Coevolution with phages does not influence the evolution of bacterial mutation rates in soil

Coevolution with phages drive the evolution of high bacterial mutation rates in vitro, but the relevance of this finding to natural populations is unclear. Here, we investigated how coevolution affects mutation rate evolution in soil, in the presence and absence of the rest of the natural microbial community. Although mutation rate on average increased threefold, neither coevolving phages nor the rest of natural community significantly affected mutation rates. Our results suggest that features of the soil over and above directly interacting organisms constrain the evolution of strong mutators, helping to explain their relatively low frequency compared with some laboratory and clinical settings.     

Within-host competition and drug resistance in the human malaria parasite Plasmodium falciparum

Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures.     

A trade-off between oxidative stress resistance and DNA repair plays a role in the evolution of elevated mutation rates in bacteria

The dominant paradigm for the evolution of mutator alleles in bacterial populations is that they spread by indirect selection for linked beneficial mutations when bacteria are poorly adapted. In this paper, we challenge the ubiquity of this paradigm by demonstrating that a clinically important stressor, hydrogen peroxide, generates direct selection for an elevated mutation rate in the pathogenic bacterium Pseudomonas aeruginosa as a consequence of a trade-off between the fidelity of DNA repair and hydrogen peroxide resistance. We demonstrate that the biochemical mechanism underlying this trade-off in the case of mutS is the elevated secretion of catalase by the mutator strain. Our results provide, to our knowledge, the first experimental evidence that direct selection can favour mutator alleles in bacterial populations, and pave the way for future studies to understand how mutation and DNA repair are linked to stress responses and how this affects the evolution of bacterial mutation rates.     

Evolution of a single niche specialist in variable environments

The pattern (space versus time) and scale (relative to the lifetime of individuals) of environmental variation is thought to play a central role in governing the evolution of the ecological niche and the maintenance of genetic variance in fitness. To evaluate this idea, we serially propagated an initially genetically uniform population of the bacterium Pseudomonas fluorescens for a few hundred generations in environments that differed in both the pattern and scale at which two highly contrasted carbon substrates were experienced. We found that, contrary to expectations, populations often evolved into a single niche specialist adapted to the less-productive substrate in variable environments and that the genetic variance in fitness across different components of the environment was not generally higher in variable environments when compared with constant environments. We provide evidence to suggest that our results reflect a novel constraint on niche evolution imposed by the supply of beneficial mutations available to selection in variable environments.     

Host plant and population determine the fitness costs of resistance to Bacillus thuringiensis

Novel adaptations often cause pleiotropic reductions in fitness. Under optimal conditions individual organisms may be able to compensate for, or reduce, these fitness costs. Declining environmental quality may therefore lead to larger costs. We investigated whether reduced plant quality would increase the fitness costs associated with resistance to Bacillus thuringiensis in two populations of the diamondback moth Plutella xylostella. We also measured the rate of decline in resistance on two host-plant (Brassica) species for one insect population (Karak). Population X plant species interactions determined the fitness costs in this study. Poor plant quality increased the fitness costs in terms of development time for both populations. However, fitness costs seen in larval survival did not always increase as plant quality declined. Both the fitness and the stability experiment indicated that fitness costs were higher on the most suitable plant for one population. Theoretically, if the fitness cost of a mutation interacts additively with environmental factors, the relative fitness of resistant insects will decrease with environmental quality. However, multiplicative costs do not necessarily increase with declining quality and may be harder to detect when fitness parameters are more subject to variation in poorer environments.     

One shot-two pathogens blocked: Exposure of Arabidopsis to hexadecane,a long chain volatile organic compound,confers induced resistance against both Pectobacterium carotovorum and Pseudomonas syringae

Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.     

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco

Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance.     

Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger.     

Non contiguous-finished genome sequence of Pseudomonas syringae pathovar syringae strain B64 isolated from wheat

The Gram-negative gammaproteobacterium Pseudomonas syringae is one of the most wide-spread plant pathogens and has been repeatedly reported to cause significant damage to crop plantations. Research on this pathogen is very intensive, but most of it is done on isolates that are pathogenic to Arabidopsis, tomato, and bean. Here, we announce a high-quality draft genome sequence of Pseudomonas syringae pv. syringae B64 which is the first published genome of a P. syringae strain isolated from wheat up to date. The genome sequence will assist in gaining insights into basic virulence mechanisms of this pathogen which has a relatively small complement of type III effectors.     

