Eicosapentaenoic Acid Plays a Beneficial Role in Membrane Organization and Cell Division of a Cold-Adapted Bacterium,Shewanella livingstonensisAc10

Abstract

Shewanella livingstonensis Ac10, a psychrotrophic gram-negative bacterium isolated from Antarctic seawater, produces eicosapentaenoic acid (EPA) as a component of phospholipids at low temperatures. EPA constitutes about 5% of the total fatty acids of cells grown at 4°C. We found that five genes, termed orf2, orf5, orf6, orf7, and orf8, are specifically required for the synthesis of EPA by targeted disruption of the respective genes. The mutants lacking EPA showed significant growth retardation at 4°C but not at 18°C. Supplementation of a synthetic phosphatidylethanolamine that contained EPA at the sn-2 position complemented the growth defect. The EPA-less mutant became filamentous, and multiple nucleoids were observed in a single cell at 4°C, indicating that the mutant has a defect in cell division. Electron microscopy of the cells by high-pressure freezing and freeze-substitution revealed abnormal intracellular membranes in the EPA-less mutant at 4°C. We also found that the amounts of several membrane proteins were affected by the depletion of EPA. While polyunsaturated fatty acids are often considered to increase the fluidity of the hydrophobic membrane core, diffusion of a small hydrophobic molecule, pyrene, in the cell membranes and large unilamellar vesicles prepared from the lipid extracts was very similar between the EPA-less mutant and the parental strain. These results suggest that EPA in S. livingstonensis Ac10 is not required for bulk bilayer fluidity but plays a beneficial role in membrane organization and cell division at low temperatures, possibly through specific interaction between EPA and proteins involved in these cellular processes.Long-chain -3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) occur in organisms from bacteria to humans as the acyl group of phospholipids in the membrane. These fatty acids have been attracting a great deal of attention, mainly because they have beneficial effects on human health (1, 5, 9). Many biophysical studies have been conducted with model membranes and have revealed that PUFAs significantly alter the basic properties of lipid bilayers such as fluidity, acyl chain order, phase behavior, elastic compressibility, and permeability (31). However, despite accumulating information on the properties of PUFA-containing bilayers, information on the physiological role of PUFAs and their molecular mode of action in living cells is very limited.Various marine gram-negative bacteria that inhabit cold and high-pressure environments such as the polar regions and the deep sea produce EPA and DHA as components of their membrane phospholipids (18). These bacteria, which belong to the genera of Shewanella, Photobacterium, Moritella, Colwellia, and Psychromonas, have a gene cluster solely dedicated to the synthesis of PUFA (3,18,21,26). The gene cluster involved in the synthesis of PUFA was first cloned from Shewanella pneumatophori SCRC-2738 (formerly Shewanella sp. strain SCRC-2738) (36). Five genes in the cluster, showin...

Piezotolerance of the Respiratory Terminal Oxidase Activity of the PiezophilicShewanella violaceaDSS12 as Compared with Non-PiezophilicShewanellaSpecies

“…Fax: +81-3-5317-9433; E-mail: htamegai@chs.nihon-u.ac.jp psychrophile, Shewanella livingstonensis Ac10, the amount of EPA in the membrane lipid increased when the organism was cultured at lower temperatures, such as 4 C, and an EPA-deficient mutant of Ac10 was found to be cold-sensitive. 11,12) S. violacea DSS12 contains high levels of EPA (15% of total fatty acids) in its membrane, 2) and the EPA-deficient mutant of S. violacea showed slow growth (Kawamoto et al, manuscript in preparation). In addition, UFAs might play important roles in the piezophilic property of the bacteria.…”

Occurrence of a Bacterial Membrane Microdomain at the Cell Division Site Enriched in Phospholipids with Polyunsaturated Hydrocarbon Chains

“…Bacterial Strain and Growth Conditions-The EPA-less mutant of S. livingstonensis Ac10 used in this study was constructed by the disruption of orf5, one of the genes required for EPA synthesis (13). We previously found that this EPA-less mutant is longer than the wild-type strain because of retardation of cell division after DNA replication; this longer bacterial length facilitates microscopic analysis of the phospholipid subcellular localization, as fluorescence microscopic analysis of the much shorter wild-type strain cells is difficult.…”

“…1; see supplemental "Experimental Procedures" for the synthesis). In these phospholipids, OLA (9-cis-18:1) was introduced into the sn-1 position because OLA and OLA-containing phospholipids were shown to have neutral effects on the phenotype of the EPA-less mutant (13,15).…”

“…DHA interacts with membrane-associated G protein-coupled receptors to support their function (10 -12). In EPA-producing bacteria, EPA plays a beneficial role in cell division (13,14). However, the molecular mechanisms of EPA and DHA action are not well defined, in particular, their roles in coordinating the spatial organization of the phospholipids that contain them in biological membranes.…”

“…The Gram-negative bacterium Shewanella livingstonensis Ac10, which produces EPA as an sn-2-acyl chain of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) (13,15), is an excellent model organism for studies on in vivo EPA function for the following reasons (13,(15)(16)(17). (i) The genes coding for EPA synthase are known, enabling the construction of an EPA-less mutant by disruption of these genes.…”