The effect of cooling rate, freeze-drying suspending fluid and culture age on the preservation of Campylobacter pylori

The effects of freezing rate, suspending fluid and age of culture on the ability of four strains of Campylobacter pylori to survive and recover from freeze-drying were examined. Freeze-drying by standard procedures generally resulted in an overall loss in viability of between 3 and 7 log units. The exact cause of poor recovery by C. pylori was not established but strain differences were detected, with NCTC 11637 (type strain) surviving better than NCTC 11638 and NCTC 11639. Recovery of the poorest growing strain (NE 26695) was notably more erratic. The largest loss in viability occurred at the primary drying stage. Losses resulting from freezing and secondary drying were less marked and the rate of freezing had only a marginal effect on recovery. Nineteen different freeze-drying suspending fluids were investigated. Overall the best recovery results were obtained with 5% inositol-broth (or horse serum) plus 25% glucose, at pH 7.0, in which loss of viability was typically about 4 log units. Other factors, such as age of culture and number of viable bacteria in the before-dry suspension, did not have a significant effect on survival. We conclude from these results that C. pylori can survive freeze-drying, albeit in small numbers, but the degree of recovery is apparently largely strain dependent.     

Genetic structure of three marine fishes from the Gulf of Pagasitikos (Greece) based on allozymes, RAPD, and mtDNA RFLP markers

In the present work we used three molecular techniques (allozymes, RAPDs and mtDNA RFLPs) in order to study the genetic structure of three commercial marine species (Mullus surmuletus, Mullus barbatus, and Pagellus erythrinus). Each species was sampled from three locations within the Gulf of Pagasitikos, Greece and from two neighbouring locations outside the Gulf (Trikeri and Alonissos). Values of genetic heterozygosity and nucleotide diversity for all populations studied were similar or above the mean values observed in marine fishes. None of the three types of molecular markers used revealed diagnostic patterns, which could allow the allocation of individuals to one of the populations. The analyses revealed that the three populations within Pagasitikos were homogenous representing thus a panmictic stock. However, there were evidences of genetic population subdivision between localities from inside and outside of the Pagasitikos Gulf. The results provide essential information for the design of a sustainable management plan of the Gulf of Pagasitikos and its demersal fish resources.     

Genome Scale Reconstruction of a Salmonella Metabolic Model: COMPARISON OF SIMILARITY AND DIFFERENCES WITH A COMMENSAL Escherichia coli STRAIN*

Salmonella are closely related to commensal Escherichia coli but have gained virulence factors enabling them to behave as enteric pathogens. Less well studied are the similarities and differences that exist between the metabolic properties of these organisms that may contribute toward niche adaptation of Salmonella pathogens. To address this, we have constructed a genome scale Salmonella metabolic model (iMA945). The model comprises 945 open reading frames or genes, 1964 reactions, and 1036 metabolites. There was significant overlap with genes present in E. coli MG1655 model iAF1260. In silico growth predictions were simulated using the model on different carbon, nitrogen, phosphorous, and sulfur sources. These were compared with substrate utilization data gathered from high throughput phenotyping microarrays revealing good agreement. Of the compounds tested, the majority were utilizable by both Salmonella and E. coli. Nevertheless a number of differences were identified both between Salmonella and E. coli and also within the Salmonella strains included. These differences provide valuable insight into differences between a commensal and a closely related pathogen and within different pathogenic strains opening new avenues for future explorations.Salmonella is a major cause of human and animal enteric disease. Salmonella consists of two species, bongori and enterica, and the latter can be further divided into subspecies (I-VI). The majority of human and animal infections are caused by S. enterica subspecies I, of which Salmonella typhimurium and Salmonella enteritidis are the most prevalent causes of human inflammatory gastroenteritis, often referred to as food poisoning (1). The recent availability of genome sequences of bacterial pathogens, including Salmonella, provides an opportunity to interrogate these organisms using a systems biology approach. By contrasting the genotype-phenotype relationship of pathogens such as Salmonella against closely related commensals such as an Escherichia coli K12 insights can be revealed into how these pathogens have adapted to their environmental niche(s). Salmonella and E. coli K12 share ∼85% of their genome (2–6). DNA microarray and genome sequencing studies have highlighted regions of the genome that are conserved between these closely related bacteria and those that are different. Many of the differences are attributable to the acquisition of virulence factors, although a significant proportion of their genome codes is for metabolic genes (2–8).A genome scale model consists of a stoichiometric reconstruction of all reactions known to act in the metabolism of an organism along with a set of accompanying constraints on the flux of each reaction in the system (9, 10). These models define the organism''s global metabolic space, network structural properties, and flux distribution potential (9, 10). Therefore constraint-based models can help predict cellular phenotypes given particular environmental conditions. Genome scale models have been useful in understanding the metabolic properties of a variety of organisms including E. coli, Bacillus subtilis, Pseudomonas putida, and Lactobacillus (9–12). Genome scale models can be validated in various ways such as continuous culture experiments, substrate utilization assays, specific gene mutations, and isotopic carbon measurements. The high through-put phenotype microarray (PM)³ system that is available through Biolog (Hayward, CA) is ideal to use for substrate utilization assays as it provides a comprehensive large-scale phenotyping technology to assess gene function at the cellular level (13).The aim of this work was to construct a Salmonella genome scale model. The model highlights the similarities and differences between pathogenic bacteria such as S. typhimurium and S. enteritidis and the commensal E. coli K12 laboratory strains. The model was validated using the PM system and literature-derived (i.e. bibliomic) information. The substrate utilization assays also highlighted current knowledge gaps that will require further experimental data that can be used in the future for refining and extending the model.     

