Knockout of ykcB, a putative glycosyltransferase, leads to vancomycin resistance in Bacillus subtilis

Abstract

ABSTRACTVancomycin resistance of gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused vancomycin resistance in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to β-lactams, and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had: i) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose, ii) a decreased amount of lipoteichoic acid, and iii) decreased biofilm formation ability. These phenotypes and vancomycin resistance were abolished by knockout of ykcC, a ykcB-operon partner involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin resistance in both wild-type B. subtilis and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to resistance to vancomycin, decreased biofilm formation ability, and increased pathogenicity to silkworms.IMPORTANCEAlthough vancomycin is effective against gram-positive bacteria, vancomycin-resistant bacteria is a major public health concern. While the vancomycin resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well-studied, they remain unclear in other gram-positive bacteria. In the present study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin resistance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in gram-positive bacteria.