An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5 alpha and EQ1

Aims: To examine Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 for known pathogenic mechanisms. Methods and Results: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d postinfection. Conclusions: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. Significance and Impact of the Study: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum

The genome structure of Colletotrichum lindemuthianum in a set of diverse isolates was investigated using a combination of physical and molecular approaches. Flow cytometric measurement of genome size revealed significant variation between strains, with the smallest genome representing 59% of the largest. Southern-blot profiles of a cloned fungal telomere revealed a total chromosome number varying from 9 to 12. Chromosome separations using pulsed-field gel electrophoresis (PFGE) showed that these chromosomes belong to two distinct size classes: a variable number of small (< 2.5 Mb) polymorphic chromosomes and a set of unresolved chromosomes larger than 7 Mb. Two dispersed repeat elements were shown to cluster on distinct polymorphic minichromosomes. Single-copy flanking sequences from these repeat-containing clones specifically marked distinct small chromosomes. These markers were absent in some strains, indicating that part of the observed variability in genome organization may be explained by the presence or absence, in a given strain, of dispensable genomic regions and/or chromosomes.

Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation

LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consis- tently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data high- light the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.

Influence of host plant heterogeneity on the distribution of a birch aphid

1. The spatial and temporal abundance of the aphid Euceraphis betulae was investigated in relation to heterogeneity in host plant ( Betula pendula) vigour and pathogenic stress. The performance of aphids feeding on vigorous and stressed foliage was also examined. 2. The plant stress and plant vigour hypotheses have been suggested as opposing ways in which foliage quality influences herbivore abundance. In many plants, however, vigorous growing foliage co-exists with stressed or damaged foliage. 3. There was a negative correlation between branch growth ( vigour) and branch stress ( leaf chlorosis), with the most vigorous branches displaying little or no stress, and the most stressed branches achieving poor growth. There was a similar negative correlation between vigour and stress at the level of individual trees, which themselves represented a continuum in quality. 4. At the beginning of the season, E. betulae were intermittently more abundant on vigorous branches than on branches destined to become stressed, but aphids became significantly more abundant on stressed branches later in the season, when symptoms of stress became apparent. Similar patterns of aphid abundance were seen on vigorous and stressed trees in the following year. 5. Euceraphis betulae performance was generally enhanced when feeding on naturally stressed B. pendula leaves, but there was some evidence for elevated potential reproduction when feeding on vigorous leaves too. 6. Overall, plant stress probably influences E. betulae distribution more than plant vigour, but the temporal and spatial variability in plant quality suggests that plant vigour could play a role in aphid distribution early in the season.

