A new structural class of bacterial thioester domains reveals a slipknot topology

This work was supported by the MRC, UK grant MR/K001485 for MJB, USL; the BBSRC, UK grant BB/J00453 and the John Innes Foundation for MJB; The Royal Society of Edinburgh and the Carnegie Trust for OKM.An increasing number of surface‐associated proteins identified in Gram‐positive bacteria are characterized by intramolecular cross‐links in structurally conserved thioester, isopeptide, and ester domains (TIE proteins). Two classes of thioester domains (TEDs) have been predicted based on sequence with, to date, only representatives of Class I structurally characterized. Here, we present crystal structures of three Class II TEDs from Bacillus anthracis, vancomycin‐resistant Staphylococcus aureus, and vancomycin‐resistant Enterococcus faecium. These proteins are structurally distinct from Class I TEDs due to a β‐sandwich domain that is inserted into the conserved TED fold to form a slipknot structure. Further, the B. anthracis TED domain is presented in the context of a full‐length sortase‐anchored protein structure (BaTIE). This provides insight into the three‐dimensional arrangement of TIE proteins, which emerge as very abundant putative adhesins of Gram‐positive bacteria.Publisher PDFPeer reviewe

Phase separation and rotor self-assembly in active particle suspensions

Adding a non-adsorbing polymer to passive colloids induces an attraction between the particles via the `depletion' mechanism. High enough polymer concentrations lead to phase separation. We combine experiments, theory and simulations to demonstrate that using active colloids (such as motile bacteria) dramatically changes the physics of such mixtures. First, significantly stronger inter-particle attraction is needed to cause phase separation. Secondly, the finite size aggregates formed at lower inter-particle attraction show unidirectional rotation. These micro-rotors demonstrate the self assembly of functional structures using active particles. The angular speed of the rotating clusters scales approximately as the inverse of their size, which may be understood theoretically by assuming that the torques exerted by the outermost bacteria in a cluster add up randomly. Our simulations suggest that both the suppression of phase separation and the self assembly of rotors are generic features of aggregating swimmers, and should therefore occur in a variety of biological and synthetic active particle systems.Comment: Main text: 6 pages, 5 figures. Supplementary information: 5 pages, 4 figures. Supplementary movies available from httP://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1116334109/-/DCSupplementa

Evidence for steric regulation of fibrinogen binding to staphylococcus aureus fibronectin-binding protein A (FnBPA)

Background: Staphylococcus aureus fibronectin-binding protein A (FnBPA) binds fibronectin and fibrinogen at adjacent sites. Results: The fibrinogen-binding mechanism is similar but not identical to homologous bacterial proteins. Ternary complex formation by intact fibronectin and fibrinogen on adjacent FnBPA sites could not be demonstrated. Conclusion: Fibrinogen-binding is sterically regulated by fibronectin binding. Significance: Steric regulation might result in targeting of S. aureus to fibrin clots. ABSTRACT The adjacent fibrinogen (Fg)- and fibronectin (Fn)- binding sites on Fn-binding protein A (FnBPA), a cell-surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo-form and in complex with a Fg-peptide. The Fg-binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg- and the most N-terminal Fn-binding sites are non-overlapping but in close proximity. While Fg and a sub-domain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg- and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues

When are active Brownian particles and run-and-tumble particles equivalent? Consequences for motility-induced phase separation

Active Brownian particles (ABPs, such as self-phoretic colloids) swim at fixed speed $v$ along a body-axis ${\bf u}$ that rotates by slow angular diffusion. Run-and-tumble particles (RTPs, such as motile bacteria) swim with constant \u until a random tumble event suddenly decorrelates the orientation. We show that when the motility parameters depend on density $\rho$ but not on ${\bf u}$, the coarse-grained fluctuating hydrodynamics of interacting ABPs and RTPs can be mapped onto each other and are thus strictly equivalent. In both cases, a steeply enough decreasing $v(\rho)$ causes phase separation in dimensions $d=2,3$, even when no attractive forces act between the particles. This points to a generic role for motility-induced phase separation in active matter. However, we show that the ABP/RTP equivalence does not automatically extend to the more general case of \u-dependent motilities

Assembly of microbial communities in replicate nutrient-cycling model ecosystems follows divergent trajectories, leading to alternate stable states

We studied in detail the reproducibility of community development in replicate nutrient‐cycling microbial microcosms that were set up identically and allowed to develop under the same environmental conditions. Multiple replicate closed microcosms were constructed using pond sediment and water, enriched with cellulose and sulphate, and allowed to develop over several months under constant environmental conditions, after which their microbial communities were characterized using 16S rRNA gene sequencing. Our results show that initially similar microbial communities can follow alternative – yet stable – trajectories, diverging in time in a system size‐dependent manner. The divergence between replicate communities increased in time and decreased with larger system size. In particular, notable differences emerged in the heterotrophic degrader communities in our microcosms; one group of steady state communities was enriched with Firmicutes, while the other was enriched with Bacteroidetes. The communities dominated by these two phyla also contained distinct populations of sulphate‐reducing bacteria. This biomodality in community composition appeared to arise during recovery from a low‐diversity state that followed initial cellulose degradation and sulphate reduction

Differential Dynamic Microscopy of Bacterial Motility

We demonstrate 'differential dynamic microscopy' (DDM) for the fast, high throughput characterization of the dynamics of active particles. Specifically, we characterize the swimming speed distribution and the fraction of motile cells in suspensions of Escherichia coli bacteria. By averaging over ~10^4 cells, our results are highly accurate compared to conventional tracking. The diffusivity of non-motile cells is enhanced by an amount proportional to the concentration of motile cells.Comment: 4 pages, 4 figures. In this updated version we have added simulations to support our interpretation, and changed the model for the swimming speed probability distribution from log-normal to a Schulz distribution. Neither modification significantly changes our conclusion

Dynamic density shaping of light driven bacteria

Many motile microorganisms react to environmental light cues with a variety of motility responses guiding cells towards better conditions for survival and growth. The use of spatial light modulators could help to elucidate the mechanisms of photo-movements while, at the same time, providing an efficient strategy to achieve spatial and temporal control of cell concentration. Here we demonstrate that millions of bacteria, genetically modified to swim smoothly with a light controllable speed, can be arranged into complex and reconfigurable density patterns using a digital light projector. We show that a homogeneous sea of freely swimming bacteria can be made to morph between complex shapes. We model non-local effects arising from memory in light response and show how these can be mitigated by a feedback control strategy resulting in the detailed reproduction of grayscale density images.Comment: 8 pages, 6 figures, eLife 2018 (accepted

Enhanced diffusion of nonswimmers in a three-dimensional bath of motile bacteria

We show, using differential dynamic microscopy, that the diffusivity of non-motile cells in a three-dimensional (3D) population of motile E. coli is enhanced by an amount proportional to the active cell flux. While non-motile mutants without flagella and mutants with paralysed flagella have quite different thermal diffusivities and therefore hydrodynamic radii, their diffusivities are enhanced to the same extent by swimmers in the regime of cell densities explored here. Integrating the advective motion of non-swimmers caused by swimmers with finite persistence-length trajectories predicts our observations to within 2%, indicating that fluid entrainment is not relevant for diffusion enhancement in 3D.Comment: 5 pages, 3 figure