Structure and function of a spectrin-like regulator of bacterial cytokinesis

© 2014 Macmillan Publishers Limited. All rights reserved. Bacterial cell division is facilitated by a molecular machine - the divisome - that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family

In Vitro Oxidative Crosslinking of Recombinant Barnacle Cyprid Cement Gland Proteins

Barnacle adhesion is a focus for fouling-control technologies as well as the development of bioinspired adhesives, although the mechanisms remain very poorly understood. The barnacle cypris larva is responsible for surface colonisation. Cyprids release cement from paired glands that contain proteins, carbohydrates and lipids, although further compositional details are scant. Several genes coding for cement gland-specific proteins were identified, but only one of these showed database homology. This was a lysyl oxidase-like protein (lcp_LOX). LOX-like enzymes have been previously identified in the proteome of adult barnacle cement secretory tissue. We attempted to produce recombinant LOX in E. coli, in order to identify its role in cyprid cement polymerisation. We also produced two other cement gland proteins (lcp3_36k_3B8 and lcp2_57k_2F5). lcp2_57k_2F5 contained 56 lysine residues and constituted a plausible substrate for LOX. While significant quantities of soluble lcp3_36k_3B8 and lcp2_57k_2F5 were produced in E. coli, production of stably soluble lcp_LOX failed. A commercially sourced human LOX catalysed the crosslinking of lcp2_57k_2F5 into putative dimers and trimers, and this reaction was inhibited by lcp3_36k_3B8. Inhibition of the lcp_LOX:lcp2_57k_2F5 reaction by lcp3_36k_3B8 appeared to be substrate specific, with no inhibitory effect on the oxidation of cadaverine by LOX. The results demonstrate a possible curing mechanism for barnacle cyprid cement and, thus, provide a basis for a more complete understanding of larval adhesion for targeted control of marine biofouling and adhesives for niche applications

Real-time compression feedback for patients with in-hospital cardiac arrest: a multi-center randomized controlled clinical trial

Objective: To determine if real-time compression feedback using a non-automated hand-held device improves patient outcomes from in-hospital cardiac arrest (IHCA). Methods: We conducted a prospective, randomized, controlled, parallel study (no crossover) of patients with IHCA in the mixed medical–surgical intensive care units (ICUs) of eight academic hospitals. Patients received either standard manual chest compressions or compressions performed with real-time feedback using the Cardio First Angel™ (CFA) device. The primary outcome was sustained return of spontaneous circulation (ROSC), and secondary outcomes were survival to ICU and hospital discharge. Results: One thousand four hundred fifty-four subjects were randomized; 900 were included. Sustained ROSC was significantly improved in the CFA group (66.7% vs. 42.4%, P < 0.001), as was survival to ICU discharge (59.8% vs. 33.6%) and survival to hospital discharge (54% vs. 28.4%, P < 0.001). Outcomes were not affected by intra-group comparisons based on intubation status. ROSC, survival to ICU, and hospital discharge were noted to be improved in inter-group comparisons of non-intubated patients, but not intubated ones. Conclusion: Use of the CFA compression feedback device improved event survival and survival to ICU and hospital discharge

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner