Model metabolic strategy for heterotrophic bacteria in the cold ocean based on Colwellia psychrerythraea 34H Jeffrey J. Czajkaa, Mary H. Abernathya, Veronica T. Benitesb,c, Edward E. K. Baidoob,c, Jody W. Demingd,1, and Yinjie J. Tanga,1 aDepartment of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130; bTechnology Division, Joint BioEnergy Institute, Emeryville, CA 94608; cBiological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and dSchool of Oceanography, University of Washington, Seattle, WA 98105 Contributed by Jody W. Deming, October 11, 2018 (sent for review May 9, 2018; reviewed by John P. Bowman and Lyle G. Whyte) Colwellia psychrerythraea 34H is a model psychrophilic bacterium represents the cold dark ocean in its lack of proteorhodopsins, found in the cold ocean—polar sediments, sea ice, and the deep photoactive proteins used by many bacteria in the surface ocean sea. Although the genomes of such psychrophiles have been se- to generate supplemental energy (13). Genomic studies of 34H quenced, their metabolic strategies at low temperature have not and other strains of C. psychrerythraea have revealed many met- been quantified. We measured the metabolic fluxes and gene ex- abolic pathways that are important in the geochemical cycling of pression of 34H at 4 °C (the mean global-ocean temperature and a nutrients in cold marine environments (14), including those for normal-growth temperature for 34H), making comparative analy- hydrocarbon degradation and denitrification (14, 15). For exam- C. psychrerythraea ses at room temperature (above its upper-growth temperature of ple, an amplified genome of , present in the Escherichia coli Colwellia-rich microbial community that responded at depth to the 18 °C) and with mesophilic . When grown at 4 °C, Deepwater Horizon 34H utilized multiple carbon substrates without catabolite repression oil spill (12, 16, 17), has the potential to de- or overflow byproducts; its anaplerotic pathways increased flux net- grade ethane and propane (components of natural gas), suggesting a possible role in bioremediation of cold environments. Addi- work flexibility and enabled CO fixation. In glucose-only medium, 2 tionally, 34H has the capacity to produce industrially relevant the Entner–Doudoroff (ED) pathway was the primary glycolytic route; compounds, such as polyhdyroxyalkanoate and polyamides (14) in lactate-only medium, gluconeogenesis and the glyoxylate shunt be- and unique cryoprotectants (18). E. coli MICROBIOLOGY came active. In comparison, , cold stressed at 4 °C, had rapid Able to grow at temperatures as low as −12 °C (19), 34H has glycolytic fluxes but no biomass synthesis. At their respective normal- been used to study cold-adapted proteins (20) and enzymes (21), growth temperatures, intracellular concentrations of TCA cycle metab- extracellular polysaccharide substances (18, 22), and motility and olites (α-ketoglutarate, succinate, malate) were 4–17 times higher in chemohalotaxis at subzero temperatures (23, 24). Recently, in- 34H than in E. coli, while levels of energy molecules (ATP, NADH, vestigation of cold-adapted enzymes has increased in importance NADPH) were 10- to 100-fold lower. Experiments with E. coli mutants due to the potential economic and ecological advantages they supported the thermodynamic advantage of the ED pathway at cold can provide over their higher-temperature-requiring counterparts temperature. Heat-stressed 34H at room temperature (2 hours) in industrial processes (25). Previous studies of cold-adapted revealed significant down-regulation of genes associated with glyco- microbes have revealed a variety of molecular adaptations that lytic enzymes and flagella, while 24 hours at room temperature caused allow their activity and survival under extreme conditions, including irreversible cellular damage. We suggest that marine heterotrophic bacteria in general may rely upon simplified metabolic strategies to Significance overcome thermodynamic constraints and thrive in the cold ocean. Colwellia psychrerythraea 34H is a cold-adapted marine bac- marine psychrophile | metabolic flux | ED pathway | short-chain fatty terium that represents a genus and species cosmopolitan to the acids | gluconeogensis cold ocean. To our knowledge, metabolic flux studies of an obligate psychrophile like 34H are not available. We charac- any bioprocesses rely on the innate metabolic strengths of terized the physiology and metabolism of 34H, at normal- Mnonmodel environmental microorganisms. From this per- growth temperature of 4 °C and upper-stress condition of spective, microbiologists are exploring new microbes from ex- room temperature (23 °C), by integrating metabolic flux studies treme environments