Identification of the CRP regulon using in vitro and in vivo transcriptional profiling

The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that controls transcription initiation from more than 100 promoters by binding to a specific DNA sequence within cognate promoters. Many genes in the CRP regulon have been predicted simply based on the presence of DNA-binding sites within gene promoters. In this study, we have exploited a newly developed technique, run-off transcription/microarray analysis (ROMA) to define CRP-regulated promoters. Using ROMA, we identified 176 operons that were activated by CRP in vitro and 16 operons that were repressed. Using positive control mutants in different regions of CRP, we were able to classify the different promoters into class I or class II/III. A total of 104 operons were predicted to contain Class II CRP-binding sites. Sequence analysis of the operons that were repressed by CRP revealed different mechanisms for CRP inhibition. In contrast, the in vivo transcriptional profiles failed to identify most CRP-dependent regulation because of the complexity of the regulatory network. Analysis of these operons supports the hypothesis that CRP is not only a regulator of genes required for catabolism of sugars other than glucose, but also regulates the expression of a large number of other genes in E.coli. ROMA has revealed 152 hitherto unknown CRP regulons

Household energy use: Applying behavioural economics to understand consumer decision-making and behaviour

AbstractHousehold energy conservation has emerged as a major challenge and opportunity for researchers, practitioners and policymakers. Consumers also seem to be gaining greater awareness of the value and need for sustainable energy practices, particularly amid growing public concerns over greenhouse gas emissions and climate change. Yet even with adequate knowledge of how to save energy and a professed desire to do so, many consumers still fail to take noticeable steps towards energy efficiency and conservation. There is often a sizeable discrepancy between peoples’ self-reported knowledge, values, attitudes and intentions, and their observable behaviour—examples include the well-known ‘knowledge-action gap’ and ‘value-action gap’. But neither is household energy consumption driven primarily by financial incentives and the rational pursuit of material interests. In fact, people sometimes respond in unexpected and undesirable ways to rewards and sanctions intended to shift consumers’ cost–benefit calculus in favour of sustainable behaviours. Why is this so? Why is household energy consumption and conservation difficult to predict from either core values or material interests? By drawing on critical insights from behavioural economics and psychology, we illuminate the key cognitive biases and motivational factors that may explain why energy-related behaviour so often fails to align with either the personal values or material interests of consumers. Understanding these psychological phenomena can make household and community responses to public policy interventions less surprising, and in parallel, can help us design more cost-effective and mass-scalable behavioural solutions to encourage renewable and sustainable energy use among consumers

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background: Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. \ud \ud Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome. \ud \ud Conclusion: We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains

Patient perspectives on the causes and prevention of rehospitalisation for exacerbations of chronic obstructive pulmonary disease: A qualitative study

Background: Chronic obstructive pulmonary disease (COPD) is the umbrella term for a group of lung diseases which are degenerative and marked by progressively worsening symptoms including: breathlessness; fatigue; acute exacerbation; multiple comorbidities; and eventually death. Acute exacerbations of COPD (AECOPD) resulting in hospitalisation may be responsible for up to 25 percent of reductions in lung function. In New Zealand an estimated 23% of those discharged from hospital after an initial episode of AECOPD are readmitted within 30 days, a situation which is both costly and disadvantageous to the patient. Objective: The aim of this study was to understand patients’ perspectives on the causes and prevention of re-hospitalisation for AECOPD. Method: Data for this study was collected from a subgroup of participants who had been recruited as part of a feasibility study using randomised controlled trial methods to explore the effectiveness of a novel self-management intervention, called “Taking Charge of COPD”. All participants were initially recruited in hospital after an episode of severe AECOPD. The subgroup of participants in this qualitative study were interviewed 12 months later and ask about their views and experiences regarding what had helped or hindered them to stay well and out of hospital. Grounded theory was used to analyse results and construct concept. Data were also collected on disease specific health status (using the Chronic COPD Questionnaire), depression and anxiety (using the Hospital Depression and Anxiety Scale), and number of moderate episodes (requiring antibiotics or steroids) or severe episodes of AECOPD (requiring hospitalisation) during the prior 12 months. Results: Twelve participants were interviewed (mean age 69.3 years (SD 13.6); range 29 – 84; 6 female, 2 Māori, 2 Pacific, 7 New Zealand European). These participants had experienced between 0 and 7 episodes of moderate AECOPD (average 1.8) and 0 to 3 episodes of severe AECOPD (average 0.7) in the 12 months since their original hospitalisation. Three main concepts were identified to describe the participants’ views on what helped or hinder them to stay well and out of hospital: 1) Being Proactive – which encompasses practical steps participants took to reduce AECOPD; 2) Being Positive – which describes the importance of a positive mindset; and 3) Taking Charge – the concept of believing in oneself. Impacting on each of these was the influence of significant others, particularly family and friends. Conclusion: This research expands our understanding of how patients manage COPD and adds a patient’s perspective to the current knowledge on how to prevent AECOPD. Programmes which promote self-efficacy and positive mental health would be beneficial additions to AECOPD prevention, as could the inclusion of family or significant others in health planning/treatment plans.

