σ^P-NagA-L1/L2 Regulatory Circuit Involved in ΔompA_299-356Mediated Increase in β-Lactam Susceptibility in Stenotrophomonas maltophilia

OmpA, the most abundant porin in Stenotrophomonas maltophilia KJ, exists as a two-domain structure with an N-terminal domain of β-barrel structure embedded in the outer membrane and a C-terminal domain collocated in the periplasm. KJΔOmpA_299-356, an ompA mutant of S. maltophilia KJ with a truncated OmpA devoid of 299 to 356 amino acids (aa), was able to stably embed in the outer membrane. KJΔOmpA_299-356 was more susceptible to β-lactams than wild-type KJ. We aimed to elucidate the mechanism underlying the ΔompA_299-356-mediated increase in β-lactam susceptibility (abbreviated as “ΔOmpA_299-356 phenotype”). KJΔOmpA_299-356 displayed a lower ceftazidime (CAZ)-induced β-lactamase activity than KJ. Furthermore, KJ2, a L1/ L2 β-lactamases-null mutant, and KJ2ΔOmpA_299-356, a KJ2 mutant with truncated OmpA devoid of299 to 356 aa, had comparable β-lactam susceptibility. Both lines of evidence indicate that decreased β-lactamase activity contributes to the ΔOmpA_299-356 phenotype. We analyzed the transcriptome results of KJ and KJΔOmpA_299-356, focusing on PG homeostasis-associated genes. Among the 36 genes analyzed, the nagA gene was upregulated 4.65-fold in KJΔOmpA_299-356. Deletion of the nagA gene from the chromosome of KJΔOmpA_299-356 restored β-lactam susceptibility and CAZ-induced β-lactamase activity to wild-type levels, verifying that nagA-upregulation in KJΔOmpA_299-356 contributes to the ΔOmpA_299-356 phenotype. Furthermore, transcriptome analysis revealed that rpoE (Smlt3555) and rpoP (Smlt3514) were significantly upregulated in KJΔOmpA_299-356. The deletion mutant construction, β-lactam susceptibility, and β-lactamase activity analysis demonstrated that σ^P, but not σ^E, was involved in the ΔOmpA_299-356 phenotype. A real-time quantitative (qRT-PCR) assay confirmed that nagA is a member of the σ^P regulon. The involvement of the σ^P-NagA-L1/L2 regulatory circuit in the ΔOmpA_299-356 phenotype was manifested.