for advantageous characteristics (1). Advances (tracking 13C-labeled compounds) and genetic analyses (tran- in sequencing techniques, genomic analyses, and metabolic analyses scriptomics). Results from these system-level analyses reveal have aided species characterization by providing the functional unique metabolic features under cold salty conditions, which output of microorganisms and revealing their genotype-to- broaden our understanding of microbial ecology in the cold phenotype regulations. For some environments, the functional ocean, currently vulnerable to global warming. Specific find- metabolisms of representative species (mainly thermophiles and ings have relevance to bioremediation of pollutants from the mesophiles) have been characterized in detail (2–5). For cold en- petroleum industry, increasingly active in polar seas, and to vironments, few studies have addressed the metabolic pathways of biomanufacturing of cold-adapted enzymes. cold-adapted microorganisms (psychrophiles) directly, the emphasis being on genomic and diversity analyses instead (6–8). However, Author contributions: J.J.C., J.W.D., and Y.J.T. designed research; J.J.C., M.H.A., V.T.B., and most of Earth’s biosphere is cold (>75% volumetrically is below E.E.K.B. performed research; V.T.B. and E.E.K.B. contributed new reagents/analytic tools; 5 °C), particularly the microbially dominated ocean with its mean J.J.C., M.H.A., V.T.B., E.E.K.B., J.W.D., and Y.J.T. analyzed data; and J.J.C., J.W.D., and Y.J.T. wrote the paper. temperature of 4 °C (9). In this study, Colwellia psychrerythraea 34H (henceforth, 34H), Reviewers: J.P.B., University of Tasmania; and L.G.W., McGill University. a psychrophilic extremophile (unable to grow above 18 °C) of The authors declare no conflict of interest. ecological importance in the cold ocean, was used as a model Published under the PNAS license. species for investigating the metabolism of cold-adapted micro- 1To whom correspondence may be addressed. Email: [email protected] or yinjie.tang@ bial life. This Gram-negative, heterotrophic gammaproteo- wustl.edu. bacterium, originally isolated from arctic marine sediments (10), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is considered cosmopolitan to polar regions and the cold deep 1073/pnas.1807804115/-/DCSupplemental. sea (11, 12). Although ice adapted in many ways (11), 34H also www.pnas.org/cgi/doi/10.1073/pnas.1807804115 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 elevated amounts of unsaturated fatty acids in the membrane reactions which are important for replenishing TCA cycle me- (keeping it from rigidifying in the cold), an overall higher content tabolites. The 34H genome shows genes that potentially encode of polar amino acids (affecting protein thermolability), enzymes enzymes for the anaplerotic reactions [phosphoenolpyruvate with high specific activity at low temperatures (10, 19, 25), and (PEP)+CO2 <=> oxaloacetate (OAA) and pyruvate (PYR)+ antifreeze molecules (18, 22). However, to our knowledge, a CO2 <=> OAA or malate] annotated as PEP decarboxylase comprehensive metabolic characterization of a psychrophilic (cps4595), malate dehydrogenase (cps0331 and cps4262), and oxa- extremophile is not available. With climate change (26, 27) and loacetate decarboxylase (cps1048∼1050). We considered the possi- concerns about pollutants from resource-extraction industries, which bility that these enzymes may have plasticity for both forward and continue to develop in the deep sea and newly ice-free Arctic re- reverse reactions, and thus investigated the ability of 34H to in- 12 gions, microbial metabolic responses in cold environments is an corporate atmospheric CO2 into its TCA cycle metabolites after emerging frontier. Using 34H as a model marine psychrophile, this growing with U-13C (fully labeled) glucose. The resulting significant research elucidates physiological functions, biomass composition, (P value = 0.0294) dilution (>10%) of labeled carbons in OAA and functional metabolism by quantifying metabolites, using 13C- cycle-derived amino acids relative to amino acids from non-TCA metabolicfluxanalyses(13C-MFA), and sequencing transcriptomes cycle precursors (<5% dilution) suggests that 34H incorporates [RNA-sequencing (RNA-Seq)] under normal-growth cold temper- CO2 into its TCA cycle metabolites via anaplerotic reactions; these ature (4 °C) and thermally stressful room temperature (above its reactions were included in the metabolic network (Fig. 1A). upper-growth temperature of 18 °C). Dynamic 13C-labeling tech- 13C-fingerprinting was performed to probe the activity of the niques (28) and metabolic modeling (29) in particular have been glyoxylate shunt, which bypasses