The transcription factor CwiR is important for envelope integrity in Burkholderia thailandensis

The bacterial cell envelope, particularly the outer membrane of Gram-negative species, is essential for survival and intrinsic resistance to antibiotics. However, the regulatory mechanisms that maintain envelope integrity have only been partially elucidated. Members of the genus Burkholderia are notorious for their resistance to antimicrobial agents, including Burkholderia thailandensis , which is closely related to highly pathogenic members of this genus, but less virulent in humans, and therefore a commonly used surrogate for studying virulence mechanisms. A conserved operon comprising BTH_I2558 - I2561 was characterized here; BTH_I2558 encodes a member of the multiple antibiotic resistance (MarR) family of transcription factors, which was named CwiR based on inferred roles in maintenance of cell wall integrity, and the other genes are predicted to encode a tripartite efflux pump. Comparing the transcriptomes of WT B. thailandensis and a d cwiR strain in which BTH_I2558 was disrupted, we found differential expression of 273 genes in the d cwiR strain, including the adjacent efflux pump genes and several genes involved in cell wall integrity. Using quantitative mass spectrometry, numerous proteins were found to accumulate differentially in d cwiR cells, including proteins involved in cell wall biosynthesis. The d cwiR strain exhibited increased sensitivity to several antibiotics, and it exhibited increased dye uptake, suggesting a compromised outer membrane. Analysis of reads mapping to the divergent BTH_I2557 and BTH_I2558 genes suggests that CwiR is important for appropriate transcription start site selection for both genes, and that a truncated cwiR mRNA is produced in the d cwiR strain. While MarR-family proteins are typically autoregulatory, the altered start site selection in cells lacking CwiR suggests that absence of CwiR binding to the cwiR promoter may not lead to accumulation of functional protein and an immediate return to the repressed state. Taken together, our data suggest that the MarR-family transcription factor CwiR is a key regulator of cell envelope integrity in B. thailandensis . Disruption of cwiR alters accumulation of numerous envelope-associated proteins, increases membrane permeability, and enhances antibiotic susceptibility, revealing a previously unrecognized regulatory pathway that contributes to envelope homeostasis.