Plant Cell Wall Degradation by Saprophytic Bacillus subtilis Strains: Gene Clusters Responsible for Rhamnogalacturonan Depolymerization

Abstract

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...

Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization

“…Its strong binding to peptidoglycan suggests that it is anchored to the bacterial surface, but its actual location is unknown. EXLX1 might promote subtle modification of the plant cell wall, perhaps aiding plant cell wall breakdown by enzymes secreted by B. subtilis (32). A similar synergism between EXLX1 and peptidoglycan hydrolases could also explain the slightly more efficient autolysis of wild-type B. subtilis compared with the EXLX1 Ϫ mutant.…”

Selection of Bacillus species for targeted in situ release of prebiotic galacto-rhamnogalacturonan from potato pulp in piglets

“…Here the bacteria were grown aerobically in both a rich and a minimal medium. Pectin was added to both growth media, because it has been shown previously to induce expression of genes involved in pectin degradation (Ochiai et al 2007). …”

A Novel Structural Fold in Polysaccharide Lyases

“…Protein Expression and Purification-Protein expression and purification were conducted as described previously (24,37 (200 pg). The N-terminal 37 amino acid residues published in the DNA/ protein data base (YesW, 620 amino acid residues, GenPept accession no.…”

“…The crystal structure of a family PL-4 RG lyase, RhgB, has been determined from Aspergillus aculeatus KSM 510 (33), although its structure and function relationship remains to be clarified. Family PL-11 RG lyases Rgl11A from Cellvibrio japonicus (34), Rgl11Y from Clostridium cellulolyticum (35), and YesW and YesX from B. subtilis (24) have been characterized enzymatically, but no structural analysis of these enzymes has, to our knowledge, been reported. Because family PL-11 enzymes show no significant homology to other proteins, including polysaccharide lyases, their structural fold exhibits a novel type that has not been observed in polysaccharide lyases analyzed so far.…”