Benjamin Cooley

The Vps/VacJ ABC transporter is required for intercellular spread of shigella flexneri

ABSTRACT The Vps/VacJ ABC transporter system is proposed to function in maintaining the lipid asymmetry of the outer membrane. Mutations in vps or vacJ in Shigella flexneri resulted in increased sensitivity to lysis by the detergent sodium dodecyl sulfate (SDS), and the vpsC mutant showed minor differences in its phospholipid profile compared to the wild type. vpsC mutants were unable to form plaques in cultured epithelial cells, but this was not due to a failure to invade, to replicate intracellularly, or to polymerize actin via IcsA for movement within epithelial cells. The addition of the outer membrane phospholipase gene pldA on a multicopy plasmid in a vpsC or vacJ mutant restored its resistance to SDS, suggesting a restoration of lipid asymmetry to the outer membrane. However, the pldA plasmid did not restore the mutants ability to form plaques in tissue culture cells. Increased PldA levels also failed to restore the mutants phospholipid profile to that of the wild type. We propose a dual function of the Vps/VacJ ABC transporter system in S. flexneri in both the maintenance of lipid asymmetry in the outer membrane and the intercellular spread of the bacteria between adjacent epithelial cells.

The extracellular polysaccharide Pel makes the attachment of P. aeruginosa to surfaces symmetric and short-ranged

Biofilms are surface-mounted, multicellular communities of microbes. Biofilms are often associated with chronic infections that resist treatment, evade the immune system, and damage host tissue. An essential characteristic of the biofilm state is that constituent organisms are bound in a polymeric matrix. This matrix gives the system spatial structure and clusters bacteria near each other, facilitating intercellular interactions. The Pseudomonas aeruginosa strain PAO1 is widely studied as a model biofilm-forming organism. The polymeric matrix of PAO1 biofilms is dominated by two bacteria-produced extracellular polymers, Pel and Psl. We use a combination of optical and atomic force microscopy to examine the roles of these polymers in very early biofilm development. In agreement with other researchers, we find that Psl mediates strong attachment to a glass surface. We find that Pel alone can mediate some attachment, but not as permanent as that mediated by Psl. Unexpectedly, we find that Pel promotes symmetric attachment, in the form of rod-shaped bacteria lying down flat on the surface, and that the presence of Pel makes attachment forces more short-ranged than they are with Psl alone. We suggest that these effects may result from synergistic interactions of Pel with the Psl polymeric matrix.

Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development

Significance It is well established that mechanical inputs, such as strain and elasticity, can be sensed by eukaryotic cells and can impact phenotype and behavior. In contrast, very little is known about how prokaryotes may respond to mechanical inputs. Here, we show that bacteria can sense shear and can respond by initiating biofilms. This is an important advance in fundamental microbiology and mechanobiology. Biofilms are difficult to prevent using extant approaches. Our knowledge points the way to a hitherto-undeveloped type of antibiofilm surface that thwarts mechanosensing by not sustaining sufficiently high shear. This would prevent bacteria from sensing surface attachment, activating cyclic-di-GMP signaling, and forming a biofilm. Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For Pseudomonas aeruginosa, it has long been known that intracellular levels of the signal cyclic-di-GMP increase upon surface adhesion and that this is required to begin biofilm development. However, what cue is sensed to notify bacteria that they are attached to the surface has not been known. Here, we show that mechanical shear acts as a cue for surface adhesion and activates cyclic-di-GMP signaling. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We show that the envelope protein PilY1 and functional type IV pili are required mechanosensory elements. An analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides describes our data well.

Asymmetry and inequity in the inheritance of a bacterial adhesive

We thank Professor Matthew Parsek (University of Washington, Seattle) for his generous gift of bacterial PAO1 strains.Wealso thank Professor Marvin Whiteley (University of Texas at Austin) forWTandΔpsl polysaccharide preparations. SIM imaging (for figure 1) was performed in the Microscopy Core Facility within the Institute for Cellular and Molecular Biology atUTAustin, with the assistance of Julie Hayes. This work was funded by startup funds fromUTAustin and a gift from ExxonMobil to VDG, and by a grant from the Human Frontiers Science Program (HFSP RGY0081/2012-GORDON).