Abstract
Bacteroides spp. are gram-negative, gut commensals that shape the enteric landscape by producing outer membrane vesicles (OMVs) that degrade dietary fibers and traffic immunomodulatory biomolecules. Understanding the mechanism behind OMV biogenesis in Bacteroides spp. is necessary to determine their role in the gut. Recent studies showed that mutation of dual membrane-spanning anti-sigma factor 1 (Dma1) increased OMV production in Bacteroides thetaiotaomicron (Bt) by modulating the expression of its downstream regulon. Additional members of the Dma family have been identified, but very little is known regarding their roles in Bt. Here, we investigate the role of Dma2 in controlling OMV biogenesis in Bt. We employ biochemical and proteomic analyses to show that mutation of dma2 increases OMV production. This induction is dependent on the expression of its cognate sigma factor, das2, but the precise mechanism by which dma2 increases OMV biogenesis remains elusive. Transcriptome analyses revealed that Δdma2 displays decreased expression of select polysaccharide utilization loci (PULs) that primarily target host-associated glycans. Follow-up comparative proteomics showed that the PUL repertoire was most impacted in the OMV fraction. In vitro growth assessments confirmed that Δdma2 exhibits delayed growth in the presence of select host-associated glycans. In vivo co-colonization studies in mice revealed that Δdma2 is outcompeted by the wild-type in the gut, which indicates that Dma2 is a key determinant of colonization fitness in Bt. Altogether, these findings expand our knowledge of the Dma family's role in OMV biogenesis and demonstrate their importance in Bacteroides physiology. IMPORTANCE: Dual membrane-spanning anti-sigma factors (Dma) are a novel class of regulatory proteins found solely among Bacteroidota. Previous studies demonstrated the importance of Dma1 in vesiculation, but the overall role of the Dma family in Bacteroides physiology remains poorly understood. Here, we show that Dma2 modulates vesiculation and the expression of select polysaccharide utilization loci (PULs) that target host-associated glycans in vitro. Mouse studies revealed that Dma2 is an important fitness determinant in vivo when competing against kin bacteria. This work begins characterizing the multifaceted involvement of Dma2 in OMV biogenesis, PUL regulation, and colonization fitness.