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A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium "Candidatus Pelagibacter sp." Strain HTCC7211.

ABSTRACT: In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, nonphosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GT_cp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211, a member of the SAR11 clade. Our results showed that GT_cp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol (DAG) as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg²⁺ notably changes the enzyme's affinity for UDP-glucose, which improves its catalytic efficiency. Homology modeling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GT_cp with its GT-B fold. A phylogenetic analysis showed that GT_cp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling but also describe an efficient enzymatic tool for the future synthesis of bioactive molecules. IMPORTANCE The bilayer formed by membrane lipids serves as the containment unit for living microbial cells. In the marine environment, it has been firmly established that phytoplankton and heterotrophic bacteria can replace phospholipids with nonphosphorus sugar-containing glycoglycerolipids in response to phosphorus limitation. However, little is known about how these glycoglycerolipids are synthesized. Here, we determined the biochemical characteristics of a glycolipid glycosyltransferase (GT_cp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211. GT_cp and its homologs form a group in the GT4 glycosyltransferase family and can synthesize neutral glycolipids (monoglucosyl-1,2-diacyl-sn-glycerol [MGlc-DAG] and monogalactosyl [MGal]-DAG) and monoglucuronic acid diacylglycerol (MGlcA-DAG). We also uncovered the key residues for DAG binding through molecular docking, site-direct mutagenesis, and subsequent enzyme activity assays. Our data provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling.

SUBMITTER: Wei T

PROVIDER: S-EPMC8231724 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 μm3 Strikingly, the periplasmic space occupied ∼20% to 50% of the total cell volume in log-phase cells and ∼50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/μm3 Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of "Candidatus Pelagibacter ubique," the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria.

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A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium "Candidatus Pelagibacter sp." Strain HTCC7211.

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