This study demonstrates that prumycin is a major factor in SD-32's suppression of cucumber powdery mildew disease. Our findings shed light for the first time on prumycin's role in biocontrol by Bacillus against this disease. © 2017 Society of Chemical Industry.

Paenibacillus sp. W-61 is capable of utilizing water-insoluble xylan for carbon and energy sources and has three xylanase genes, xyn1, xyn3, and xyn5. Xyn1, Xyn3, and Xyn5 are extracellular enzymes of the glycoside hydrolase (GH) families 11, 30, and 10, respectively. Xyn5 contains several domains including those of carbohydrate-binding modules (CBMs) similar to a surface-layer homologous (SLH) protein. This study focused on the role of Xyn5, localized on the cell surface, in water-insoluble xylan utilization. Electron microscopy using immunogold staining revealed Xyn5 clusters over the entire cell surface. Xyn5 was bound to cell wall fractions through its SLH domain. A ⌬xyn5 mutant grew poorly and produced minimal amounts of Xyn1 and Xyn3 on water-insoluble xylan. A Xyn5 mutant lacking the SLH domain (Xyn5⌬SLH) grew poorly, secreting Xyn5⌬SLH into the medium and producing minimal Xyn1 and Xyn3 on water-insoluble xylan. A mutant with an intact xyn5 produced Xyn5 on the cell surface, grew normally, and actively synthesized Xyn1 and Xyn3 on water-insoluble xylan. Quantitative reverse transcription-PCR showed that xylobiose, generated from water-insoluble xylan decomposition by Xyn5, is the most active inducer for xyn1 and xyn3. Luciferase assays using a Xyn5-luciferase fusion protein suggested that xylotriose is the best inducer for xyn5. The cell surface Xyn5 appears to play two essential roles in water-insoluble xylan utilization: (i) generation of the xylo-oligosaccharide inducers of all the xyn genes from water-insoluble xylan and (ii) attachment of the cells to the substrate so that the generated inducers can be immediately taken up by cells to activate expression of the xyn system.

Paenibacillus sp. strain W-61, which can utilize xylan as the sole source of carbon and energy, produces extracellular xylanases 1 and 3 (Xyn1 and Xyn3) and cell surface xylanase 5. In this study we found that lppX, immediately downstream of xyn1, encodes a lipoprotein located on the outer layer of the cytoplasmic membrane and that the LppX lipoprotein is essential for the secretion of active Xyn1 across the cytoplasmic membranes. In Escherichia coli, wild-type LppX was destined for the inner layer of the outer membrane. Mutant LppX(C19A), in which Cys-19, a possible lipomodification residue, is replaced with Ala, was located in the periplasm without being anchored to the membranes. Another mutant, LppX(S20D S21D), with substitutions of Asp for Ser-20 and Ser-21 (conversion to an Asp-Asp signal for sorting to the inner membrane), resided on the outer layer of the inner membrane, demonstrating that LppX has the sorting property of a lipoprotein. E. coli harboring both xyn1 and lppX secreted active Xyn1 into the periplasm. In contrast, E. coli carrying xyn1 alone failed to do so, accumulating inactive Xyn1 in the cytoplasmic membranes. Exogenous LppX(C19A) liberated the inactive Xyn1, which had been stagnating in the inner membrane, into the medium as an active enzyme. Thus, we propose that LppX is a novel type of lipoprotein that assists Xyn1 in making the proper fold necessary for traveling across the cytoplasmic membranes to be secreted as an active enzyme.

The xylanolytic bacterium Paenibacillus sp. strain W-61 encodes three extracellular xylanase genes, xyn1, xyn3, and xyn5. In this study, we identified a transcriptional activator required for transcription of the xyn3 gene in strain W-61. The activator, AxyR, contained the highly homologous AraC-type DNA binding domain and required xylobiose, xylotriose, or xylotetraose as cofactor for binding to the xyn3 promoter region.