In Escherichia coli, the expression of the RpoS regulon is known to be crucial for survival in liquid cultures during stationary phase. By measuring cell viability and by transcriptome analysis, here we show that rpoS cells as well as wild-type cells survive when they form colonies on solid media.

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ESCHERICHIA coli possess seven RNA polymerase -factors, which compete for association with the core polymerase subunit, each coordinating the transcription of a set of genes, which allows fine control of adaptation to different physiological conditions (RECORD et al. 1996). In cells that approach stationary phase, the -factor ³⁸ (RpoS) competes with the primary -factor ⁷⁰ (RpoD) for association with the core polymerase subunit. To date 100 genes have been reported to belong to the RpoS regulon whose induction results in physiological and morphological modifications that increase resistance to various stresses such as heat shock, cold shock, acid shock, oxidative stress, osmotic stress, and UV light (STORZ and HENGGE-ARONIS 2000).

Paradoxically, mutations and even inactivation of the rpoS gene might be tolerated or advantageous in nature, despite the partial or complete loss of RpoS-dependent resistance to environmental challenges, as suggested by the high degree of allelic variation of the rpoS genes from E. coli natural isolates (HERBELIN et al. 2000; JORGENSEN et al. 2000). Several studies have shown that the fitness of rpoS mutants depends on environmental conditions (FERENCI 2001; FARRELL and FINKEL 2003). However, the influence of the structured environment on fitness of the rpoS mutant is unknown. In nature, E. coli is often found in structured environments, e.g., microcolonies, aggregates, or biofilms. Hence, in this study we have tested how a structured environment influences the survival of an E. coli rpoS mutant to determine whether the selection of rpoS alleles found in nature can occur in such environments.

While the decrease in the number of colony forming units (CFUs) obtained from planktonic cells between 1 day (D1) and 7 days (D7) in culture was 200-fold for rpoS mutant and 10-fold for wild-type strain (wt), there was little difference in colonies between D1 and D7 for each strain as well as between the two strains (Table 1a). Furthermore, the measurements of the cell viability in aging colonies, as well as of the colony area, show that the rpoS mutant had difficulties relative to wt during the first 24 hr (D1) but there was no difference between the two strains after 7 days (D7; Table 1b), indicating better survival of rpoS cells at D7 than at D1, and/or increased cell turnover. To identify the functions that might be responsible for fitness modifications of rpoS mutants in structured environments, we established genome-wide transcription profiles of cells in aging colonies of wt and rpoS strains using macroarray technology.

View this table: In this window In a new window   TABLE 1 Growth and death within wild-type and rpoS colonies

 
The transcriptome analysis supports the observation that the rpoS colonies have more difficulties at D1 compared to wt colonies (Tables 1b and 2). For example, at D1, we observed a lower expression of genes coding for translation apparatus [ribosomal proteins (50S and 30S subunits)] in rpoS than in wt colonies, which may indicate that the rpoS cells have a lower growth rate than wt cells (GAUSING 1977). Furthermore, at D1, we observed a higher expression of the oxyR and soxR genes (coding for transcriptional regulators involved in the response to hydrogen peroxide and superoxide, respectively; STORZ and IMLAY 1999) in rpoS colonies than in wt colonies, indicating that oxidative stress might be the major problem for rpoS cells (Table 2). The induction of these two stress responses in rpoS mutant may be the consequence of the deficiency in RpoS-mediated protection against oxidants (Dps, KatE; HENGGE-ARONIS 2002) and/or an increase in production of reactive oxygen species. Moreover, some genes that are not regulated by OxyR but are induced by H₂O₂ treatment (ZHENG et al. 2001)—such as the ydcH (unknown function) and the yfiA gene encoding a protein associated with 70S ribosomes in stationary phase (MAKI et al. 2000)—were also upregulated in rpoS compared to wt at D1. The decrease in viability of rpoS cells at D1 could also result from the absence of downregulation of the ompF gene (coding for major outer membrane porin; LIU and FERENCI 2001), which is observed in wt. The downregulation of the ompF gene reduces membrane permeability and consequently the entry of toxic compounds into cells (MARTINEZ-MARTINEZ et al. 2000).