Bacterial antimicrobial metal ion resistance

Metals such as mercury, arsenic, copper and silver have been used in various forms as antimicrobials for thousands of years with until recently, little understanding of their mode of action. The discovery of antibiotics and new organic antimicrobial compounds during the twentieth century saw a general decline in the clinical use of antimicrobial metal compounds, with the exception of the rediscovery of the use of silver for burns treatments and niche uses for other metal compounds. Antibiotics and new antimicrobials were regarded as being safer for the patient and more effective than the metal-based compounds they supplanted. Bacterial metal ion resistances were first discovered in the second half of the twentieth century. The detailed mechanisms of resistance have now been characterized in a wide range of bacteria. As the use of antimicrobial metals is limited, it is legitimate to ask: are antimicrobial metal resistances in pathogenic and commensal bacteria important now? This review details the new, rediscovered and 'never went away' uses of antimicrobial metals; examines the prevalence and linkage of antimicrobial metal resistance genes to other antimicrobial resistance genes; and examines the evidence for horizontal transfer of these genes between bacteria. Finally, we discuss the possible implications of the widespread dissemination of these resistances on re-emergent uses of antimicrobial metals and how this could impact upon the antibiotic resistance problem

Draft genome sequences of 14 Escherichia coli phages isolated from cattle slurry

The diversity of bacteriophages in slurry from dairy cows remains largely unknown. Here, we report the draft genome sequences of 14 bacteriophages isolated from dairy cow slurry using Escherichia coli K-12 MG1655 as a host

Functional dairy protein supplements for elite athletes

Elite athletes require a greater dietary protein intake than recreationally active people to maintain optimal muscular function. The timing of protein ingestion relative to exercise is critical to maximizing its physiological impact on skeletal muscles. Sports protein supplements provide a convenient means of supplying athletes with an adequate and timely source of quality dietary protein. There is now strong evidence that not all dietary proteins are equipotent in their effects on various aspects of athletic performance and specific protein isolates can provide benefits to athletes beyond simple supply of nutritional amino acids. Thus, there is an opportunity to develop new functional protein supplements to maximize athletic performance. This paper outlines the clinical evidence for the benefits of dairy proteins in sports performance and describes the development of new dairy protein supplements to build muscle strength, and to expedite recovery of strength following muscle-damaging eccentric exercise.<br /

High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq

Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences

Comparative genomics of bacteriophage of the genus Seuratvirus

Despite being more abundant and having smaller genomes than their bacterial host, relatively few bacteriophages have had their genomes sequenced. Here, we isolated 14 bacteriophages from cattle slurry and performed de novo genome sequencing, assembly, and annotation. The commonly used marker genes polB and terL showed these bacteriophages to be closely related to members of the genus Seuratvirus. We performed a core-gene analysis using the 14 new and four closely related genomes. A total of 58 core genes were identified, the majority of which has no known function. These genes were used to construct a core-gene phylogeny, the results of which confirmed the new isolates to be part of the genus Seuratvirus and expanded the number of species within this genus to four. All bacteriophages within the genus contained the genes queCDE encoding enzymes involved in queuosine biosynthesis. We suggest these genes are carried as a mechanism to modify DNA in order to protect these bacteriophages against host endonucleases

Multidrug resistant, extended spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from a dairy farm

Escherichia coli strains were isolated from a single dairy farm as a sentinel organism for the persistence of antibiotic resistance genes in the farm environment. Selective microbiological media were used to isolate 126 E. coli isolates from slurry and faeces samples from different farm areas. Antibiotic resistance profiling for 17 antibiotics (seven antibiotic classes), showed 57.9% of the isolates were resistant to between 3 and 15 antibiotics. The highest frequency of resistance was to ampicillin (56.3%), and the lowest to imipenem (1.6%), which appeared to be an unstable phenotype and was subsequently lost. Extended spectrum beta-lactamase resistance (ESBL) was detected in 53 isolates and blaCTX-M, blaTEM and blaOXA genes were detected by PCR in twelve, four and two strains, respectively. Phenotypically most isolates showing resistance to cephalosporins were AmpC rather than ESBL, a number of isolates having both activities. Phenotypic resistance patterns suggested co-acquisition of some resistance genes within subsets of the isolates. Genotyping using ERIC PCR demonstrated these were not clonal, and therefore co-resistance may be associated with mobile genetic elements. These data show a snapshot of diverse resistance genes present in the E. coli population reservoir, including resistance to historically used antibiotics as well as cephalosporins in contemporary use