Iron Uptake and Homeostasis in Microorganisms

Preface. Iron is considered to be a minor element employed, in a variety of forms, by nearly all living organisms. In some cases, it is utilised in large quantities, for instance for the formation of magnetosomes within magnetotactic bacteria or during use of iron as a respiratory donor or acceptor by iron oxidising or reducing bacteria. However, in most cases the role of iron is restricted to its use as a cofactor or prosthetic group assisting the biological activity of many different types of protein. The key metabolic processes that are dependent on iron as a cofactor are numerous; they include respiration, light harvesting, nitrogen fixation, the Krebs cycle, redox stress resistance, amino acid synthesis and oxygen transport. Indeed, it is clear that Life in its current form would be impossible in the absence of iron. One of the main reasons for the reliance of Life upon this metal is the ability of iron to exist in multiple redox states, in particular the relatively stable ferrous (Fe2+) and ferric (Fe3+) forms. The availability of these stable oxidation states allows iron to engage in redox reactions over a wide range of midpoint potentials, depending on the coordination environment, making it an extremely adaptable mediator of electron exchange processes. Iron is also one of the most common elements within the Earth’s crust (5% abundance) and thus is considered to have been readily available when Life evolved on our early, anaerobic planet. However, as oxygen accumulated (the ‘Great oxidation event’) within the atmosphere some 2.4 billion years ago, and as the oceans became less acidic, the iron within primordial oceans was converted from its soluble reduced form to its weakly-soluble oxidised ferric form, which precipitated (~1.8 billion years ago) to form the ‘banded iron formations’ (BIFs) observed today in Precambrian sedimentary rocks around the world. These BIFs provide a geological record marking a transition point away from the ancient anaerobic world towards modern aerobic Earth. They also indicate a period over which the bio-availability of iron shifted from abundance to limitation, a condition that extends to the modern day. Thus, it is considered likely that the vast majority of extant organisms face the common problem of securing sufficient iron from their environment – a problem that Life on Earth has had to cope with for some 2 billion years. This struggle for iron is exemplified by the competition for this metal amongst co-habiting microorganisms who resort to stealing (pirating) each others iron supplies! The reliance of micro-organisms upon iron can be disadvantageous to them, and to our innate immune system it represents a chink in the microbial armour, offering an opportunity that can be exploited to ward off pathogenic invaders. In order to infect body tissues and cause disease, pathogens must secure all their iron from the host. To fight such infections, the host specifically withdraws available iron through the action of various iron depleting processes (e.g. the release of lactoferrin and lipocalin-2) – this represents an important strategy in our defence against disease. However, pathogens are frequently able to deploy iron acquisition systems that target host iron sources such as transferrin, lactoferrin and hemoproteins, and thus counteract the iron-withdrawal approaches of the host. Inactivation of such host-targeting iron-uptake systems often attenuates the pathogenicity of the invading microbe, illustrating the importance of ‘the battle for iron’ in the infection process. The role of iron sequestration systems in facilitating microbial infections has been a major driving force in research aimed at unravelling the complexities of microbial iron transport processes. But also, the intricacy of such systems offers a challenge that stimulates the curiosity. One such challenge is to understand how balanced levels of free iron within the cytosol are achieved in a way that avoids toxicity whilst providing sufficient levels for metabolic purposes – this is a requirement that all organisms have to meet. Although the systems involved in achieving this balance can be highly variable amongst different microorganisms, the overall strategy is common. On a coarse level, the homeostatic control of cellular iron is maintained through strict control of the uptake, storage and utilisation of available iron, and is co-ordinated by integrated iron-regulatory networks. However, much yet remains to be discovered concerning the fine details of these different iron regulatory processes. As already indicated, perhaps the most difficult task in maintaining iron homeostasis is simply the procurement of sufficient iron from external sources. The importance of this problem is demonstrated by the plethora of distinct iron transporters often found within a single bacterium, each targeting different forms (complex or redox state) of iron or a different environmental condition. Thus, microbes devote considerable cellular resource to securing iron from their surroundings, reflecting how successful acquisition of iron can be crucial in the competition for survival. The aim of this book is provide the reader with an overview of iron transport processes within a range of microorganisms and to provide an indication of how microbial iron levels are controlled. This aim is promoted through the inclusion of expert reviews on several well studied examples that illustrate the current state of play concerning our comprehension of how iron is translocated into the bacterial (or fungal) cell and how iron homeostasis is controlled within microbes. The first two chapters (1-2) consider the general properties of microbial iron-chelating compounds (known as ‘siderophores’), and the mechanisms used by bacteria to acquire haem and utilise it as an iron source. The following twelve chapters (3-14) focus on specific types of microorganism that are of key interest, covering both an array of pathogens for humans, animals and plants (e.g. species of Bordetella, Shigella, , Erwinia, Vibrio, Aeromonas, Francisella, Campylobacter and Staphylococci, and EHEC) as well as a number of prominent non-pathogens (e.g. the rhizobia, E. coli K-12, Bacteroides spp., cyanobacteria, Bacillus spp. and yeasts). The chapters relay the common themes in microbial iron uptake approaches (e.g. the use of siderophores, TonB-dependent transporters, and ABC transport systems), but also highlight many distinctions (such as use of different types iron regulator and the impact of the presence/absence of a cell wall) in the strategies employed. We hope that those both within and outside the field will find this book useful, stimulating and interesting. We intend that it will provide a source for reference that will assist relevant researchers and provide an entry point for those initiating their studies within this subject. Finally, it is important that we acknowledge and thank wholeheartedly the many contributors who have provided the 14 excellent chapters from which this book is composed. Without their considerable efforts, this book, and the understanding that it relays, would not have been possible. Simon C Andrews and Pierre Cornelis

Folsomia candida (Collembola): A "standard" soil arthropod

Folsomia candida Willem 1902, a member of the order Collembola (colloquially called springtails), is a common and widespread arthropod that occurs in soils throughout the world. The species is parthenogenetic and is easy to maintain in the laboratory on a diet of granulated dry yeast. F. candida has been used as a "standard" test organism for more than 40 years for estimating the effects of pesticides and environmental pollutants on nontarget soil arthropods. However. it has also been employed as a model for the investigation of numerous other phenomena such as cold tolerance, quality as a prey item, and effects of microarthropod grazing on pathogenic fungi and mycorrhizae of plant roots. In this comprehensive review. aspects of the life history, ecology, and ecotoxicology of F candida are covered. We focus on the recent literature, especially studies that have examined the effects of soil pollutants on reproduction in F candida using the protocol published by the International Standards Organization in 1999.