Microalgae fiber optic biosensors for herbicide monitoring using sol–gel technology

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol–gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol–gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 μg L⁻¹) and the broadest dynamic calibration range (19–860 μg L⁻¹) with IC₅₀ 125 ± 14 μg L⁻¹. Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.     

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N¹-glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N¹-(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.     

Abberant α-Synuclein Confers Toxicity to Neurons in Part through Inhibition of Chaperone-Mediated Autophagy

Background

The mechanisms through which aberrant α-synuclein (ASYN) leads to neuronal death in Parkinson''s disease (PD) are uncertain. In isolated liver lysosomes, mutant ASYNs impair Chaperone Mediated Autophagy (CMA), a targeted lysosomal degradation pathway; however, whether this occurs in a cellular context, and whether it mediates ASYN toxicity, is unknown. We have investigated presently the effects of WT or mutant ASYN on the lysosomal pathways of CMA and macroautophagy in neuronal cells and assessed their impact on ASYN-mediated toxicity.

Methods and Findings

Novel inducible SH-SY5Y and PC12 cell lines expressing human WT and A53T ASYN, as well as two mutant forms that lack the CMA-targeting motif were generated. Such forms were also expressed in primary cortical neurons, using adenoviral transduction. In each case, effects on long-lived protein degradation, LC3 II levels (as a macroautophagy index), and cell death and survival were assessed. In both PC12 and SH-SY5Y cycling cells, induction of A53T ASYN evoked a significant decrease in lysosomal degradation, largely due to CMA impairment. In neuronally differentiated SH-SH5Y cells, both WT and A53T ASYN induction resulted in gradual toxicity, which was partly dependent on CMA impairment and compensatory macroautophagy induction. In primary neurons both WT and A53T ASYN were toxic, but only in the case of A53T ASYN did CMA dysfunction and compensatory macroautophagy induction occur and participate in death.

Conclusions

Expression of mutant A53T, and, in some cases, WT ASYN in neuronal cells leads to CMA dysfunction, and this in turn leads to compensatory induction of macroautophagy. Inhibition of these lysosomal effects mitigates ASYN toxicity. Therefore, CMA dysfunction mediates aberrant ASYN toxicity, and may be a target for therapeutic intervention in PD and related disorders. Furthermore, macroautophagy induction in the context of ASYN over-expression, in contrast to other settings, appears to be a detrimental response, leading to neuronal death.     

Microbial 1-butanol production: Identification of non-native production routes and in silico engineering interventions

The potential of engineering microorganisms with non-native pathways for the synthesis of long-chain alcohols has been identified as a promising route to biofuels. We describe computationally derived predictions for assembling pathways for the production of biofuel candidate molecules and subsequent metabolic engineering modifications that optimize product yield. A graph-based algorithm illustrates that, by culling information from BRENDA and KEGG databases, all possible pathways that link the target product with metabolites present in the production host are identified. Subsequently, we apply our recent OptForce procedure to pinpoint reaction modifications that force the imposed product yield in Escherichia coli. We demonstrate this procedure by suggesting new pathways and genetic interventions for the overproduction of 1-butanol using the metabolic model for Escherichia coli. The graph-based search method recapitulates all recent discoveries based on the 2-ketovaline intermediate and hydroxybutyryl-CoA but also pinpointes one novel pathway through thiobutanoate intermediate that to the best of our knowledge has not been explored before.     

Numerical analysis of electrophoretic protein patterns of Campylobacter laridis and allied thermophilic campylobacters from the natural environment

Twenty-one strains comprising Campylobacter laridis (nine), nalidixic acid sensitive campylobacters (NASC) (four), and urease-positive thermophilic campylobacters (UPTC) (eight) were characterized by one-dimensional SDS-PAGE of cellular proteins. The UPTC and NASC strains included six from river water, two from mussels and four from sea water. The type strains of three other Campylobacter species were included for reference. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 21 strains formed nine clusters at the 80% similarity (S) level. The typical C. laridis strains were restricted to two phenons (2 and 5); the atypical strains being distributed among the remaining phenons. In the second analysis, which excluded the principal protein bands (40-48.5 kD range), the 21 strains formed five clusters at the 80% S level. The typical C. laridis strains were relatively homogeneous and fell into a single phenon (2) within which two subgroups were discernable. The atypical strains were more heterogeneous with respect to background protein pattern, with representatives appearing in all five phenons. An electropherotyping scheme comprising six electropherotypes, and based on both analyses is proposed. The high within-group S level and separation from reference strains of Campylobacter in the second analysis, suggested that UPTC and NASC strains belonged within C. laridis possibly as biovars.     

Isoprenoid quinone composition of representatives of the genus Campylobacter

The isoprenoid quinone composition of 17 strains representing nine species or sub-species of the genus Campylobacter was investigated. All strains produced similar respiratory quinone patterns consisting of unsaturated menaquinones with six isoprene units and a novel unidentified quinone. Mass spectral analysis indicate the unknown compound has six isoprene units and a formula C₄₂H₅₈O₂. The present study indicates respiratory quinones may be useful generic markers for Campylobacter.