Genomic and Proteomic Analyses of the Agarolytic System Expressed bySaccharophagus degradans2-40

Abstract

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a ␤-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surfaceassociated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an ␣-neoagarobiose hydrolase to release galactose from agarose.Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a rod-shaped, aerobic, marine bacterium isolated from the surface of decomposing saltwater cord grass, Spartina alterniflora, in the lower Chesapeake Bay (3). S. degradans 2-40 is related to a group of marine ␥-subgroup proteobacteria capable of degrading complex polysaccharides (CPs) (14, 16), a critical function in the marine food web. S. degradans 2-40 is unique among these bacteria due to its ability to utilize CPs of algal, higher plant, fungal, and animal origins, such as agar, alginate, cellulose, chitin, ␤-glucan, laminarin, pectin, pullulan, starch, and xylan, as sole carbon and energy sources (3,15,20,23). The mechanism by which this bacterium degrades these normally recalcitrant substrates has been established only for the chitinolytic system (20).Agar, a cell wall constituent of many red algae (Rhodophyta), exists in nature as a mixture of unsubstituted and substituted agarose polymers that form an agarocolloid gel (10, 12). Agarose is composed of repeating neoagarobiose units (3-6-anhydro-L-galactose-␣1-3-D-galactose) joined by ␤1-4 bonds that form a helix in aqueous environments. The galactose moieties of the repeating neoagarobiose units can be methylated, pyruvated, sulfonated, or glycosylated to form various substituted derivatives with different gelling and solubility characteristics.Up to 70% of the algal cell wall can be agar polymers. The remaining material consists of other galactans and embedded xylan and cellulose microfibri...

Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

“…For example, Z. galactanivorans utilizes mainly GH16 ␤-agarases (42) to degrade agar, whereas the estuarine bacterium S. degradans utilizes a mixture of GH86, GH50, and GH16 (43). All of these organisms also contain at least one gene encoding a conserved GH117 (supplemental Table 1), raising the question whether GH117 enzymes are the predominant enzymes to break the ␣-glycosidic bond in agarose or whether there are alternative endo-acting ␣-agarases that may play a significant role in agar degradation.…”

Enhanced Agarose and Xylan Degradation for Production of Polyhydroxyalkanoates by Co-Culture of Marine Bacterium, Saccharophagus degradans and Its Contaminant, Bacillus cereus

“…In the first approach, the batch growth kinetics and PHA synthesis ability of Sde 2-40, B. cereus, and the co-culture were examined after being grown on xylan or agarose as the sole carbon source in minimal media. In the second approach, the biosynthesis of PHA in Sde2-40, B. cereus, and the co-culture was investigated by two-step batch culture under nitrogen source (NH 4 Cl and yeast extract) limitation using agarose and xylan as the sole source of carbon. Inoculum (30 mL) was added to sterilized medium (300 mL) in a 500 mL capacity flask.…”

“…Inoculum (30 mL) was added to sterilized medium (300 mL) in a 500 mL capacity flask. First, cells were left in balanced nutrient environments for 100 h at 30 • C. After recovering biomass aseptically by centrifugation and washing with 2.3% (w/v) sterile NaCl solution, cells were added to sterilized medium (300 mL) without NH 4 Cl and yeast extract as nitrogen sources in a 500 mL flask and incubated for 48 h at 30 • C.…”

“…Saccharophagus degradans 2-40 (Sde 2-40; early name Microbulbifer degradans 2-40), a motile gamma proteobacterium of marine origin, uniquely utilises different carbon sources and degrades a variety of complex polysaccharides such as agar, alginic acid, carrageenan, cellulose, chitin, glucan, laminarin, pectin, pullulan, starch, and xylans [1][2][3][4][5][6]. Sde 2-40 can powerfully degrade polysaccharides from plant and marine sources.…”

“…Xylan, the principal constituent of plant cell wall hemicellulose, is copiously occurring polysaccharide next to cellulose [9]. Sde 2-40 has the capability of utilizing agarose and xylan for growth [1,4,6].…”