Tumescenamide C, a cyclic lipodepsipeptide from Streptomyces sp. KUSC_F05, exerts antimicrobial activity against the scab-forming actinomycete Streptomyces scabiei.

The antimicrobial activity of tumescenamide C against the scab-forming S. scabiei NBRC13768 was confirmed with a potent IC50 value (1.5 µg/mL). Three tumescenamide C-resistant S. scabiei strains were generated to compare their gene variants. All three resistant strains contained nonsynonymous variants in genes related to cellobiose/cellotriose transport system components; cebF1, cebF2, and cebG2, which are responsible for the production of the phytotoxin thaxtomin A. Decrease in thaxtomin A production and the virulence of the three resistant strains were revealed by the LC/MS analysis and necrosis assay, respectively. Although the nonsynonymous variants were insufficient for identifying the molecular target of tumescenamide C, the cell wall component wall teichoic acid (WTA) was observed to bind significantly to tumescenamide C. Moreover, changes in the WTA contents were detected in the tumescenamide C-resistant strains. These results imply that tumescenamide C targets the cell wall system to exert antimicrobial effects on S. scabiei.

Lack of Vacuolar H+ -Pyrophosphatase and Cytosolic Pyrophosphatases Causes Fatal Developmental Defects in Arabidopsis thaliana.

The cytosolic level of inorganic pyrophosphate (PPi) is finely regulated, with PPi hydrolyzed primarily by the vacuolar H+-pyrophosphatase (H+-PPase, VHP1/FUGU5/AVP1) and secondarily by five cytosolic soluble pyrophosphatases (sPPases; PPa1-PPa5) in Arabidopsis thaliana. Loss-of-function mutants of H+-PPase (fugu5s) have been reported to show atrophic phenotypes in their rosette leaves when nitrate is the sole nitrogen source in the culture medium. For this phenotype, two questions remain unanswered: why does atrophy depend on physical contact between shoots and the medium, and how does ammonium prevent such atrophy. To understand the mechanism driving this phenotype, we analyzed the growth and phenotypes of mutants on ammonium-free medium in detail. fugu5-1 showed cuticle defects, cell swelling, reduced ß-glucan levels, and vein malformation in the leaves, suggesting cell wall weakening and cell lethality. Based on the observation in the double mutants fugu5-1 ppa1 and fugu5-1 ppa4 of more severe atrophy compared to fugu5-1, the nitrogen-dependent phenotype might be linked to PPi metabolism. To elucidate the role of ammonium in this process, we examined the fluctuations of sPPase mRNA levels and the possibility of alternative PPi-removing factors, such as other types of pyrophosphatase. First, we found that both the protein and mRNA levels of sPPases were unaffected by the nitrogen source. Second, to assess the influence of other PPi-removing factors, we examined the phenotypes of triple knockout mutants of H+-PPase and two sPPases on ammonium-containing medium. Both fugu5 ppa1 ppa2 and fugu5 ppa1 ppa4 had nearly lethal embryonic phenotypes, with the survivors showing striking dwarfism and abnormal morphology. Moreover, fugu5 ppa1+/- ppa4 showed severe atrophy at the leaf margins. The other triple mutants, fugu5 ppa1 ppa5 and fugu5 ppa2 ppa4, exhibited death of root hairs and were nearly sterile due to deformed pistils, respectively, even when grown on standard medium. Together, these results suggest that H+-PPase and sPPases act in concert to maintain PPi homeostasis, that the existence of other PPi removers is unlikely, and that ammonium may suppress the production of PPi during nitrogen metabolism rather than stimulating PPi hydrolysis.