Asc1 Supports Cell-Wall Integrity Near Bud Sites by a Pkc1 Independent Mechanism

Background: The yeast ribosomal protein Asc1 is a WD-protein family member. Its mammalian ortholog, RACK1 was initially discovered as a receptor for activated protein C kinase (PKC) that functions to maintain the active conformation of PKC and to support its movement to target sites. In the budding yeast though, a connection between Asc1p and the PKC signaling pathway has never been reported. Methodology/Principal Findings: In the present study we found that asc1-deletion mutant (asc1D) presents some of the hallmarks of PKC signaling mutants. These include an increased sensitivity to staurosporine, a specific Pkc1p inhibitor, and susceptibility to cell-wall perturbing treatments such as hypotonic- and heat shock conditions and zymolase treatment. Microscopic analysis of asc1D cells revealed cell-wall invaginations near bud sites after exposure to hypotonic conditions, and the dynamic of cells ’ survival after this stress further supports the involvement of Asc1p in maintaining the cell-wall integrity during the mid-to late stages of bud formation. Genetic interactions between asc1 and pkc1 reveal synergistic sensitivities of a double-knock out mutant (asc1D/pkc1D) to cell-wall stress conditions, and high basal level of PKC signaling in asc1D. Furthermore, Asc1p has no effect on the cellular distribution or redistribution of Pkc1p at optimal or at cell-wall stress conditions. Conclusions/Significance: Taken together, our data support the idea that unlike its mammalian orthologs, Asc1p act

agr-Mediated Dispersal of Staphylococcus aureus Biofilms

The agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies have suggested a role for the agr system in S. aureus biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. Here, we report that repression of agr is necessary to form a biofilm and that reactivation of agr in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains possessing divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Through genetic analysis, a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases displayed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Shifts in soil microbial community structure, nitrogen cycling and the concomitant declining N availability in ageing primary boreal forest ecosystems

Plant growth in boreal forests is commonly limited by a low supply of nitrogen, a condition that may be aggravated by high tree below-ground allocation of carbon to ectomycorrhizal (ECM) fungi and associated microorganisms. These in turn immobilise N and reduce its availability to plants as boreal ecosystems develop. Here, we studied a boreal forest ecosystem chronosequence created by new land rising out of the sea due to iso-static rebound along the coast of northern Sweden. We used height over the ocean to estimate ecosystem age and examined its relationship to soil microbial community structure and the gross turnover of N. The youngest soils develop with meadows by the coast, followed by a zone of N2-fixing alder trees, and primary boreal conifer forest on ground up to 560 years old. The young soils in meadows contained little organic matter and microbial biomass per unit area. Nitrogen was turned over at low rates when expressed per area (m-2), but specific rates (per gram soil carbon (C)) were the highest found along the transect. In the zone with alder, the amounts of soil C and microbial biomass were much higher (bacterial biomass had doubled and fungal biomass quadrupled). Rates of gross N mineralisation (expressed on an area basis) were highest, but the retention of added labelled NH4+ was lowest in this soil as compared to other ages. The alder zone also had the largest extractable pools of inorganic N in soil and highest N % in plant foliage. In the older conifer forest ecosystems the amounts of soil C and N, as well as biomass of both bacteria and fungi increased. Data on organic matter 14C suggested that the largest input of recently fixed plant C occurred in the younger coniferous forest ecosystems. With increasing ecosystem age, the ratio of microbial C to total soil C was constant, whereas the ratio of microbial N to total soil N increased and gross N mineralization declined. Simultaneously, plant foliar N % decreased and the natural abundance of 15N in the soil increased. More specifically, the difference in δ15N between plant foliage and soil increased, which is related to relatively greater retention of 15N relative to 14N by ECM fungi as N is taken up from the soil and some N is transferred to the plant host. In the conifer forest, where these changes were greatest, we found increased fungal biomass in the F- and H-horizons of the mor-layer, in which ECM fungi are known to dominate (the uppermost horizon with litter and moss is dominated by saprotrophic fungi). Hence, we propose that the decreasing availability of N to the plants and the subsequent decline in plant production in ageing boreal forests is linked to high tree belowground C allocation to ECM fungi, a strong microbial sink for available soil N

Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH4+-N per gram of soil) and high (200 μg NH4+-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil