HtrA Contributes to Biofilm Formation in Mycobacterium smegmatis 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 ΔhtrA 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.