Abstract
Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium Vibrio splendidus One strain, V. splendidus 13B01, exhibited high extracellular alginate lyase activity compared with other V. splendidus strains. To identify the enzymes responsible for this high extracellular activity, we compared V. splendidus 13B01 with the previously characterized V. splendidus 12B01, which has low extracellular activity and lacks two alginate lyase genes present in V. splendidus 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to V. splendidus 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions.
Highlights
Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment
We focused on V. splendidus 13B01 due to its high extracellular alginate lyase activity
We focused on the high extracellular lyase activity exhibited by some strains but not others
Summary
Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Glycans play a central role in the marine carbon cycle because they represent 30 – 80% of the total carbon content in algal matter, yet measurements of residual algal biomass in sinking particles obtained with sediment traps indicate that Ͼ99% of the autotrophic biomass that is produced at the surface, including the glycans, disappears throughout the water column [5,6,7] These results suggest that microbes, key decomposers of organic matter, have the capacity to efficiently deconstruct the wide variety of glycans produced by algae in the sea. The V. splendidus strains all exhibited similar levels of intracellular and membrane-bound alginate lyase activity, suggesting that some strains acquired additional lyases with high extracellular activity These results are in line with a previous bioinformatics study revealing very high genotypic microdiversity of extracellular CAZymes, even among very closely related microbes [14]. Our results demonstrate that differences in the enzyme repertoires between closely related strains can be used to rapidly pinpoint key proteins with beneficial functions
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