Dual Membrane-spanning Anti-Sigma 2 Controls OMV biogenesis and Colonization Fitness in Bacteroides thetaiotaomicron

Bacteroides spp. are Gram-negative, gut commensals that shape the enteric landscape by producing o uter m embrane v esicles (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 D ual M embrane-spanning A nti-sigma factor 1 increased OMV production in Bacteroides thetaiotaomicron ( Bt ) by regulating members 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 D ual M embrane-spanning A nti-sigma factor 2 (Dma2 ) in controlling OMV biogenesis in Bt . We employ biochemical and proteomic analyses to show that mutation of dma2 increases OMV production in a manner that is dependent on the expression of its cognate sigma factor, das2 . The precise mechanism by which dma2 increases OMV biogenesis remains elusive. However, transcriptome analyses revealed that Δdma2 has decreased expression of select p olysaccharide u tilization 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 showed that Δdma2 exhibits delayed growth in the presence of select host-associated glycans. 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 demonstrates their importance in Bacteroides physiology.

D ual m embrane-spanning a nti-sigma factors (Dma) are a novel class of regulatory system found solely amongst Bacteroidota. Previous studies have demonstrated the role of Dma1 in vesiculation, but the overall role of the Dma family in Bacteroides physiology remains poorly understood. Here, we demonstrate that Dma2 modulates vesiculation and regulates the expression of select p olysaccharide u tilization loci (PULs) that target host-associated glycans. In vivo studies revealed that Dma2 is an important fitness determinant when competing against kin bacteria. This work begins characterizing the multifaceted involvement of Dma2 in OMV biogenesis, PUL regulation, and colonization fitness.