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Rapid acquisition of Gigapascal-high-pressure resistance by Escherichia coli.
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MedLine Citation:
PMID:  21264062     Owner:  NLM     Status:  MEDLINE    
Pressure and temperature are important environmental variables that influence living systems. However, while they vary over a considerable range on Earth and other planets, it has hardly been addressed how straightforwardly and to what extent cellular life can acquire resistance to extremes of these parameters within a defined genomic context and a limited number of generations. Nevertheless, this is a very pertinent question with respect to the penetration of life in allegedly inhospitable environments. In this study, directed evolution was used to reveal the potential of the nonsporulating and mesophilic model bacterium Escherichia coli to develop the ability to survive exposure to high temperature or pressure. While heat resistance could only marginally be increased, our data show that piezoresistance could readily and reproducibly be extended into the GPa range, thereby greatly exceeding the currently recognized maximum for growth or survival.
Dietrich Vanlint; Rachael Mitchell; Edward Bailey; Filip Meersman; Paul F McMillan; Chris W Michiels; Abram Aertsen
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-01-25
Journal Detail:
Title:  mBio     Volume:  2     ISSN:  2150-7511     ISO Abbreviation:  MBio     Publication Date:  2011  
Date Detail:
Created Date:  2011-01-25     Completed Date:  2011-08-30     Revised Date:  2013-06-30    
Medline Journal Info:
Nlm Unique ID:  101519231     Medline TA:  MBio     Country:  United States    
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Languages:  eng     Pagination:  e00130-10     Citation Subset:  IM    
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Copyright © 2011 Vanlint et al.
Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M²S), Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Leuven, Belgium.
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MeSH Terms
Adaptation, Physiological
Biological Evolution
Escherichia coli / genetics,  growth & development,  physiology*
Hot Temperature
Microbial Viability*

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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Journal Information
Journal ID (nlm-ta): mBio
Journal ID (hwp): mbio
Journal ID (pmc): mbio
Journal ID (publisher-id): mBio
ISSN: 2150-7511
Publisher: American Society of Microbiology, 1752 N St., N.W., Washington, DC
Article Information
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Copyright © 2011 Vanlint et al.
Received Day: 3 Month: 12 Year: 2010
Accepted Day: 29 Month: 12 Year: 2010
Electronic publication date: Day: 25 Month: 1 Year: 2011
collection publication date: Season: Jan–Feb Year: 2011
Volume: 2 Issue: 1
E-location ID: e00130-10
ID: 3025523
PubMed Id: 21264062
Publisher Id: mBio00130-10
DOI: 10.1128/mBio.00130-10

Rapid Acquisition of Gigapascal-High-Pressure Resistance by Escherichia coli Alternate Title:GPa-High-Pressure Resistance in Escherichia coli
Dietrich Vanlinta
Rachael Mitchellb
Edward Baileyb
Filip Meersmanbc
Paul F. McMillanb
Chris W. Michielsa
Abram Aertsena
Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M²S), Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Leuven, Belgiuma;
University College London, Department of Chemistry, Christopher Ingold Laboratories, London, United Kingdomb; and
Department of Chemistry, Katholieke Universiteit Leuven, Leuven, Belgiumc
Correspondence: Address correspondence to Abram Aertsen,
[edited-by] Editor Sankar Adhya, National Cancer Institute


The known edges of our biosphere are typically delineated by extremophilic bacteria and archaea that manage to resist the severe physical and/or chemical conditions encountered in these environments. As such, studies of microorganisms from marine hydrothermal vents show that the currently recognized upper temperature and pressure limits for growth are 121°C (1) and 120 MPa (2), respectively. Besides the existence of extremophiles, however, another and less documented reflection of bacterial adaptability is the capacity to develop resistance against inhospitable environments within a defined genomic content and a limited number of generations. Such directed evolution of bacterial resistance could yield clues as to the possibility of the existence or persistence of microbial life in extreme environments that so far have been inaccessible or unexplored by current observational techniques.

In this context, we have examined how rapidly and to what extent a mesophilic and nonsporulating model bacterium such as Escherichia coli might adapt to lethal heat or high pressure (HP), two of the most important and general physical constraints on life. We followed a directed-evolution strategy in which an E. coli culture was iteratively challenged with progressively increasing temperatures or pressures. Between consecutive exposures to heat or HP, survivors were allowed to resuscitate and grow under standard conditions and thus automatically became enriched in the resulting population. After a number of such cycles, a single clone was purified from the population in order to compare its resistance profile to that of the original parent strain. The outcomes of these selection regimens are depicted in Fig. 1A and B for development of heat and HP resistance, respectively.

The results immediately reveal that the developed heat resistance was only marginal, shifting the upper survival temperature by approximately 1.03-fold from 58.5 to 60.0°C (Fig. 1A, compare green and red bars). However, the developed piezoresistance was extraordinary, and the upper pressure limit for survival readily increased at least 3-fold from 600 MPa to 2 GPa (Fig. 1B, compare green and black bars). In fact, as many as ca. 1 in 104 cells of the HP-resistant population readily survived a 2-GPa treatment. While signs of alleged enzymatic activity in E. coli cells at 1.4 GPa have been reported previously (3, 4), these results provide the first irrevocable evidence of microbial survival at ≥2 GPa.

