Xiaolong Liu

Bacteria of the phylum Bacteroidota are major human commensals and pathogens in addition to being abundant members of the wider biosphere. Bacteroidota move by gliding and they export proteins using the Type 9 Secretion System (T9SS). Here we discover that gliding motility and the T9SS share an unprecedented mechanism of energisation in which outer membrane proteins are covalently attached by disulfide bonds to a moving internal track structure that propels them laterally through the membrane. We determined the structure of an exemplar Bacteroidota mobile track by obtaining the cryoEM structure of a 3 MDa circular mini-track from Porphyromonas gingivalis . Our discoveries identify a mechanistic and evolutionary link between gliding motility and T9SS-dependent protein transport.

In Gram-negative bacteria, the outer membrane is the first line of defence against antimicrobial agents and immunological attacks ¹ . A key part of outer membrane biogenesis is the insertion of outer membrane proteins by the β-barrel-assembly machinery (BAM) ^2–4 . Here we report the cryo-electron microscopy structure of a BAM complex isolated from Flavobacterium johnsoniae , a member of the Bacteroidota, a phylum that includes key human commensals and major anaerobic pathogens. This BAM complex is extensively modified from the canonical Escherichia coli system and includes an extracellular canopy that overhangs the substrate folding site and a subunit that inserts into the BAM pore. The novel BamG and BamH subunits that are involved in forming the extracellular canopy are required for BAM function and are conserved across the Bacteroidota, suggesting that they form an essential extension to the canonical BAM core in this phylum. For BamH, isolation of a suppressor mutation enables the separation of its essential and non-essential functions. The need for a highly remodelled and enhanced BAM complex reflects the unusually complex membrane proteins found in the outer membrane of the Bacteroidota.

Bacteria of the phylum Bacteroidota are major human commensals and pathogens in addition to being abundant members of the wider biosphere. Bacteroidota move by gliding and they export proteins using the Type 9 Secretion System (T9SS). Here we discover that gliding motility and the T9SS share a novel mechanism of energisation in which outer membrane proteins are covalently attached by disulfide bonds to a moving internal track structure which propels them through the membrane. We determined the structure of a representative Bacteriodota mobile track by obtaining the cryoEM structure of a 3 MDa circular mini-track from Porphyromonas gingivalis . Our discoveries identify a novel mechanistic and evolutionary link between gliding motility and T9SS-dependent protein transport.

In Gram-negative bacteria the outer membrane (OM) is the first line of defence against antimicrobial agents and immunological attacks. A key part of OM biogenesis is insertion of OM proteins (OMPs) by the β-barrel–assembly machinery (BAM). Here we report the cryo-electron microscopy (cryoEM) structure of a BAM complex isolated from Flavobacterium johnsoniae , a member of the phylum Bacteroidota that includes key human commensals and major anaerobic pathogens. This BAM complex is radically different from the canonical Escherichia coli system and includes an extensive extracellular canopy that overhangs the substrate folding site and a subunit that inserts into the BAM pore. We find that two of the novel subunits involved in forming the extracellular canopy are essential for BAM function. For one of these subunits isolation of a suppressor mutation allows the separation of their essential and non-essential functions. The need for a highly remodelled and enhanced BAM complex reflects the unusually complex membrane proteins found in the OM of the Bacteroidota.

Insertion of new material into theEscherichia colipeptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA.In vivocrosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.

Rod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a change in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the “on state” of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.