Genetic Basis of Evolutionary Adaptation by Escherichia coli to Stressful Cycles of Freezing, Thawing and Growth

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary-phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure sensitive and took up more PI than the parent strain, with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY, and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment, and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure sensitive, was unaffected in membrane resealing, implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA, and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.Consumer demand for minimally processed, additive-free foods that have a long shelf life has stimulated research into new nonthermal methods of food preservation. High hydrostatic pressure (HHP) can inactivate nonsporing food-borne pathogens and spoilage organisms with minimal effects on food quality and is thus regarded as one of the more promising of the new technologies. The operation of HHP machinery requires relatively low energy consumption, produces little waste, and therefore, has a low impact on the environment (13,37,46). The use of HHP in the food industry is gradually increasing and is now being applied to a wide range of food products, including oysters, crabs and other crustaceans, cured hams, guacamole, salsas, fruit juices, desserts, and prepared meals (50).In order to ensure the safety of pressure-treated foods, it is important not only to have quantitative information on the intrinsic pressure resistance of different microorganisms but also to understand the basis of cellular pressure resistance and how this is affected by conditions during growth, pressure treatment, and recovery. Three main processes are currently believed to contribute to the inactivation of pressure-treated cells. These are protein denaturation, oxidative damage, and loss of membrane integrity (34).In all bacteria examined so far, pressure resistance is highly depende...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Role of rpoS in the Development of Cell Envelope Resilience and Pressure Resistance in Stationary-Phase Escherichia coli

Charoenwong

Andrews

Mackey

2011

“…E. coli is often used to study bacterial adaptation (49), experimental evolution (9,36), and speciation (11,28,33,43). Comparisons between representative genomes continue to provide valuable information about these processes (58), and as genetic data accumulate, our understanding of how bacteria adapt to change and evolve is clarified.…”

mentioning

confidence: 99%

Cryptic Lineages of the Genus Escherichia

Walk

Alm

Gordon

et al. 2009

250

244

Extended multilocus sequence typing (MLST) analysis of atypical Escherichia isolates was used to identify five novel phylogenetic clades (CI to CV) among isolates from environmental, human, and animal sources. Analysis of individual housekeeping loci showed that E. coli and its sister clade, CI, remain largely indistinguishable and represent nascent evolutionary lineages. Conversely, clades of similar age (CIII and CIV) were found to be phylogenetically distinct. When all Escherichia lineages (named and unnamed) were evaluated, we found evidence that Escherichia fergusonii has evolved at an accelerated rate compared to E. coli, CI, CIII, CIV, and CV, suggesting that this species is younger than estimated by the molecular clock method. Although the five novel clades were phylogenetically distinct, we were unable to identify a discriminating biochemical marker for all but one of them (CIII) with traditional phenotypic profiling. CIII had a statistically different phenotype from E. coli that resulted from the loss of sucrose and sorbitol fermentation and lysine utilization. The lack of phenotypic distinction has likely hindered the ability to differentiate these clades from typical E. coli, and so their ecological significance and importance for applied and clinical microbiology are yet to be determined. However, our sampling suggests that CIII, CIV, and CV represent environmentally adapted Escherichia lineages that may be more abundant outside the host gastrointestinal tract.

“…As such, our data underscore that, aside from absolute resistance, resuscitation speed itself is an evolvable trait. Previous work showed that E. coli can also improve its resuscitation time in response to recurring freeze-thaw cycles (32,33), suggesting that selection for faster recovery is a more general phenomenon that can be triggered by several stresses. Interestingly, most recent evidence seems to suggest that E. coli can evolve to increase the time it needs or takes before growth resumption to avoid being killed by antibiotics (34).…”

Section: Discussionmentioning

confidence: 99%

Stress-Induced Evolution of Heat Resistance and Resuscitation Speed in Escherichia coli O157:H7 ATCC 43888

Gayán

Cambré

Michiels

et al. 2016

The development of resistance in foodborne pathogens to food preservation techniques is an issue of increasing concern, especially in minimally processed foods where safety relies on hurdle technology. In this context, mild heat can be used in combination with so-called nonthermal processes, such as high hydrostatic pressure (HHP), at lower individual intensities to better retain the quality of the food. However, mild stresses may increase the risk of (cross-)resistance development in the surviving population, which in turn might compromise food safety. In this investigation, we examined the evolution of Escherichia coli O157:H7 strain ATCC 43888 after recurrent exposure to progressively intensifying mild heat shocks (from 54.0°C to 60.0°C in 0.5°C increments) with intermittent resuscitation and growth of survivors. As such, mutant strains were obtained after 10 cycles of selection with ca. 10 6 -fold higher heat resistance than that for the parental strain at 58.0°C, although this resistance did not extend to temperatures exceeding 60.0°C. Moreover, these mutant strains typically displayed cross-resistance against HHP shock and displayed signs of enhanced RpoS and RpoH activity. Interestingly, additional cycles of selection maintaining the intensity of the heat shock constant (58.5°C) selected for mutant strains in which resuscitation speed, rather than resistance, appeared to be increased. Therefore, it seems that resistance and resuscitation speed are rapidly evolvable traits in E. coli ATCC 43888 that can compromise food safety. IMPORTANCEIn this investigation, we demonstrated that Escherichia coli O157:H7 ATCC 43888 rapidly acquires resistance to mild heat exposure, with this resistance yielding cross-protection to high hydrostatic pressure treatment. In addition, mutants of E. coli ATCC 43888 in which resuscitation speed, rather than resistance, appeared to be improved were selected. As such, both resistance and resuscitation speed seem to be rapidly evolvable traits that can compromise the control of foodborne pathogens in minimal processing strategies, which rely on the efficacy of combined mild preservation stresses for food safety. Minimal processing of foods is based on the combination of mild preservation methods (or hurdles) for maximizing retention of the sensorial and nutritional properties of the food while maintaining the appropriate level of food safety and shelf life (1, 2). Methods such as mild heating, acidification, and the use of natural antimicrobial compounds, high hydrostatic pressure (HHP), pulsed electric fields, ultrasound, and irradiation are some of the techniques commonly used in hurdle approaches (2-4). While the efficacy of minimal processing relies on the additive, or even synergistic, lethal or growth-inhibitory effects of such mild hurdles, the mild intensity might nevertheless pose the risk of increasing resistance to the corresponding treatments. More specifically, rare mutant strains with an increased stress resistance that can spontaneously emerge in a population ...