Implication of the ^E Regulon Members OmpO and ^N in the ompA_299-356-Mediated Decrease of Oxidative Stress Tolerance in Stenotrophomonas maltophilia

The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJ Delta OmpA(299-356), an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by Delta ompA(299-356). The transcriptomes of wild-type S. maltophilia and the KJ Delta OmpA(299-356) mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJ Delta OmpA(299-356). KJ Delta OmpA(299-356) complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, sigma factor expression levels were examined based on the transcriptome results. The expression levels of three sigma factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJ Delta OmpA(299-356). Next, the involvement of the three sigma factors in the Delta ompA(299-356)-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the Delta ompA(299-356)-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated sigma(E) decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the Delta ompA(299-356)-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation.IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal beta-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) sigma factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating sigma(P) and sigma(E) expression levels. The outcomes of sigma(P) and sigma(E) activation are different and are linked to beta-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.