Organic Solvents as Probes for the Structure and Function of the Bacterial Membrane: Effects of Ethanol on the Wild Type and an Ethanol-Resistant Mutant of Escherichia coli K-12

The effects of ethanol on the growth of a wild-type Escherichia coli K-12 are described. These effects include a reduction of the steady-state growth rate and an interference with the division process. They appear as an immediate response to the addition of ethanol and are rapidly reversed by removal of ethanol. Mutants were selected that could grow at a concentration of ethanol that stopped wild-type growth. The growth of one of the mutants we studied (strain S9L100) is stimulated by the presence of ethanol, methanol, or dimethyl sulfoxide. This strain exhibits pleiotropic growth defects including abnormal cell division and morphology. It also appears to have an altered lac permease function which is not due to a mutation in the Y gene itself. We conclude that this mutant has an altered membrane and that the membrane defect may be the cause of the abnormal growth properties. The use of compounds which serve as general membrane perturbants and mutants resistant to these perturbants form a system accessible to both genetic and physical-biochemical techniques.     

Soil–plant–atmosphere conditions regulating convective cloud formation above southeastern US pine plantations

Loblolly pine trees (Pinus taeda L.) occupy more than 20% of the forested area in the southern United States, represent more than 50% of the standing pine volume in this region, and remove from the atmosphere about 500 g C m per year through net ecosystem exchange. Hence, their significance as a major regional carbon sink can hardly be disputed. What is disputed is whether the proliferation of young plantations replacing old forest in the southern United States will alter key aspects of the hydrologic cycle, including convective rainfall, which is the focus of the present work. Ecosystem fluxes of sensible () and latent heat (LE) and large‐scale, slowly evolving free atmospheric temperature and water vapor content are known to be first‐order controls on the formation of convective clouds in the atmospheric boundary layer. These controlling processes are here described by a zero‐order analytical model aimed at assessing how plantations of different ages may regulate the persistence and transition of the atmospheric system between cloudy and cloudless conditions. Using the analytical model together with field observations, the roles of ecosystem and LE on convective cloud formation are explored relative to the entrainment of heat and moisture from the free atmosphere. Our results demonstrate that cloudy–cloudless regimes at the land surface are regulated by a nonlinear relation between the Bowen ratio and root‐zone soil water content, suggesting that young/mature pines ecosystems have the ability to recirculate available water (through rainfall predisposition mechanisms). Such nonlinearity was not detected in a much older pine stand, suggesting a higher tolerance to drought but a limited control on boundary layer dynamics. These results enable the generation of hypotheses about the impacts on convective cloud formation driven by afforestation/deforestation and groundwater depletion projected to increase following increased human population in the southeastern United States.     

Cold air drainage flows subsidize montane valley ecosystem productivity

In mountainous areas, cold air drainage from high to low elevations has pronounced effects on local temperature, which is a critical driver of many ecosystem processes, including carbon uptake and storage. Here, we leverage new approaches for interpreting ecosystem carbon flux observations in complex terrain to quantify the links between macro‐climate condition, drainage flows, local microclimate, and ecosystem carbon cycling in a southern Appalachian valley. Data from multiple long‐running climate stations and multiple eddy covariance flux towers are combined with simple models for ecosystem carbon fluxes. We show that cold air drainage into the valley suppresses local temperature by several degrees at night and for several hours before and after sunset, leading to reductions in growing season respiration on the order of ~8%. As a result, we estimate that drainage flows increase growing season and annual net carbon uptake in the valley by >10% and >15%, respectively, via effects on microclimate that are not be adequately represented in regional‐ and global‐scale terrestrial ecosystem models. Analyses driven by chamber‐based estimates of soil and plant respiration reveal cold air drainage effects on ecosystem respiration are dominated by reductions to the respiration of aboveground biomass. We further show that cold air drainage proceeds more readily when cloud cover and humidity are low, resulting in the greatest enhancements to net carbon uptake in the valley under clear, cloud‐free (i.e., drought‐like) conditions. This is a counterintuitive result that is neither observed nor predicted outside of the valley, where nocturnal temperature and respiration increase during dry periods. This result should motivate efforts to explore how topographic flows may buffer eco‐physiological processes from macroscale climate change.     

T cell infiltration into class II MHC-disparate allografts and acute rejection is dependent on the IFN-gamma-induced chemokine Mig.

Direct evidence that cytokines with chemoattractant properties for leukocytes, chemokines, recruit alloantigen-primed T cells into transplanted allografts has been lacking. We present evidence that neutralization of a single chemokine inhibits T cell infiltration into class II MHC-disparate murine allografts and acute rejection. The chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma (Mig) are expressed in allogeneic skin grafts during the late stages of acute rejection. Survival of class II MHC-disparate B6.H-2bm12 allografts is prolonged from day 14 to day 55 posttransplant when C57BL/6 recipients are given a short course treatment with an antiserum to Mig. This treatment also inhibits T cell and macrophage infiltration into the allografts. B6.H-2bm12 allografts are also not rejected by IFN-gamma-/- C57BL/6 recipients. Injection of Mig directly into B6.H-2bm12 grafts on IFN-gamma-deficient recipients restores T cell infiltration and rejection. Therefore, the inability of IFN-gamma-deficient recipients to reject the class II MHC-disparate allografts is due to the lack of intraallograft Mig production and alloantigen-primed T cell recruitment to the graft. These results indicate for the first time the potential utility of chemokine neutralization strategies in preventing T cell infiltration into allografts and abrogating acute rejection.     