View this table: In this window In a new window   TABLE 2 Genes with altered expression in rpoS compared to wt colonies

 
Between D1 and D7, the fraction of dead cells within wt and rpoS colonies reached 60%. The difficulties faced by both strains were reflected by the increase in expression of RpoS-independent genes involved in stress resistance and DNA repair: e.g., the ³² (RpoH)/RpoN-regulated heat-shock genes ibpA, ibpB (KITAGAWA et al. 2002), the psp genes (phage shock proteins; MODEL et al. 1997), some SOS genes (dinJ, dinG; COURCELLE et al. 2001), and the nfo gene encoding endonuclease IV (HOSFIELD et al. 1999). We also observed, but only in rpoS, an increase in transcripts of RpoH-regulated genes (groES and htgA; MISSIAKAS and RAINA 1997), as well as the sbcB, recB (respectively, the exodeoxyribonuclease I and the exonuclease V subunit; SEIGNEUR et al. 1999), xseB (exonuclease subunit; VISWANATHAN et al. 2001), sodA (manganese superoxide dismutase; COMPAN and TOUATI 1993), and ung (uracil-N-glycosylase; D'SOUZA and HARRISON 2003) genes. Therefore, rpoS cells in aging colonies appear to face various stresses that damage DNA and proteins. However, the fact that the rpoS strain does not show an increase in mortality relative to wt at D7 suggests that it succeeds in coping with the stresses encountered in aging colonies.

Remarkably, the expression of genes coding for ribosomal proteins globally decreases between D1 and D7 in the wt strain, while it increases in rpoS colonies (Figure 1). A similar tendency was observed for genes coding for RNA (rpoB, rpoC, and rpoH) and DNA (holC) polymerase subunits (RECORD et al. 1996). Finally, the expression of fis (APPLEMAN et al. 1998), a gene coding for a protein required for adaptation of cells to rapid growth conditions, increases in rpoS D7 colonies relative to rpoS D1, as well as compared to wt D7. The resulting increase in transcription, protein, and DNA synthesis in aging rpoS colonies may simply be a side effect due to the absence of RpoS regulation. However, this global change may be the result of rpoS cell turnover, as some cells grow and divide while others die.

View larger version (21K): In this window In a new window Download PPT slide   FIGURE 1.— Fold change in transcript abundance of genes coding for ribosomal proteins. The two graphs compare expression of the 60 genes encoding ribosomal proteins in the rpoS strain to wt strain: top after 1 day (D1; blue) and bottom after 7 days (D7; green). The mRNA ratios for each gene between D1 and D7 in the wt strain [(wtD7/wtD1) or if wtD1 > wtD7 (–wtD1/wtD7)] are reported on the x-axis. mRNA ratios of the same genes for D1 and D7 between the wt strain and the rpoS strain [(rpoS/wt) or if wt > rpoS (–wt/rpoS)] are reported on the y-axis. At D1, most of the values are in the bottom left quadrant, corresponding to wt D7 < wt D1 and rpoS < wt. At D7, most of the values are in the top left quadrant, corresponding to wt D7 < wtD1 and rpoS > wt.

 
In conclusion, our study shows that RpoS-independent stress responses may be sufficient to ensure the survival of an rpoS population in aging colonies, which may not be the case in planktonic cells where the level of stress may be higher, particularly in oxidative stress (DUKAN and NYSTROM 1998). Furthermore, besides providing a collective defense against antagonists (MA and EATON 1992), the cellular aggregation may facilitate access to resources that cannot be efficiently utilized by isolated cells in suspension. This may be the case for reutilization of nutrients from the dead cells concentrated inside the colony (SHAPIRO 1992). Under such conditions, rpoS cells might use the available nutrients more efficiently than wt as suggested by the significant differences in transcriptional modifications of energy-producing pathways between the two strains. A compensatory transcription in rpoS colonies is suggested by the expression of genes involved in energy salvage: transcription of the psp operon and genes belonging to the tricarboxilic acid and the glyoxylate cycles (CRONAN and LAPORTE 1996), which are, respectively, RpoN and RpoD dependent, was higher in rpoS than in wt D7 colonies (Table 2). Therefore, it may be that the structured environments are favoring the observed enrichment for modified or null rpoS alleles in nature by providing an environmental niche in which the handicap resulting from the lack of various RpoS-dependent functions is attenuated through compensation.

We thank Bénédicte Gérard for help in transcriptome analysis, and Marie-Agnès Petit, Miroslav Radman, Eric Stewart, and Marin Vulic for critical reading of the manuscript. This work was supported by a grant from the Indo-French Center for the Promotion of Advanced Research.

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