coliBL21(DE3) pLysS cells was diluted to a cell density of 0.1 at 600 nm in LB medium containing kanamycin (50 g/ml) and shaken vigorously at 37C. cadherin and cadherin-like multiple domains contribute to protein-protein interactions that may mediate cell-cell contact in the marine environment. Saccharophagus degradansstrain 2-40 is the first free-living marine bacterium demonstrated to be capable of degrading cellulose of algal origin and higher plant material (29,31).S. degradans2-40 was isolated from decaying salt marsh cord grass,Spartina alterniflora, in the Chesapeake Bay watershed (3,12). It is a pleomorphic, Gram-negative, aerobic, motile gammaproteobacterium, uniquely degrading at L-Hexanoylcarnitine least 10 different complex polysaccharides, including agar, chitin, alginic acid, cellulose, -glucan, laminarin, pectin, pullulan, starch, and xylan (9,10,15,16,30). These enzymatic capabilities suggest that the bacteriumS. degradansmay have a significant role in the marine carbon cycle, mediating the degradation ERYF1 of complex polysaccharides from plants, algae, and invertebrates (30,31). TheS. degradans2-40 genome has been sequenced (http://genome.jgi-psf.org/finished_microbes/micde/micde.home.html; GenBank accession numbersCP000282andNC_007912). It has 4,008 genes in a single replicon consisting of 5.06 Mb. The genome codes for 15 polypeptides longer than 2,000 amino acids (aa), ranging from 274 to 1 1,600 kDa. Each contains multiple domains and motifs that are reported to bind calcium ions and mediate protein-protein interactions (30,31). They are acidic, pI 3.5 to 4.9, and have a secretion signal; cadherin (CA) and cadherin-like (CADG) domains are prevalent. Cadherins are a superfamily of transmembrane glycoproteins that mediate, Ca2+-dependent cell-cell adhesion in all solid tissues throughout the animal kingdom (25). At the molecular level, homotypic adhesion between cells arises from homophilic interactions between cadherin extracellular domains repeated in tandem (18,19). Each of these domains, consisting of approximately 110 amino acids, forms a -sandwich with Greek-key folding topology. The cadherin domains are mostly distributed in the metazoan lineage. According to the SMART database (accessed July L-Hexanoylcarnitine 2009), there are 23,340 CA domains in 3,673 proteins, and of these 3,481 (94.7%) are metazoan proteins and only 186 (5.06%) are bacterial proteins (six other proteins with CA domains belong toArchaea). Genomic analysis showed that CA domains and CADG domains are not L-Hexanoylcarnitine uncommon in bacteria (7). Focusing on the proteins Sde_3323 and Sde_0798, we tested the notion that they would interact as in metazoans, binding via calcium-dependent homophilic and heterophilic interactions. We also examined the possibility that they would directly interact with theS. degradans2-40 cell surface, playing a role in protein-protein interactions and cellular aggregation. == MATERIALS AND METHODS == == Strains, media, and culture conditions. == S. degradansstrain 2-40 or ATCC 43961 (Sde 2-40; formerlyMicrobulbifer degradansstrain 2-40) was isolated from decaying salt marsh cord grass,S. alterniflora, in the Chesapeake Bay watershed (3).Escherichia colistrain XL-1 Blue was used for plasmid constructions and strain BL21(DE3) pLysS was used for protein overexpression.S. degradans2-40 was grown in minimal medium containing (per liter) 23 g of Instant L-Hexanoylcarnitine Ocean salts (Aquarium Systems, Mentor, OH), 5 g of NH4Cl, 2 g of carbon source, and 50 mM Tris-HCl (pH 7.6); 20 g of agar per liter was used to prepare solid medium. The carbon sources used in this study included agar (Acumedia Manufacturers, Inc. Lansing, MI), agarose (Hispanagar, Burgos, Spain), cellulose (Sigma Aldrich, Munich, Germany), cellobiose (Sigma Aldrich, Munich, Germany), chitin from crab shells (practical grade; Sigma Aldrich, Munich, Germany), lichenan (Sigma Aldrich, Munich, Germany), pectin (Sigma Aldrich, Germany), starch (Sigma Aldrich, Munich, Germany), xylan from oat spelt (Sigma Aldrich, Munich, Germany), xylan from birch wood (Sigma Aldrich, Munich, Germany), sea broth 2216 (Difco Laboratories, MD), and blood sugar (Sigma Aldrich, Munich, Germany). All civilizations had been incubated at 30C.E. coliXL1-Blue and BL21(DE3) pLysS strains had been grown up in Luria-Bertani (LB) broth or agar supplemented with 50 g/ml kanamycin and incubated at 37C. == Cloning and molecular biology protocols. == All DNA manipulations and regular molecular biology protocols.