DltX of Bacillus thuringiensis Is Essential for D-Alanylation of Teichoic Acids and Resistance to Antimicrobial Response in Insects

Abstract

The <i>dlt</i> operon of Gram-positive bacteria is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TAs). Addition of D-alanine to TAs reduces the negative charge of the cell envelope thereby preventing cationic antimicrobial peptides (CAMPs) from reaching their target of action on the bacterial surface. In most gram-positive bacteria, this operon consists of five genes <i>dltXABCD</i> but the involvement of the first ORF (<i>dltX</i>) encoding a small protein of unknown function, has never been investigated. The aim of this study was to establish whether this protein is involved in the D-alanylation process in <i>Bacillus thuringiensis</i>. We, therefore constructed an in frame deletion mutant of <i>dltX</i>, without affecting the expression of the other genes of the operon. The growth characteristics of the <i>dltX</i> mutant and those of the wild type strain were similar under standard <i>in vitro</i> conditions. However, disruption of <i>dltX</i> drastically impaired the resistance of <i>B. thuringiensis</i> to CAMPs and significantly attenuated its virulence in two insect species. Moreover, high-performance liquid chromatography studies showed that the <i>dltX</i> mutant was devoid of D-alanine, and electrophoretic mobility measurements indicated that the cells carried a higher negative surface charge. Scanning electron microscopy experiments showed morphological alterations of these mutant bacteria, suggesting that depletion of D-alanine from TAs affects cell wall structure. Our findings suggest that DltX is essential for the incorporation of D-alanyl esters into TAs. Therefore, DltX plays a direct role in the resistance to CAMPs, thus contributing to the survival of <i>B. thuringiensis</i> in insects. To our knowledge, this work is the first report examining the involvement of <i>dltX</i> in the D-alanylation of TAs.