Abstract
<jats:title>Abstract</jats:title> <jats:p> Despite growing interest in the MreBCD morphogenetic complex as a potential antimicrobial target, its function in <jats:italic>Pseudomonas aeruginosa</jats:italic> remains poorly understood. While previous studies using the MreB inhibitor A22 have established its role in cell shape maintenance and pilus regulation, the impact of <jats:italic>mreB</jats:italic> deletion has not been comprehensively investigated. Using genetic and microscopy-based approaches, we show that deletion of <jats:italic>mreB</jats:italic> is viable in <jats:italic>P. aeruginosa,</jats:italic> but results in spherical cells that lose all forms of motility despite retaining flagella. Importantly, we uncover a previously overlooked polar effect of the in-frame <jats:italic>mreB</jats:italic> deletion on the downstream <jats:italic>mreCD</jats:italic> genes and show, using CRISPRi-mediated silencing, that <jats:italic>mreCD</jats:italic> expression is essential for viability. <jats:italic>ΔmreB</jats:italic> mutants also display increased sensitivity to β-lactam antibiotics and enhanced initial surface attachment, yet form more compact biofilms with reduced dispersal. In mixed-culture biofilms, spherical <jats:italic>ΔmreB</jats:italic> cells are outcompeted by rod-shaped wild-type cells and remain confined to the biofilm base. The identification of natural <jats:italic>P. aeruginosa</jats:italic> isolates carrying truncated <jats:italic>mreB</jats:italic> alleles further indicates that loss of MreB function can be tolerated in natural populations. Together, our findings reveal important contributions of the MreBCD system to viability, morphogenesis, motility and biofilm development in <jats:italic>P. aeruginosa</jats:italic> , providing new insights into bacterial adaptation and informing the development of targeted antimicrobial strategies. </jats:p>