UC San Diego UC San Diego Previously Published Works Title Adaptive laboratory evolution of Escherichia coli K-12 MG1655 for growth at high hydrostatic pressure. Permalink https://escholarship.org/uc/item/8hf561zk Journal Frontiers in microbiology, 5(JAN) ISSN 1664-302X Authors Marietou, Angeliki Nguyen, Alice TT Allen, Eric E et al. Publication Date 2014 DOI 10.3389/fmicb.2014.00749 Peer reviewed eScholarship.org Powered by the California Digital Library University of California ORIGINAL RESEARCH ARTICLE published: 07 January 2015 doi: 10.3389/fmicb.2014.00749 Adaptive laboratory evolution of Escherichia coli K-12 MG1655 for growth at high hydrostatic pressure Angeliki Marietou †,AliceT.T.Nguyen†, Eric E. Allen and Douglas H. Bartlett* Marine Biology Research Division, Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA Edited by: Much of microbial life on Earth grows and reproduces under the elevated hydrostatic Mark Alexander Lever, Swiss pressure conditions that exist in deep-ocean and deep-subsurface environments. In this Federal Institute of Technology in study adaptive laboratory evolution (ALE) experiments were conducted to investigate the Zürich, Switzerland possible modification of the piezosensitive Escherichia coli for improved growth at high Reviewed by: Aude Picard, Harvard University, pressure. After approximately 500 generations of selection, a strain was isolated that USA acquired the ability to grow at pressure non-permissive for the parental strain. Remarkably, Christian Tamburini, Mediterranean this strain displayed growth properties and changes in the proportion and regulation of Institute of Oceanography, France unsaturated fatty acids that indicated the acquisition of multiple piezotolerant properties. *Correspondence: These changes developed concomitantly with a change in the gene encoding the acyl Douglas H. Bartlett, Marine Biology Research Division, Center for carrier protein, which is required for fatty acid synthesis. Marine Biotechnology and Biomedicine, Scripps Institution of Keywords: adaptive laboratory evolution, Escherichia coli, high pressure, piezophile, acpP Oceanography, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0202, USA e-mail: [email protected] †These authors have contributed equally to this work. INTRODUCTION (Yayanos and Pollard, 1969; Welch et al., 1993). Interestingly, Elevated hydrostatic pressure promotes reduced system volumes a moderate pressure of 30 MPa was sufficient to suppress gene and volume changes of activation in chemical equilibria and transcription (Sato et al., 1996). Protein synthesis was com- rates of reactions, respectively (Meersman and McMillan, 2014). pletely inhibited at 68 MPa; aminoacyl-tRNA binding, ribosome Microorganisms growing preferentially at elevated hydrostatic translocation, and ribosome stability have all been implicated as pressures exist in large portions of Earth’s biosphere including the basis of high pressure impacts on translation (Schwarz and deep-sea and deep-subsurface locations (Meersman et al., 2013). Landau, 1972; Groß and Jaenicke, 1990; Alpas et al., 2003). Cell Despite the fact that the growth attributes of these piezophiles division was affected at pressures ranging from 20 to 50 MPa appear to require relatively little evolutionary change (Prieur resulting in elongation of the cells and a filamentous morphology et al., 2009), increased hydrostatic pressure exerts pervasive effects since cell division is affected prior to cell growth and accumu- on many aspects of cellular function and the adaptations required lation of biomass (Zobell and Cobet, 1963). Pressure of 10 MPa remain incompletely defined (Lauro et al., 2008). Arguably the and higher affect flagellation by disrupting filament polymeriza- best understood adaptation is the need for bacterial piezophiles tion and flagellum rotation (Meganathan and Marquis, 1973). to maintain a sufficient proportion of unsaturated fatty acids in Other structural changes under high pressure include a compact their membranes as a means of tuning phase, viscosity, and/or nucleoid structure (Welch et al., 1993). ion permeability (Bartlett, 2002; Kawamoto et al., 2011). High pressure growth of E. coli at 30 or 50 MPa induces a Escherichia coli is the most studied piezosensitive microor- cascade of stress responses with the concomitant regulation of ganism with the ability to grow to pressures of up to 50 MPa a series of genes; such cascade networks include heat and cold (Zobell and Cobet, 1963). It has been successfully used as a stress responses (Welch et al., 1993; Welch and Bartlett, 1998; model organism to investigate the effects of increasing pressure Aertsen et al., 2004a; Ishii et al., 2005). Interestingly, heat shock on