Project Summary
Bacteroides spp. are Gram-negative, gut commensals that comprise ~40% of the human gut microbiota.
Previous studies have shown that Bacteroides spp. produce large quantities of outer membrane vesicles (OMVs)
believed to shape the enteric landscape. Bacteroides OMVs have been shown to modulate host immunity and
function as “public goods” to digest dietary and host glycans. However, the physiological relevance of
Bacteroides OMVs in the gut remains controversial because the mechanism by which OMVs are produced in
Gram-negative bacteria is currently unknown. To begin unraveling the mechanism of OMV biogenesis in
Bacteroides thetaiotaomicron (Bt), I designed a system to enable the identification of Bt mutants with altered
OMV production. This was achieved by developing an OMV reporter consisting of BACOVA_04502 (Inulinase,
INL), a protein previously found to be enriched in Bt OMVs, fused to Nanoluciferase (Nluc). Once constructed, I
used my reporter strain to conduct a transposon mutagenesis screen to identify genes that modulate OMV
biogenesis. Given that INL-Nluc is almost exclusively present in OMVs, mutants displaying abnormal levels of
Nluc activity corresponded to mutants producing abnormal levels of OMVs. While screening, I identified four
independent transposon mutants in the gene BT_4721. I demonstrate that BT¿4721 produces significantly more
OMVs than the wild type (WT). BT_4721 is annotated as an OMP_b-brl_2 domain-containing protein of unknown
function and is encoded in an operon with a putative extracytoplasmic function (ECF)-type sigma factor,
BT_4720. ECF-type sigma factors are a family of transcriptional regulators that modulate gene expression in
response to extracytoplasmic signals. These are typically encoded adjacent to their cognate anti-sigma factor,
which negatively regulates the activity of the sigma factor. Indeed, disruption of BT_4720 reverts the phenotype
of BT¿4721. Structural analyses predict that BT_4721 encodes an eight stranded ß-barrel domain, an intrinsically
disordered region, and an alpha helical transmembrane region. The predicted domain architecture suggests that
BT_4721 spans both the inner and outer membranes to function as a cell surface receptor and anti-sigma factor,
all in a single polypeptide. In addition, RNA sequencing of BT¿4721 revealed three genes of unknown function,
BT_1287, BT_4005, and BT_4719, are most significantly upregulated. BT_4005 and BT_4719 are both
annotated as NigD-like proteins, and I show that BT_4005 is necessary for the hypervesiculation phenotype
observed in BT_4721. Interestingly, BT_4005 is encoded in the chromosome next to genes involved in the
synthesis of lipids and lipopolysaccharides. Based on my findings, we hypothesize that BT_4721 is a novel anti-
sigma factor that modulates OMV biogenesis through its sigma factor-controlled regulon. In Aim 1, various
methods will be employed to confirm that BT_4721 functions as a structurally novel anti-sigma factor. In Aim 2,
I will determine the role of BT_4005 in inducing OMV production. Results from this proposal will provide critical
information to develop our understanding of OMV biogenesis in Bt.
