Convergent evolution in the supercoiling of prokaryotic flagellar filaments.

The supercoiling of bacterial and archaeal flagellar filaments is required for motility. Archaeal flagellar filaments have no homology to their bacterial counterparts and are instead homologs of bacterial type IV pili. How these prokaryotic flagellar filaments, each composed of thousands of copies of identical subunits, can form stable supercoils under torsional stress is a fascinating puzzle for which structural insights have been elusive. Advances in cryoelectron microscopy (cryo-EM) make it now possible to directly visualize the basis for supercoiling, and here, we show the atomic structures of supercoiled bacterial and archaeal flagellar filaments. For the bacterial flagellar filament, we identify 11 distinct protofilament conformations with three broad classes of inter-protomer interface. For the archaeal flagellar filament, 10 protofilaments form a supercoil geometry supported by 10 distinct conformations, with one inter-protomer discontinuity creating a seam inside of the curve. Our results suggest that convergent evolution has yielded stable superhelical geometries that enable microbial locomotion.

Cellular Stoichiometry of Chemotaxis Proteins in Sinorhizobium meliloti.

Chemotaxis systems enable microbes to sense their immediate environment, moving toward beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti, a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to various degrees. The 10-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that consist of only CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti, instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti, which utilizes a phosphate sink mechanism based on CheA retrophosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased compared to CheA, indicative of variations in the adaptation system of S. meliloti Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems.IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti contributes greatly to host-plant growth by fixing atmospheric nitrogen. The provision of nitrogen as ammonium by S. meliloti leads to increased biomass production of its legume host alfalfa and diminishes the use of environmentally harmful chemical fertilizers. To better understand the role of chemotaxis in host-microbe interaction, a comprehensive catalogue of the bacterial chemotaxis system is vital, including its composition, function, and regulation. The stoichiometry of chemotaxis proteins in S. meliloti has very few similarities to the systems in Escherichia coli and Bacillus subtilis In addition, total amounts of proteins are significantly lower. S. meliloti exhibits a chemotaxis system distinct from known models by incorporating new proteins as exemplified by the phosphate sink mechanism.

An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano.

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.

Bundle of care to drive improvements in palliative and end-of-life care (PEOLC) in an acute tertiary hospital.

OBJECTIVES: There is little evidence to suggest the best model of palliative and end-of-life care (PEOLC) in an acute care hospital. We introduced a bundle of care to drive improvements in PEOLC; this bundle included three full-time nursing positions providing a palliative care clinical consult service with physician backup, as well as educating staff, using the NSW Resuscitation Plan and the Last-Days-of-Life Toolkit. METHODS: Two audits were performed at John Hunter Hospital, a tertiary hospital in Newcastle, Australia, each sampling from all deaths in a 12-month period, one prior to and one after the bundle of care was introduced. Sampling was stratified into deaths that occurred within 4-48 hours of admission and after 48 hours. Key outcomes/data points were recorded and compared across the two time periods. RESULTS: Statistically significant improvements noted included: lower mortality on the wards after 48 hours of admission, better recognition of the dying patient, increased referral to palliative care nurses and physicians, reduction in the number of medical emergency team calls and increase in the use of comfort care and resuscitation plans. Currently, 73% of patients have their end-of-life wishes observed as per their advance care directive. CONCLUSION: A bundle of care involving dedicated nurses with physician backup providing a consult service and education is an effective method for driving improvements in PEOLC.

Neck and capsid architecture of the robust Agrobacterium phage Milano.

Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of the Agrobacterium phage Milano. The Milano neck 1 protein connects the 12-fold symmetrical neck to a 5-fold vertex of the icosahedral capsid. Comparison of Milano neck 1 homologs leads to four proposed classes, likely evolved from the simplest one in siphophages to more complex ones in myo- and podophages. Milano neck is surrounded by the atypical collar, which covalently crosslinks the tail sheath to neck 1. The Milano capsid is decorated with three types of proteins, a minor capsid protein (mCP) and two linking proteins crosslinking the mCP to the major capsid protein. The extensive network of disulfide bonds within and between neck, collar, capsid and tail provides an exceptional structural stability to Milano.

The epidemiology of overtransfusion of red cells in trauma resuscitation patients in the context of a mature massive transfusion protocol.

PURPOSE: Packed red blood cell (PRBC) transfusion remains an integral part of trauma resuscitation and an independent predictor of unfavourable outcomes. It is often administered urgently based on clinical judgement. These facts put trauma patients at high risk of potentially dangerous overtransfusion. We hypothesised that trauma patients are frequently overtransfused and overtransfusion is associated with worse outcomes. METHODS: Trauma patients who received PRBCs within 24 h of admission were identified from the trauma registry during the period January 1 2011-December 31 2018. Overtransfusion was defined as haemoglobin concentration of greater than or equal to 110 g/L at 24 h post ED arrival (± 12 h). Demographics, injury severity, injury pattern, shock severity, blood gas values and outcomes were compared between overtransfused and non-overtransfused patients. RESULTS: From the 211 patients (mean age 45 years, 71% male, ISS 27, mortality 12%) who met inclusion criteria 27% (56/211) were overtransfused. Patients with a higher pre-hospital systolic blood pressure (112 vs 99 mmHg p < 0.01) and a higher initial haemoglobin concentration (132 vs 124 p = 0.02) were more likely to be overtransfused. Overtransfused patients received smaller volumes of packed red blood cells (5 vs 7 units p = 0.049), fresh frozen plasma (4 vs 6 units p < 0.01) and cryoprecipitate (6 vs 9 units p = 0.01) than non-overtransfused patients. CONCLUSION: More than a quarter of patients in our cohort were potentially given more blood products than required without obvious clinical consequences. There were no clinically relevant associations with overtransfusion.