cell processes and structures. E. coli belongstotheclassof pre-treatment improved the pressure resistance of E. coli in sub- Gammaproteobacteria which includes a large proportion of the sequent high pressure treatment, suggesting that heat shock pro- characterized piezophiles, while as a foodborne pathogen has teins might play a protective role (Aertsen et al., 2004a). Finally, been subjected to high pressure pasteurization processes poten- sub-lethal pressure treatments (100–500 MPa) increased expres- tially promoting the emergence of piezoresistant strains (Vanlint sion of stress sigma factors and genes involved in spontaneous et al., 2012). High pressure affects many cellular processes mutations and triggered the SOS and oxidative stress responses in E. coli, including replication, transcription, and translation. (Aertsen et al., 2004b, 2005; Malone et al., 2006). Recently, Previous studies have demonstrated DNA synthesis inhibition at a genome wide screening approach confirmed the pleiotropic 50 MPa while RNA synthesis was abolished completely at 77 MPa effects of pressure on E. coli identifying a series of genes required www.frontiersin.org January 2015 | Volume 5 | Article 749 | 1 Marietou et al. E. coli growth at high pressure for growth at high pressure, including genes involved in DNA to cause the steel ball to break the glass cover slip and mix the replication, cell division, cytoskeleton, and cell envelope physiol- fixative with the culture at in situ pressure conditions. ogy (Black et al., 2013). For epifluorescence microscopy the fixed cells were immobi- In this report we investigate the feasibility of employing adap- lized onto a 0.2 μm polycarbonate membrane (EMD Millipore) tive laboratory evolution (ALE) techniques to drive the pheno- and stained using 4,6 -diamidino-2-phenylindole (DAPI) type of a model mesophile in the direction of piezophily. ALE is nucleic acid stain (Vector Laboratories, Inc). The stained samples a powerful tool that relies on the Darwinian principles of muta- were viewed at 1000 fold magnification on an Olympus BX51 flu- tion and selection (Pourmir and Johannes, 2012). In some cases orescence microscope (Olympus). TEM samples were post fixed the selections involve a single selective step (Vanlint et al., 2011), with 1% osmium and 2% of uranyl acetate and embedded in in others a few hundred to a few thousand generations (Lee Ducurpan at 60◦C for 36 h. Ultra-thin sections (60 nm) were cut et al., 2011), and in the extreme case of the long-term studies with a diamond knife on Leica Ultracut UCT Microtome and post of Lenski and colleagues they may proceed for more than 50,000 stained with uranyl acetate and lead. Images were captured on FEI generations (Barrick et al., 2009). Examples of ALE experiments Tecnai spirit at 80 KV. selecting for more extremophilic characteristics in mesophiles include increased salt tolerance (Zhou et al., 2013), increased FATTY ACID ANALYSIS radiation resistance (Harris et al., 2009), the conversion of a Fatty acid analysis was performed by MIDI Labs (Newark, DE) on mesophile into a facultative thermophile (Blaby et al., 2012)and frozen cell pellets harvested during the early logarithmic phase of the selection for survival to exposures of more than 2 gigapascals growth. Saponification, methylation, extraction, and base wash of pressure (Hauben et al., 1997). It should be noted that resis- were performed before the fatty acid methyl esters were ana- tance to ultra-high pressure does not confer improved growth at lyzed on an Agilent/HP 6890 gas chromatograph. Using a pattern elevated pressures (Hauben et al., 1997; Lauro et al., 2008). In this recognition software, Sherlock MIS, the fatty acid composition of study, following 505 generations of selection we have isolated a each sample was compared to a stored database. The software was strain with the ability to grow at 62 MPa and alter membrane able to identify each component of the analyzed sample, produc- fatty acid composition in response to pressure. A mutation has ing a composition report including the relative amount (%) of the been identified in a gene involved in lipid metabolism that could named fatty acids. contribute in the improved piezotolerance of the newly isolated strain. DNA PURIFICATION AND GENETIC ANALYSIS Cells growing at exponential phase were centrifuged at 14,000 × MATERIALS AND METHODS g for 2 min and the cell pellets were used for DNA purification. GROWTH CONDITIONS The Wizard Genomic DNA purification kit (Promega) was used E. coli K-12 MG1655 was grown in triplicate lineages (A, B, and C) according to the manufacturer’s instructions. The purified DNA under fermentative conditions at 37◦C using Luria Bertani (LB) was used as template