Biological control of black vine weevil (Otiorhynchus sulcatus, Coleoptera: Curculionidae) by entomopathogenic bacteria and their cell-free toxic metabolites

Biocontrol agents such as Xeiwrhabduf, nemalophilci and X. nematophila ssp. bovienii and their cell-free protein toxin complexes were lethal to larvae of O. sulcatus when applied to potting compost in the absence of plants. Similarly, strawberry plants infected with 0. sulcaitfi larvae were protected from damage by applications of both cell suspensions of the bacteria and solutions of their cell-free toxic metabolites, indicating that it is the protein toxins, which are responsible for the lethal effects observed. These toxic metabolites were found more effective against 0. sulccitus larvae when treated in soil microflora. Insect mortality is increased by increasing temperature and bacterial concentration. The toxins remained pathogenic for several months when stored in potting soil either at 15 or 20°C, however, bacterial cells were not as persistent as the toxins. It is therefore suggested that these bacteria and their toxic metabolites can he applied in soil for insect pest control.

The isolation of a single spore isolate of Pasteuria penetrans and its pathogenicity on Meloidogyne javanica

We have obtained a single spore isolate of Pasteuria penetrans, derived by allowing a single spore to attach to a second-stage juvenile (J2) of the root-knot nematode Meloidogyne javanica. By analysing DNA sequences at three different loci we have obtained evidence that the isolate is, indeed, genetically pure. We compared the ability of the single spore isolate and the parent population from which it was selected to attach to and parasitise both the original population of M. javanica on which it was isolated and a single egg mass line derived from it. There was no difference in the attachment of spores of the single spore isolate to juveniles compared to the parental population, although there were higher numbers of both attaching to J2 of the single egg mass line compared to its parental population. Judging from the numbers of egg masses and Pasteuria-infected females, the single spore isolate was less pathogenic to the parental population of M. javanica than was the parental spore population.

Why high seed densities within buried mesh bags may overestimate depletion rates of soil seed banks

1. Estimates of seed bank depletion rates are essential for modelling and management of plant populations. The seed bag burial method is often used to measure seed mortality in the soil. However, the density of seeds within seed bags is higher than densities in natural seed banks, which may elevate levels of pathogens and influence seed mortality. The aim of this study was to quantify the effects of fungi and seed density within buried mesh bags on the mortality of seeds. Striga hermonthica was chosen as the study species because it has been widely studied but different methods for measuring seed mortality in the soil have yielded contradictory estimates. 2. Seed bags were buried in soil and exhumed at regular time intervals to monitor mortality of the seeds in three field experiments during two rainy seasons. The effect of fungal activity on seed mortality was evaluated in a fungi exclusion experiment. Differences in seed-to-seed interaction were obtained by using two and four densities within the seed bags in consecutive years. Densities were created by mixing 1000 seeds with 0, 10, 100 or 1000 g of coarse sand. 3. The mortality rate was significantly lower when fungi were excluded, indicating the possible role of pathogenic fungi. 4. Decreasing the density of seeds in bags significantly reduced seed mortality, most probably because of decreased seed-to-seed contamination by pathogenic fungi. 5. Synthesis and applications. Models of plant populations in general and annual weeds in particular often use values from the literature for seed bank depletion rates. These depletion rates have often been estimated by the seed bag burial method, yet seed density within seed bags may be unrealistically high. Consequently, estimates of seed mortality rates may be too high because of an overestimation of the effects of soil or seed-borne pathogens. Species that have been classified from such studies as having short-lived seed banks may need to be re-assessed using realistic densities either within seed bags or otherwise. Similarly, models of seed bank dynamics based on such overestimated depletion rates may lead to incorrect conclusions regarding the seed banks and, perhaps, the management of weeds and rare species.

