HtrA Contributes to Biofilm Formation in <i>Mycobacterium smegmatis</i> by Downregulating the Cell Wall Amidase Ami3

Mycobacterium tuberculosis , 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 Mycobacterium smegmatis . This screen identified trypsin HtrA as a critical factor for growth and biofilm formation. Deletion of htrA 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 ami3 recapitulated the biofilm and growth defects of the Δ htrA strain. Furthermore, we found that the essential role of periplasmic protease HtrA for normal growth could be suppressed by novel mutations in pmt , 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.