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.     

Disintegration of Dung Pats from Cattle Treated with the Ivermectin Anthelmintic Bolus,or the Biocontrol Agent Duddingtonia flagrans

An experiment was performed during the grazing seasons of 1998, 1999 and 2000 to study the influence of the antiparasitic drug ivermectin and the nematophagous fungus Duddingtonia flagrans on cattle dung disintegration. The faeces originated from groups of animals that were part of a separate grazing experiment where different control strategies for nematode parasite infections were investigated. Each group consisted of 10 first-season grazing cattle that were either untreated, treated with the ivermectin sustained-release bolus, or fed chlamydospores of D. flagrans. Faeces were collected monthly on 4 occasions and out of pooled faeces from each group, 4 artificial 1 kg dung pats were prepared and deposited on nylon mesh on an enclosed pasture and protected from birds. The position of the new set of pats was repeated throughout the 3 years of the study. Each year, the dung pats were weighed 4, 6, 8 and 10 weeks after deposition and immediately afterwards replaced to their initial positions.

Results showed that there was no difference in faecal pat disintegration between groups. However, the time-lag between deposition and complete disintegration of the faeces varied significantly between deposition occasions. Dung pats disappeared within 2 weeks (visual observation) when subjected to heavy rainfall early after deposition, whereas an extended dry period coincided with faeces still remaining 12 months after deposition.

     

Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC

Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator ( agr ) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans- autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection.     

An internal domain of Exo70p is required for actin-independent localization and mediates assembly of specific exocyst components

The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.     

Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions

Background  

The pan-genome of a bacterial species consists of a core and an accessory gene pool. The accessory genome is thought to be an important source of genetic variability in bacterial populations and is gained through lateral gene transfer, allowing subpopulations of bacteria to better adapt to specific niches. Low-cost and high-throughput sequencing platforms have created an exponential increase in genome sequence data and an opportunity to study the pan-genomes of many bacterial species. In this study, we describe a new online pan-genome sequence analysis program, Panseq.     

Predictors of early and late stroke following cardiac surgery

Background:

Much is known about the short-term risks of stroke following cardiac surgery. We examined the rate and predictors of long-term stroke in a cohort of patients who underwent cardiac surgery.

Methods:

We obtained linked data for patients who underwent cardiac surgery in the province of Ontario between 1996 and 2006. We analyzed the incidence of stroke and death up to 2 years postoperatively.

Results:

Of 108 711 patients, 1.8% (95% confidence interval [CI] 1.7%–1.9%) had a stroke perioperatively, and 3.6% (95% CI 3.5%–3.7%) had a stroke within the ensuing 2 years. The strongest predictors of both early and late stroke were advanced age (≥ 65 year; adjusted hazard ratio [HR] for all stroke 1.9, 95% CI 1.8–2.0), a history of stroke or transient ischemic attack (adjusted HR 2.1, 95% CI 1.9–2.3), peripheral vascular disease (adjusted HR 1.6, 95% CI 1.5–1.7), combined coronary bypass grafting and valve surgery (adjusted HR 1.7, 95% CI 1.5–1.8) and valve surgery alone (adjusted HR 1.4, 95% CI 1.2–1.5). Preoperative need for dialysis (adjusted odds ratio [OR] 2.1, 95% CI 1.6–2.8) and new-onset postoperative atrial fibrillation (adjusted OR 1.5, 95% CI 1.3–1.6) were predictors of only early stroke. A CHADS₂ score of 2 or higher was associated with an increased risk of stroke or death compared with a score of 0 or 1 (19.9% v. 9.3% among patients with a history of atrial fibrillation, 16.8% v. 7.8% among those with new-onset postoperative atrial fibrillation and 14.8% v. 5.8% among those without this condition).

Interpretation:

Patients who had cardiac surgery were at highest risk of stroke in the early postoperative period and had continued risk over the ensuing 2 years, with similar risk factors over these periods. New-onset postoperative atrial fibrillation was a predictor of only early stroke. The CHADS₂ score predicted stroke risk among patients with and without atrial fibrillation.Stroke remains a devastating complication following cardiac surgery, with substantial functional and economic impact.^1–3 Stroke research in cardiac surgery has focused on the immediate postoperative period;^4–9 however, most patients undergoing cardiac surgery have conditions such as hypertension, diabetes and atrial fibrillation, which place them at long-term risk of stroke.Early and late outcomes among patients undergoing cardiac surgery could be improved if the risk of postoperative stroke was defined and predictors of stroke identified. With this information, clinicians could optimize medical therapy for stroke risk factors such as hypertension,^10,11 improve the evidence-based use of oral anticoagulation in patients with atrial fibrillation and evaluate intraoperative surgical strategies (e.g., removal of the left atrial appendage¹²) in patients whose clinical characteristics predict an increased risk of stroke. We examined the rate and predictors of long-term stroke within 2 years after cardiac surgery.