Mutations in H5N1 influenza virus hemagglutinin that confer binding to human tracheal airway epithelium

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary "dead end." We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.

Characterization and origin of infection of Rhizoctonia solani associated with Brassica oleracea crops in the UK

An extensive study was conducted to determine where in the production chain Rhizoctonia solani became associated with UK module-raised Brassica oleracea plants. In total, 2600 plants from 52 crops were sampled directly from propagators and repeat sampled from the field. Additional soil, compost and water samples were collected from propagation nurseries and screened using conventional agar isolation methods. No isolates of R. solani were recovered from any samples collected from propagation nurseries. Furthermore, nucleic acid preparations from samples of soil and compost from propagation nurseries gave negative results when tested for R. solani using real-time PCR. Conversely, R. solani was recovered from 116 of 1300 stem bases collected from field crops. All the data collected suggested R. solani became associated with B. oleracea in the field rather than during propagation. Parsimony and Bayesian phylogenetic studies of ribosomal DNA suggested the majority of further classified isolates belonged to anastomosis groups 2-1 (48/57) and AG-4HGII (8/57), groups known to be pathogenic on Brassica spp. in other countries. Many R. solani isolates were recovered from symptomless plant material and the possibilities for such an association are discussed.

Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control

The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project—VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L’hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Åqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37–46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies.

Interference competition and high temperature reduce the ‘virulence’ of fig wasps and contribute to stability of a fig-wasp mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps from ovipositing in all flowers, or seed production would cease, and the mutualism would go extinct. In Ficus racemosa, we find that syconia (‘figs’) that have few foundresses (ovipositing wasps) are underexploited in the summer (few seeds, few galls, many empty ovules) and are overexploited in the winter (few seeds, many galls, few empty ovules). Conversely, syconia with many foundresses produce intermediate numbers of galls and seeds, regardless of season. We use experiments to explain these patterns, and thus, to explain how this mutualism is maintained. In the hot summer, wasps suffer short lifespans and therefore fail to oviposit in many flowers. In contrast, cooler temperatures in the winter permit longer wasp lifespans, which in turn allows most flowers to be exploited by the wasps. However, even in winter, only in syconia that happen to have few foundresses are most flowers turned into galls. In syconia with higher numbers of foundresses, interference competition reduces foundress lifespans, which reduces the proportion of flowers that are galled. We further show that syconia encourage the entry of multiple foundresses by delaying ostiole closure. Taken together, these factors allow fig trees to reduce galling in the wasp-benign winter and boost galling (and pollination) in the wasp-stressing summer. Interference competition has been shown to reduce virulence in pathogenic bacteria. Our results show that interference also maintains cooperation in a classic, cooperative symbiosis, thus linking theories of virulence and mutualism. More generally, our results reveal how frequency-dependent population regulation can occur in the fig-wasp mutualism, and how a host species can ‘set the rules of the game’ to ensure mutualistic behavior in its symbionts.

Population performance of collembolans feeding on soil fungi from different ecological niches

The potential reproductive value of arbuscular mycorrhizal fungi (Gloinus intraradices and Glomus invermaium), root pathogenic fungi (Rhizoctonia solani and Fusarium culmorum) and saprotrophic fungi (Penicillium hordei and Trichoderma harzianum) were examined for the collembolans Folsomia candida Willem and Folsomia fimetaria L. Dried baker's yeast (Saccharomyces cerevisiae) was used as a reference standard food in laboratory cultures. Collembolan performance was determined as final size, fecundity and population growth rate after when fed the fungal food sources for 31 days. The mycorrhizal fungi gave the least growth and fecundity compared with the other fungi, but G. intraradices gave good fecundity for F. candida. In terms of growth, Baker's yeast was a high-quality food for both adults and juveniles of both species, but it was a poorer food in terms of fecundity of F. candida. Preference of the fungi in all possible pairwise combinations showed that although F. fimetaria did not perform well on Glomus spp. and F. candida did not grow well on Glomus spp. their preference for these fungi did not reflect this. The highest fecundity was seen with the root pathogen F. culmorum. Different quality indicators such as the C:N ratio of the fungal food sources as well as other biological parameters are discussed in relation to their reproductive value and Collembola preferential feeding. (c) 2007 Elsevier Ltd. All rights reserved.