The Monofunctional Catalase KatE of Xanthomonas axonopodis pv. citri Is Required for Full Virulence in Citrus Plants

BACKGROUND: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). METHODOLOGY/PRINCIPAL FINDINGS: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. CONCLUSIONS: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac

LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones

BACKGROUND The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. RESULTS Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. CONCLUSION The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue

A study protocol of a randomised controlled trial incorporating a health economic analysis to investigate if additional allied health services for rehabilitation reduce length of stay without compromising patient outcomes

Background Reducing patient length of stay is a high priority for health service providers. Preliminary information suggests additional Saturday rehabilitation services could reduce the time a patient stays in hospital by three days. This large trial will examine if providing additional physiotherapy and occupational therapy services on a Saturday reduces health care costs, and improves the health of hospital inpatients receiving rehabilitation compared to the usual Monday to Friday service. We will also investigate the cost effectiveness and patient outcomes of such a service. Methods/Design A randomised controlled trial will evaluate the effect of providing additional physiotherapy and occupational therapy for rehabilitation. Seven hundred and twelve patients receiving inpatient rehabilitation at two metropolitan sites will be randomly allocated to the intervention group or control group. The control group will receive usual care physiotherapy and occupational therapy from Monday to Friday while the intervention group will receive the same amount of rehabilitation as the control group Monday to Friday plus a full physiotherapy and occupational therapy service on Saturday. The primary outcomes will be patient length of stay, quality of life (EuroQol questionnaire), the Functional Independence Measure (FIM), and health utilization and cost data. Secondary outcomes will assess clinical outcomes relevant to the goals of therapy: the 10 metre walk test, the timed up and go test, the Personal Care Participation Assessment and Resource Tool (PC PART), and the modified motor assessment scale. Blinded assessors will assess outcomes at admission and discharge, and follow up data on quality of life, function and health care costs will be collected at 6 and 12 months after discharge. Between group differences will be analysed with analysis of covariance using baseline measures as the covariate. A health economic analysis will be carried out alongside the randomised controlled trial. Discussion This paper outlines the study protocol for the first fully powered randomised controlled trial incorporating a health economic analysis to establish if additional Saturday allied health services for rehabilitation inpatients reduces length of stay without compromising discharge outcomes. If successful, this trial will have substantial health benefits for the patients and for organizations delivering rehabilitation services

Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations

<p>Abstract</p> <p>Background</p> <p><it>Zymomonas mobilis </it>ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. <it>Z. mobilis </it>performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly.</p> <p>Results</p> <p>In this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point.</p> <p>Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (<it>glk, zwf, pgl, pgk, and eno</it>) and gene <it>pdc</it>, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.</p> <p>Conclusion</p> <p>High oxygen concentrations present during <it>Z. mobilis </it>fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.</p

Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2.

Two phenylalanines (FF) in an acidic tract (FFAT)-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA-E). Such motifs are found in several lipid transfer protein (LTP) families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP). Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors

The Princeton Protein Orthology Database (P-POD): A Comparative Genomics Analysis Tool for Biologists

Many biological databases that provide comparative genomics information and tools are now available on the internet. While certainly quite useful, to our knowledge none of the existing databases combine results from multiple comparative genomics methods with manually curated information from the literature. Here we describe the Princeton Protein Orthology Database (P-POD, http://ortholog.princeton.edu), a user-friendly database system that allows users to find and visualize the phylogenetic relationships among predicted orthologs (based on the OrthoMCL method) to a query gene from any of eight eukaryotic organisms, and to see the orthologs in a wider evolutionary context (based on the Jaccard clustering method). In addition to the phylogenetic information, the database contains experimental results manually collected from the literature that can be compared to the computational analyses, as well as links to relevant human disease and gene information via the OMIM, model organism, and sequence databases. Our aim is for the P-POD resource to be extremely useful to typical experimental biologists wanting to learn more about the evolutionary context of their favorite genes. P-POD is based on the commonly used Generic Model Organism Database (GMOD) schema and can be downloaded in its entirety for installation on one's own system. Thus, bioinformaticians and software developers may also find P-POD useful because they can use the P-POD database infrastructure when developing their own comparative genomics resources and database tools

Experimental detection of short regulatory motifs in eukaryotic proteins: tips for good practice as well as for bad

It has become clear in outline though not yet in detail how cellular regulatory and signalling systems are constructed. The essential machines are protein complexes that effect regulatory decisions by undergoing internal changes of state. Subcomponents of these cellular complexes are assembled into molecular switches. Many of these switches employ one or more short peptide motifs as toggles that can move between one or more sites within the switch system, the simplest being on-off switches. Paradoxically, these motif modules (termed short linear motifs or SLiMs) are both hugely abundant but difficult to research. So despite the many successes in identifying short regulatory protein motifs, it is thought that only the “tip of the iceberg” has been exposed. Experimental and bioinformatic motif discovery remain challenging and error prone. The advice presented in this article is aimed at helping researchers to uncover genuine protein motifs, whilst avoiding the pitfalls that lead to reports of false discovery. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12964-015-0121-y) contains supplementary material, which is available to authorized users