HtrA Contributes to Biofilm Formation in Mycobacterium smegmatis by Downregulating the Cell Wall Amidase Ami3

<i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, utilizes biofilm formation as a key mechanism to withstand host-derived stresses. To identify novel factors involved in this process, we performed a CRISPRi screen in the model organism <i>Mycobacterium smegmatis</i>. This screen identified trypsin HtrA as a critical factor for growth and biofilm formation. Deletion of <i>htrA</i> led to a profound upregulation of the cell wall amidase Ami3. We demonstrated that Ami3 is a crucial negative regulator of biofilm formation, as overexpression of <i>ami3</i> recapitulated the biofilm and growth defects of the Δ<i>htrA</i> strain. Furthermore, we found that the essential role of periplasmic protease HtrA for normal growth could be suppressed by novel mutations in <i>pmt</i>, a gene encoding a phosphomyoinositol mannosyltransferase, at residues F53 and N55, distinct from the previously reported D68 site. Our findings establish a novel regulatory pathway in which HtrA modulates mycobacterial biofilm formation by controlling the levels of Ami3 and reveal new genetic interactions within this network.