It is also worth noting that the HP- and heat-resistant mutants displayed only a marginal cross-resistance against heat and HP, respectively (Fig. 1), indicating that the acquired adaptations are very specific for each stress and precluding any a priori causal link between HP and heat resistance.

Importantly, GPa-HP-resistant E. coli mutants have been obtained in all four independent HP evolution experiments performed. Moreover, in addition to this reproducibility, the relative ease with which GPa resistance seems able to evolve in E. coli is further underscored by the fact that none of these independently isolated mutants displayed any obvious growth defects at atmospheric pressure (data not shown). Together, these observations seem to indicate that this bacterium has the genetic blueprint to acquire extreme levels of HP resistance when selection forces it to do so. This apparent predisposition might also be reflected by the fact that natural isolates of E. coli display a great intrinsic variability with regard to HP resistance in the 200- to 700-MPa range studied so far (5, 6, 7).

To the best of our knowledge, the level of piezoresistance that E. coli developed during this study by far exceeds the currently recognized maximum for growth (i.e., 120 MPa) (2) or survival (i.e., 800 to 900 MPa) (8, 9) for any vegetative microorganism. In fact, it was generally assumed that vegetative microorganisms would never be able to survive exposures in the GPa range, as such pressures would irreversibly denature the proteome and disrupt membrane-related processes (10). Indeed, during in vitro studies most proteins unfold at pressures of 400 to 800 MPa and protein-protein and protein-nucleic acid interactions are disrupted. Furthermore, lipid model systems of cell membranes already undergo unfavorable fluid-gel transitions in the 100- to 300-MPa range (10). However, the extent to which these processes are reversible in a cellular context is not known.

Surprisingly, as shown in Fig. 1B, the evolved HP-resistant E. coli mutant displayed similar levels of survival when exposed to 1.2 and to 2 GPa. This could indicate that, despite the significant difference in their intensities, the two pressures have similar impacts on the cell that can be countered by the resistance mechanism. However, when we compared inactivation kinetics of the HP-resistant mutant at 800 MPa with that at 2 GPa, we found that the inactivation rate at 2 GPa was significantly lower than that at 800 MPa (see Fig. S1 in the supplemental material). The latter result is clearly reflected in the corresponding decimal reduction times: D2 GPa = 16 min > D800 MPa = 6.5 min (Fig. S1). It should be noted that within the 1.2- to 2-GPa range, water undergoes subsequent transitions from liquid into ice-VI and ice-VII crystal forms, as predicted by its pressure-temperature phase diagram. However, it is unclear whether or not such phase transitions could somehow contribute to the resilience of the HP-resistant mutant at these pressures.

In summary, we report on the extraordinary adaptive potential of a mesophilic nonsporulating bacterium to GPa-high pressures. These findings can have major implications in exploring the ecological boundaries for survival or adaption of cellular life with regard to HP as a limiting factor on Earth and perhaps other celestial bodies. Further analysis of the GPa-HP-resistant bacteria artificially evolved in this study will shed new light on how cells can safeguard the integrity of their proteins and membranes at these pressures and eventually how organisms can adapt to or have evolved from HP environments.

SUPPLEMENTAL MATERIAL Text S1 Supplemental methods. Download
Click here for additional data file (mBio00130-10-s01.doc)

FIG S1 Inactivation kinetics of the isolated HP-resistant mutant of E. coli (cf. Fig. 1B) at 800 MPa (squares) and 2 GPa (triangles). Note that 0 min comprises pressure buildup and -down times. A fit of the curves yields a D800 MPa value of 6.5 min and a D2 GPa value of 16 min. Download
Click here for additional data file (mBio00130-10-sf01.doc)


Citation Vanlint, D., R. Mitchell, E. Bailey, F. Meersman, P. F. McMillan, et al. 2011. Rapid acquisition of gigapascal-high-pressure resistance by Escherichia coli. mBio 2(1):e00130-10. doi:10.1128/mBio.00130-10.


This work was supported by a doctoral fellowship of the Flemish Agency for Innovation by Science and Technology (IWT-Vlaanderen, to D.V.), a postdoctoral fellowship of the Research Foundation - Flanders (FWO-Vlaanderen, to F.M.), a Senior Research Fellowship award of the Engineering and Physical Sciences Research Council (EPSRC EP/D07357X, to P.F.M.), and a grant from the Research Fund of the Katholieke Universiteit Leuven (STRT1/10/036, to A.A.).

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[Figure ID: f1]
FIG 1  

Directed evolution of E. coli K-12 MG1655 towards heat (at atmospheric pressure) (A) or HP (at ambient temperature) (B) resistance. An axenic culture of E. coli MG1655 was iteratively exposed to progressively intensifying temperature or pressure shocks (15 min each), with a resuscitation and growth step between consecutive treatments. Survival after each treatment was determined (blue diamonds) and expressed as log(N), in which N represents the number of survivors determined by plate counts and expressed as CFU per milliliter. At the end of both selection regimens, the most heat (red bars)- and pressure (black bars)-resistant clone that had enriched in the respective cultures was isolated and its acquired heat (A) and HP (B) resistance was determined and compared to that of the original parental strain (green bars). It should be noted that these selection profiles are representative of 20 and 4 independent trials for heat and HP resistance development, respectively. Further experimental details can be found in the supplemental material (Text S1).

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