed the same double mutation as in MC4100 (M. Cashel, personal communication), so it is possible that an undocumented spoT1 mutation was incorporated into the MC4100 strain during its complex construction (Casadaban, 1976). A full genealogical analysis of the studied strains was not undertaken, however, as it is clear from the strain variation, even between the two MC4100 stocks (the TF and BS variants), that frequent changes are common in stored cultures. It is also unclear whether spoT1-like mutations are more prevalent in relA1 mutants.

The environmental influence on ppGpp regulation is affected by the same dichotomy already observed and discussed for RpoS (Ferenci, 2005), namely the different needs of the cell in response to nutrient limitation and stress resistance. As described for the MC4100TF strain and its spoT1655 derivative, or MG1655 and its spoT4100 derivative, the nature of the spoT allele that is present in E. coli simultaneously influences the level of RpoS, stress resistance and nutritional capabilities of E. coli. The benefit of high basal ppGpp levels is seen in the greater stress resistance that is conferred (Fig. 3), whereas the benefit of low ppGpp is evident from increased growth, particularly under nutrient limitation or with poor nutrients (Fig. 2a, b). Depending on the environment, the antagonistic pleiotropy between resistance and nutrition can change the cell's SPANC balance. Given that external stresses and nutritional constraints are common in natural settings, it is not surprising that mutations occur and are constantly selected in shifting environments. Indeed, the variation in spoT resembles the polymorphisms in rpoS, which are, if anything, even more extensive (Atlung et al., 2002; Ferenci, 2005).

In the past, the relationship between growth rate and ppGpp levels was discussed mainly in the context of high ppGpp resulting in a reduction in stable RNA synthesis and thereby reduced translational capacity (Sarubbi et al., 1988). After the effect of ppGpp on RpoS was discovered (Gentry et al., 1993), it was suggested that some of the ppGpp effect could be indirect and could be acting through elevated expression of the alternative sigma factor (Cashel et al., 1996). Indeed, as shown in this study, the elimination of RpoS results in faster growth on a range of substrates. Even on rich media, the inhibitory effect of artificially elevated ppGpp can be largely overcome by rpoS mutations. Hence, the growth rate effects of high ppGpp are more complex than simple control of translational processes and they probably involve competition between sigma factors as well. Adding to the complexity, both ppGpp and RpoS levels differ between strains, so the overall effect of elevated ppGpp is also likely to be strain-specific.

In summary, the physiological and regulatory roles of RpoS and ppGpp are interlinked at several levels, including strain variation. Increased ppGpp generally has a positive effect on RpoS levels, but differences in RpoS between the MC4100 and MG1655 backgrounds demonstrate that other factors besides ppGpp need to be invoked to explain the extent of rpoS changes. Previous results have pointed to considerable polymorphisms in rpoS between E. coli strains (Ferenci, 2003); these new results suggest that at least one of the genes involved in ppGpp synthesis and degradation is also subject to the same kind of selective pressures as is rpoS. In this respect, spoT and rpoS are involved in the dynamic balancing of SPANC within a bacterium in response to the nature of the immediate environment and demand for nutrients. These findings are significant for molecular studies of bacteria, because it is now known that two of the global systems that control the allocation of transcriptional resources are actually strain-specific, since ppGpp and RpoS levels are potentially non-uniform in E. coli under identical growth conditions. This has major consequences for global studies of the pattern of expression of hundreds of genes controlled directly or indirectly (by competition) at the level of RNA polymerase. Equally importantly, many of the numerous traits controlled by ppGpp (Braeken et al., 2006; Cashel et al., 1996; Magnusson et al., 2005; Nakanishi et al., 2006) are also likely to be subject to strain variation.

	ACKNOWLEDGEMENTS

 
This study was greatly supported by contributions from M. Cashel, who supplied strains, helpful comments and unpublished information on spoT mutants. This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP-Brazil) and an Australian Endeavour Research Fellowship (to B. S.), as well as the Australian Research Council (to T. F.).

Edited by: J. Green

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Received 13 March 2008; revised 14 May 2008; accepted 15 May 2008.

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