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Plant cell wall degradation is a premier event when Bacillus subtilis, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in B. subtilis strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (yesOPQRSTUVWXYZ, ytePQRST, and ybcMOPST-ybdABDE) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium, B. subtilis strain natto, fermenting soybeans also express the gene cluster including the yes series during the assimilation of soybean used as a carbon source. Among proteins encoded in the yes cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus Aspergillus aculeatus. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation.Bacillus subtilis, the best-characterized gram-positive bacterium, is widely distributed and has various high levels of potential to incorporate external DNA; produce amino acids, antibacterial agents, and industrially important enzymes; secrete large amounts of proteins; form endospores; and associate with plants. The complete genome sequence for the type strain, B. subtilis 168, was determined in 1997, and about 4,000 genes were found contained in the bacterial genome (26). The clarification of function-unknown genes is now being focused on, and essential genes in the bacterium have been identified through the construction of knockout mutants for each gene (25). Although mechanisms for DNA uptake (10), amino acid fermentation (20), antibiotic production (49), protein secretion (49), and endospore formation (44) are well analyzed, little is known about the interaction between bacilli and plants.B. subtilis is important for producing fermented foods and for composting plants (41). In Japan, this bacterium is called "Kosoukin," which means "bacteria present in dead grass," and a type of B. subtilis, strain natto, has been used for the production of "natto" for over 1,000 years. Natto is produced through the fermentation of boiled soybeans by strain natto. Natto-like fermented soybean foods such as tempe and Thua nao are also produced in Asia and Africa. In natto production, cells of strain natto sprayed on boiled soybeans degrade their cell wall...

Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a ␤-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-L-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/⌬GGG) were determined at 2.10-and 2.99-Å resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an ␣/␣-barrel ؉ anti-parallel ␤-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/⌬GGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His 311 and Tyr 365 as the catalytic base and acid, respectively. Two structural determinants, i.e. a short ␣-helix in the central ␣/␣-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.Carbohydrate-active enzymes such as glycoside hydrolases, polysaccharide lyases, glycosyl transferases, and carbohydrate esterases are categorized into more than 200 families based on amino acid sequences in the Carbohydrate-Active enZYmes (CAZy) data base (1). Polysaccharide lyases (PLs) 2 are classified into 21 PL families and they play an important role in degrading acidic polysaccharides such as polygalacturonan, rhamnogalacturonan, alginate, chondroitin, hyaluronan, heparin, heparan, and xanthan. These lyases commonly recognize uronic acid residues in polysaccharides, catalyze ␤-elimination, and produce unsaturated saccharides with CϭC double bonds at nonreducing terminal uronate residues.Alginate, produced by brown seaweeds as a major component of the cell wall matrix, is a linear polysaccharide composed of ␣-L-guluronate (G) and its C5 epimer ␤-D-mannuronate (M). Three blocks, such as poly-␣-L-guluronate (poly(G)), poly-␤-D-mannuronate (poly(M)), and heteropolymeric random sequences (poly(MG)), constitute the alginate polymer (2). The alginate degradation pathway has been characterized in some bacteria, virus, and abalone. In the bacterium Sphingomonas sp. strain A1, alginate is directly incorporated into the cytoplasm by the cell surface pit, periplasmic binding proteins, and ATPbinding cassette transporter. The incorporated alginate is subsequently depolymerized into unsaturated disaccharides, trisaccharides, and tetrasaccharides through the action of three cytoplasmic endotype alginate lyases, A1-I, A1-II, and A1-III (Fig. 1A) (3). The unsaturated oligosaccha...

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AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of AmyI-1-18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, AmyI-1-18 exhibited antimicrobial activity against nine. Its 50% growth-inhibitory concentrations (ICs50 ) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 μM, respectively. AmyI-1-18 had little or no hemolytic activity even at 500 μM, and showed negligible cytotoxicity up to 1200 μM. The degree of 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of AmyI-1-18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA-intercalating dye, were 89%, 43%, and 3%, respectively, when AmyI-1-18 was added at a concentration equal to its 4×IC50 . Therefore, the antimicrobial activity of AmyI-1-18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane-disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that AmyI-1-18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare.

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