Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in Pseudomonas putida KT2440

[Background] Accumulation of inorganic polyphosphate (polyP), a persistent trait throughout the whole Tree of Life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. The share of polyP in the tolerance of the soil bacterium Pseudomonas putida KT2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations.[Results] Cells lacking polyphosphate kinase (Ppk), which expectedly presented a low intracellular polyP level, were more sensitive to a number of harsh external conditions such as ultraviolet irradiation, addition of β-lactam antibiotics and heavy metals (Cd2+ and Cu2+). Other phenotypes related to a high-energy phosphate load (e.g., swimming) were substantially weakened as well. Furthermore, the ppk mutant was consistently less tolerant to solvents and its survival in stationary phase was significantly affected. In contrast, the major metabolic routes were not significantly influenced by the loss of Ppk as diagnosed from respiration patterns of the mutant in phenotypic microarrays. However, the catalytic vigour of the mutant decreased to about 50% of that in the wild-type strain as estimated from the specific growth rate of cells carrying the catabolic TOL plasmid pWW0 for m-xylene biodegradation. The catalytic phenotype of the mutant was restored by over-expressing ppk in trans. Some of these deficits could be explained by the effect of the ppk mutation on the expression profile of the rpoS gene, the stationary phase sigma factor, which was revealed by the analysis of a P rpoS  → rpoS‘-’lacZ translational fusion. Still, every stress-related effect of lacking Ppk in P. putida was relatively moderate as compared to some of the conspicuous phenotypes reported for other bacteria.[Conclusions] While polyP can be involved in a myriad of cellular functions, the polymer seems to play a relatively secondary role in the genetic and biochemical networks that ultimately enable P. putida to endure environmental stresses. Instead, the main value of polyP could be ensuring a reservoire of energy during prolonged starvation. This is perhaps one of the reasons for polyP persistence in live systems despite its apparent lack of essentiality.This study was supported by the BIO and FEDER CONSOLIDER-INGENIO programme of the Spanish Ministry of Science and Innovation, the MICROME and ST-FLOW Contracts of the EU, the ERANET Program and the PROMT Project of the CAM. P.I.N. is a researcher from Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) and holds a Marie Curie Actions Program grant from the EC (ALLEGRO, UE-FP7-PEOPLE-2011-IIF-300508). M.C. was the recipient of a fellowship from the University of Costa Rica.Peer Reviewe

Protocol for absolute quantification of proteins in Gram-negative bacteria based on QconCAT-based labeled peptides

Summary: Mass-spectrometry-based absolute protein quantification uses labeled quantification concatamer (QconCAT) as internal standards (ISs). To calculate the amount of protein(s), the ion intensity ratio between the analyte and its cognate IS is compared in each biological sample. The present protocol describes a systematic workflow to design, produce, and purify QconCATs and to quantify soluble proteins in Pseudomonas putida KT2440. Our methodology enables the quantification of detectable peptide and serves as a versatile platform to produce ISs for different biological systems. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Engaging patients and primary care providers in the design of novel opinion leader based interventions for acute asthma in the emergency department: a mixed methods study

Abstract Background Multifaceted interventions driven by the needs of patients and providers can help move evidence into practice more rapidly. This study engaged both patients and primary care providers (PCPs) to help design novel opinion leader (OL)-based interventions for patients with acute asthma seen in emergency departments (EDs). Methods A mixed methods design was employed. In phase I, we invited convenience samples of patients with asthma presenting to the ED and PCPs to participate in a survey. Perceptions with respect to: a) an ideal OL-profile for asthma guidance; and b) content, style and delivery methods of OL-based interventions in acute asthma directed from the ED were collected. In phase II, we conducted focus groups to further explore preferences and expectations for such interventions with attention to barriers and facilitators for implementation. Results Overall, 54 patients completed the survey; 39% preferred receiving guidance from a respirologist, 44% during their ED visit and 56% through individual discussions. In addition, 55% expressed interest in having PCP follow-up within a week of ED discharge. A respirologist was identified as the ideal OL-profile by 59% of the 39 responding PCPs. All expressed interest in receiving notification of their patients’ ED presentation, most within a week and including diagnosis and ED/post ED-treatment. Personalized, guideline-based, recommendations were considered to be the ideal content by the majority; 39% requested this guidance through a pamphlet faxed to their offices. In the focus groups, patients and PCPs recognized the importance of health professional liaisons in transitions in care; patient anxiety and PCP time constraints were identified as potential barriers for ED-educational information uptake and proper post-ED follow-up, respectively. Conclusions Engaging patients and PCPs yielded actionable information to tailor OL-based multifaceted interventions for acute asthma in the ED. We identified potential facilitators for the implementation of such interventions (e.g., patient interaction with alternative health care professionals who could facilitate transitions in asthma care between the ED and the primary care setting), and for the provision of post discharge self-management education (e.g., consideration of the first week of ED discharge as a practical time frame for this intervention). Prioritization of identified barriers (e.g., lack of PCP involvement) could be addressed by the identification of potential early adopters in practice environments (e.g., clinicians with special interest in asthma)

Governance, accountability and the datafication of early years education in England

In this paper we attempt to critically ‘make visible the flow and circulation of data’ through analysing the datafication of the early years education sector in England (children aged 2–5). The concept of datafication is used to understand the processes and impacts of burgeoning data-based governance and accountability regimes. This analysis builds upon early childhood researchers who were influenced by Foucault and others, who have noted the ways in which the surveillance and performative culture of accountability both affirms, legitimates and seduces through discourses of quality while increasingly regulating and governing the early years. Using data from three research sites (a children's centre, a primary school and a combined nursery school and children's centre) as well as an interview with a local authority early years advisor, we examine how comparative data-based accountability increasingly governed early years teachers' professionalism and pedagogies. We argue that the planned tracking of children's performance from baseline assessment at four years old to eleven years old may further govern and constrain early years professionalism as young children are reconfigured as ‘miniature centres of calculation’