Regulation of phenylacetic acid uptake is σ54 dependent in Pseudomonas putida CA-3

<p>Abstract</p> <p>Background</p> <p>Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader <it>Pseudomonas putida </it>CA-3 was subjected to random mini-Tn<it>5 </it>mutagenesis and mutants screened for altered styrene/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences.</p> <p>Results</p> <p>One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn<it>5 </it>insertion in the <it>rpoN </it>gene encoding σ54. Complementation of the D7 mutant with the wild type <it>rpoN </it>gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type <it>P. putida </it>CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne <it>paaL </it>in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the <it>paaL </it>promoter from <it>P. putida </it>CA-3 revealed two σ⁵⁴consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in <it>Pseudomonas </it>species studied to date.</p> <p>Conclusions</p> <p>In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon <it>paaL </it>gene, encoding a transport protein essential for phenylacetic acid utilisation in <it>P. putida </it>CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading <it>Pseudomonads</it>.</p

Nonlinear vertical oscillations of a particle in a sheath of a rf discharge

A new simple method to measure the spatial distribution of the electric field in the plasma sheath is proposed. The method is based on the experimental investigation of vertical oscillations of a single particle in the sheath of a low-pressure radio-frequency discharge. It is shown that the oscillations become strongly nonlinear and secondary harmonics are generated as the amplitude increases. The theory of anharmonic oscillations provides a good qualitative description of the data and gives estimates for the first two anharmonic terms in an expansion of the sheath potential around the particle equilibrium.Comment: 11 pages, 4 figure

Building an end user focused THz based ultra high bandwidth wireless access network: The TERAPOD approach

The TERAPOD project aims to investigate and demonstrate the feasibility of ultra high bandwidth wireless access networks operating in the Terahertz (THz) band. The proposed TERAPOD THz communication system will be developed, driven by end user usage scenario requirements and will be demonstrated within a first adopter operational setting of a Data Centre. In this article, we define the full communications stack approach that will be taken in TERAPOD, highlighting the specific challenges and aimed innovations that are targeted

Retarding field energy analyser ion current calibration and transmission

International audienceAccurate measurement of ion current density and ion energy distributions (IED) is often critical for plasma processes in both industrial and research settings. Retarding field energy analyzers (RFEA) have been used to measure IEDs because they are considered accurate, relatively simple and cost effective. However, their usage for critical measurement of ion current density is less common due to difficulties in estimating the proportion of incident ion current reaching the current collector through the RFEA retarding grids. In this paper an RFEA has been calibrated to measure ion current density from an ion beam at pressures ranging from 0.5 to 50.0 mTorr. A unique method is presented where the currents generated at each of the retarding grids and the RFEA upper face are measured separately, allowing the reduction in ion current to be monitored and accounted for at each stage of ion transit to the collector. From these I-V measurements a physical model is described. Subsequently, a mathematical description is extracted which includes parameters to account for grid transmissions, upper face secondary electron emission and collisionality. Pressure-dependant calibration factors can be calculated from least mean square best fits of the collector current to the model allowing quantitative measurement of ion current density

Biomechanical comparison of the pullout properties of external skeletal fixation pins in the tibiae of intact and ovariectomised ewes.

The pin-bone interface is the least stable component of the external skeletal fixator. Concerns exist regarding the ability to obtain adequate implant purchase in poor quality bone. Consequently, reduced bone quality has been viewed as a contra-indication for the use of external skeletal fixators. The aim of this study was to investigate the holding power of two different fixator pin designs in bone from entire and ovariectomised sheep. Thirty-two aged ewes were divided into two groups. Group 1 were controls, and Group 2 were ovariectomised (OVX). The ewes were sacrificed 12 months post-ovariectomy and five pairs of tibiae were harvested from each group. The holding power of cortical and cancellous fixator pins was assessed at five standardised locations on each tibia. An increase in mean cortical thickness was noted in the OVX group. The holding power of cancellous fixator pins was superior to that of cortical pins, irrespective of whether or not ovariectomy had been performed. Cancellous pins had an increased holding power in post ovariectomy bone compared to control bone. Cortical pin performance was not affected by ovariectomy. There was a lack of correlation between the incidence of insertional fractures of the far cortex and implant holding power. The results raise questions over the effectiveness of ovariectomy in establishing osteopaenic bone suitable for assessing implant performance, hence further investigations are warranted

Transforming teacher education, an activity theory analysis

This paper explores the work of teacher education in England and Scotland. It seeks to locate this work within conflicting socio-cultural views of professional practice and academic work. Drawing on an activity theory framework that integrates the analysis of these contradictory discourses with a study of teacher educators’ practical activities, including the material artefacts that mediate the work, the paper offers a critical perspective on the social organisation of university-based teacher education. Informed by Engeström’s activity theory concept of transformation, the paper extends the discussion of contradictions in teacher education to consider the wider socio-cultural relations of the work. The findings raise important questions about the way in which teacher education work within universities is organised and the division of labour between schools and universities

Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data

Background: MicroRNAs (miRNAs) are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes) are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II) promoter. However, the length of the primary transcripts and promoter organization is currently unknown. Methodology: We performed Pol II chromatin immunoprecipitation (ChIP)-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP). We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters. Conclusion: miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex. © 2009 Corcoran et al