The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment.

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Saccharophagus degradans strain 2-40 is a representative of an emerging group of marine complex polysaccharide (CP)-degrading bacteria. It is unique in its metabolic versatility, being able to degrade at least 10 distinct CPs from diverse algal, plant and invertebrate sources. The S. degradans genome has been sequenced to completion, and more than 180 open reading frames have been identified that encode carbohydrases. Over half of these are likely to act on plant cell wall polymers. In fact, there appears to be a full array of enzymes that degrade and metabolize plant cell walls. Genomic and proteomic analyses reveal 13 cellulose depolymerases complemented by seven accessory enzymes, including two cellodextrinases, three cellobiases, a cellodextrin phosphorylase, and a cellobiose phosphorylase. Most of these enzymes exhibit modular architecture, and some contain novel combinations of catalytic and/or substrate binding modules. This is exemplified by endoglucanase Cel5A, which has three internal family 6 carbohydrate binding modules (CBM6) and two catalytic modules from family five of glycosyl hydrolases (GH5) and by Cel6A, a nonreducing-end cellobiohydrolase from family GH6 with tandem CBM2s. This is the first report of a complete and functional cellulase system in a marine bacterium with a sequenced genome.

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...

T , and can be further distinguished from members of these two genera by 16S rRNA gene cluster analysis, the ability to utilize 10 different complex polysaccharides as sole carbon sources, a significantly lower G+C content and differences in fatty acid content. The three genera of complex polysaccharide-degrading, marine bacteria now encompass 20 strains from diverse marine niches.The carbon cycle in marine habitats has not yet been elucidated, as many of the micro-organisms that degrade complex polysaccharides (CPs), especially those derived from higher plants and algae, have not been identified. These include highly specialized micro-organisms that recycle CP, a critical step in the marine food web. Within the past decade a number of such bacteria have been discovered.In 1997, a Gram-negative bacterium that degrades cellulose, xylan and chitin was isolated by Gonzalez et al. (1997) from a salt marsh in Georgia, USA, and named Microbulbifer hydrolyticus IRE-31 T . In 2002, Distel et al. (2002 reported taxonomic criteria for a shipworm symbiont that degrades cellulose and found them to be sufficiently different from the Microbulbifer taxon to be accorded a new genus, Teredinibacter. Within the past 2 years, sequences from six strains related to Microbulbifer have been deposited in the GenBank database.In 1986, Andrykovitch & Marx (1988) isolated bacteria involved in the degradation of a salt marsh grass, Spartina alterniflora, found in the lower Chesapeake Bay, USA. One of these was designated strain 2-40 T . Based upon phenotypic characteristics, it was placed with the alteromonads, where it resides today in the order 'Alteromonadales' of the 'Gammaproteobacteria', in the family Alteromonadaceae. Later, based on sequence analysis of the 16S rRNA gene, its distinctive cellular morphology and its CP-degradative capabilities, 2-40 T was considered to be closely related to the genus Microbulbifer. A more meaningful classification of strain 2-40 T had been hampered by the limited availability of 16S rRNA gene sequences from closely related organisms. Now, on the basis of more than 20 available sequences, we propose a new genus, Saccharophagus, to accommodate the most versatile marine carbohydrate degrader yet identified.

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Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25°C after 3 days of incubation. Incubation at 37°C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25°C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.Salmonella enterica serovar Typhimurium is a causative agent of gastrointestinal salmonellosis in humans. Phage type 104 (definitive type 104 [DT104]) strain of S. enterica serovar Typhimurium has recently emerged with resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline in the United States (3), the United Kingdom (25), France, Denmark (2), and Japan (23). This multidrug resistance has posed a major problem in the treatment of complications resulting from these Salmonella infections. Moreover, new strains with resistance to additional antibiotics, such as trimethoprim and fluoroquinolones, are beginning to emerge (18,26). The Centers for Disease Control and Prevention has reported that DT104 was identified in 32% of the S. enterica serovar Typhimurium strains isolated from humans in 1996, an increase from 28% in 1995 and 7% in 1990. DT104 is now only the second most prevalent type of Salmonella after serovar Enteritidis phage type 4 in the United Kingdom (13).During our study on S. enterica serovar Typhimurium DT104, we have observed a rugose phenotype, which has also been described for Vibrio cholerae after serial passages in alkaline peptone media (14,19,28) or in response to starvation (17,27,29). This phenotype of V. cholerae was defined by corrugated colony morphology, which was associated with the formation of exopolysaccharide (EPS) and cell aggregation.Because there was a question of whether the rugose variant of V. cholerae was virulent, a study was performed and showed that the rugose c...

Multiple industrial and medical uses of chitin and its derivatives have been developed in recent years. The demand for enzymes with new or desirable properties continues to grow as additional uses of chitin, chitooligosaccharides, and chitosan become apparent. Microorganisms, the primary degraders of chitin in the environment, are a rich source of valuable chitin-modifying enzymes. This review summarizes many methods that can be used to isolate and characterize chitin-modifying enzymes including chitin depolymerases, chitodextrinases, chitin deacetylases, N-acetylglucosaminidases, chitin-binding proteins, and chitosanases. Chitin analogs, zymography, detection of reducing sugars, genomic library screening, chitooligosaccharide electrophoresis, degenerate PCR primer design, thin layer chromatography, and chitin-binding assays are discussed.