The rpoS Gene Is Predominantly Inactivated during Laboratory Storage and Undergoes Source-Sink Evolution in Escherichia coli Species

Abstract

The rpoS gene codes for an alternative RNA polymerase sigma factor, which acts as a general regulator of the stress response. Inactivating alleles of rpoS in collections of natural Escherichia coli isolates have been observed at very variable frequencies, from less than 1% to more than 70% of strains. rpoS is easily inactivated in nutrient-deprived environments such as stab storage, which makes it difficult to determine the true frequency of rpoS inactivation in nature. We studied the evolutionary history of rpoS and compared it to the phylogenetic history of bacteria in two collections of 82 human commensal and extraintestinal E. coli strains. These strains were representative of the phylogenetic diversity of the species and differed only by their storage conditions. In both collections, the phylogenetic histories of rpoS and of the strains were congruent, indicating that horizontal gene transfer had not occurred at the rpoS locus, and rpoS was under strong purifying selection, with a ratio of the nonsynonymous mutation rate (Ka) to the synonymous substitution rate (Ks) substantially smaller than 1. Stab storage was associated with a high frequency of inactivating alleles, whereas almost no amino acid sequence variation was observed in RpoS in the collection studied directly after isolation of the strains from the host. Furthermore, the accumulation of variations in rpoS was typical of source-sink dynamics. In conclusion, rpoS is rarely inactivated in natural E. coli isolates within their mammalian hosts, probably because such strains rapidly become evolutionary dead ends. Our data should encourage bacteriologists to freeze isolates immediately and to avoid the use of stab storage.T he rates of recombination and mutation in a genome are critical issues because the diversity generated by these mechanisms is the substrate for selection. Some regions of bacterial chromosomes are more prone to mutation (1-4) or recombination (5) than other regions, due to physical or physiological constraints. Then, selection can favor diversification, for example, that of cell surface proteins such as beta barrel porins (6), and recombination events, which can restore particular functions, as in the case of the mismatch repair (MR) genes (7,8).There is growing evidence that selection also acts on central regulators to allow adaptation by rewiring of networks. In vitro experimental models of Escherichia coli evolution have shown that adaptation occurs through modifications of global regulatory networks, which are often interconnected (9-13). These networks control DNA superhelicity (12), the stringent response (12), and the general stress response, via RpoS (9, 10, 13). RpoS is an alternative sigma factor of RNA polymerase that regulates more than 10% of E. coli genes and plays a critical role in survival under conditions of exposure to several types of stress, including acid, heat, and oxidative stress (14,15). A particular case of in vitro evolution is the inactivation of crl (which modulates the balance between different sigma f...

The General Stress Response Is Conserved in Long-Term Soil-Persistent Strains of Escherichia coli

“…Our data show that the soil environment does not preferentially select for rpoS mutations, and this is consistent with recent studies reporting that loss-of-function RpoS mutations are rare in a large collection of natural isolates of commensal, pathogenic, and environmental E. coli (32,68). Bleibtreu et al (68) reported no variation in the amino acid sequence of RpoS in the E. coli strains which had minimal laboratory handling before being sequenced and showed that storage and successive transfers resulted in the rpoS mutations.…”

“…3A). It has been recently shown that the phylogenetic grouping based on rpoS is highly consistent with phylogenetic clustering based on multilocus sequence typing (MLST), thus suggesting that rpoS is a good indicator of evolutionary history of E. coli strains (68). On this basis, strain COB585 is the most divergent of the soil-persistent strains used in study, and it is most closely related to ECC08, which is a strain collected from a beach water sample at Bayfront Park Beach, Hamilton, Canada (7).…”

“…Bleibtreu et al (68) reported no variation in the amino acid sequence of RpoS in the E. coli strains which had minimal laboratory handling before being sequenced and showed that storage and successive transfers resulted in the rpoS mutations. Spira et al (69) also reported that transfer of E. coli on LB stabs between laboratories led to mutations in rpoS.…”

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

“…For example, naturally occurring strains can differ in the amount of RpoS they produce during exponential phase (12) or stationary phase (13). All studies that have measured RpoS levels in naturally occurring strains of E. coli have detected variations (13)(14)(15)(16)(17), though the extent and cause of the variations in RpoS between strains is still a matter of some controversy (16,17).…”

Hydrophobicity of Residue 128 of the Stress-Inducible Sigma Factor RpoS Is Critical for Its Activity

“…rpoS mutations of laboratory-stocked E. coli strains maintained in stab agar, leading to nutritional starvation, were reported to be associated with a high frequency of inactivating alleles (Bleibtreu et al, 2014). In a survey of rpoS among 2,040 environmental E. coli isolates, RpoS mutants were found to be present in the environment with a frequency of 0.003 among isolates (Chiang et al, 2011).…”