Survival costs of reproduction vary with age in North American red squirrels

The costs of reproduction are expected to be higher under unfavourable conditions, so that breeding in years of low food supply should have important costs. In addition, the costs of reproduction may be contingent on the age of individuals, and young growing and old senescent individuals should suffer higher costs than the prime-age ones. We tested these predictions by investigating the costs of reproduction as a function of food availability and age in female North American red squirrels using the long-term data on survival and reproduction. We found that the costs of reproduction were independent of food supply, and we did not detect any trade-off between the current and future reproduction. We also did not detect any survival cost of reproduction for the prime-age females, but found evidence for survival costs in yearlings and old (6 years or above) females with successfully breeding individuals having a lower chance of survival compared with unsuccessful or non-breeding ones. These results supported our prediction that the costs of reproduction depended on the age of female red squirrels and were higher in young growing and old senescent individuals. Our study also indicated that, in contrast to large herbivores, heterogeneity in individual quality and viability selection in red squirrels do not affect the study of trade-offs and of the age variation in life-history traits.     

Temperature effects on parasite prevalence in a natural hybrid complex

Both host susceptibility and parasite infectivity commonly have a genetic basis, and can therefore be shaped by coevolution. However, these traits are often sensitive to environmental variation, resulting in genotype-by-environment interactions. We tested the influence of temperature on host–parasite genetic specificity in the Daphnia longispina hybrid complex, exposed to the protozoan parasite Caullerya mesnili. Infection rates were higher at low temperature. Furthermore, significant differences between host clones, but not between host taxa, and a host genotype-by-temperature interaction were observed.     

Molecular Characterization of an Ice Nucleation Protein Variant (InaQ) from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli

The ice nucleation protein (INP) of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized. The deduced amino acid sequence of InaQ shares similar protein domains with three P. syringae INPs, namely, InaK, InaZ, and InaV, which were identified as an N-terminal domain, a central repeating domain, and a C-terminal domain. The expression of the full-length InaQ and of various truncated variants was induced in Escherichia coli to analyze their transmembrane transport and surface-binding activities, while using the green fluorescence protein (GFP) as the fusion partner. With two transmembrane segments and a weak secretion signal, the N-terminal domain (InaQ-N) alone was found to be responsible for the transport process as well as for the binding to the outer membrane, whereas the C-terminal region was nonfunctional in protein transport. Increased membrane transport and surface-binding capacities were induced by a low isopropyl-β-D-thiogalactoside concentration (0.1 mmol/l) but not by culture temperatures (15 ºC to 37 ºC). Furthermore, by constructing the GFP-fused proteins with a single InaQ-N, as well as two and three tandemly aligned InaQ-N molecules, the transport and membrane-binding activities of these proteins were compared using Western blot analysis, immmunofluorescence microscopy, and assays of the GFP specific fluorescence intensity of subcellular fractions and flow cytometry, which showed that the increase of InaQ-N repeats resulted in a coordinated increase of the surface-immobilization efficiency. Therefore, the results of this study can serve as a molecular basis for improving the performance of INP-based cell surface-display systems.     

Honest sexual signalling mediated by parasite and testosterone effects on oxidative balance

Extravagant ornaments evolved to advertise their bearers'' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules (cellular proteins, lipids and DNA). Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties.     

The role of moulting in parasite defence

Parasitic infections consist of a succession of steps during which hosts and parasites interact in specific manners. At each step, hosts can use diverse defence mechanisms to counteract the parasite's attempts to invade and exploit them. Of these steps, the penetration of parasites into the host is a key step for a successful infection and the epithelium is the first line of host defence. The shedding of this protective layer (moulting) is a crucial feature in the life cycle of several invertebrate and vertebrate taxa, and is generally considered to make hosts vulnerable to parasites and predators. Here, we used the crustacean Daphnia magna to test whether moulting influences the likelihood of infection by the castrating bacterium Pasteuria ramosa. This parasite is known to attach to the host cuticula before penetrating into its body. We found that the likelihood of successful parasite infection is greatly reduced if the host moults within 12 h after parasite exposure. Thus, moulting is beneficial for the host being exposed to this parasite. We further show that exposure to the parasite does not induce hosts to moult earlier. We discuss the implications of our findings for host and parasite evolution and epidemiology.     

Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.     

The function of calreticulin in plant immunity: New discoveries for an old protein

Since its initial discovery as a high affinity Ca²⁺-binding protein in the sarcoplasmic reticulum and endoplasmic reticulum (ER), calreticulin (CRT) has been documented to be a multifunctional protein in both animal and plant cells. This protein is well recognized as a Ca²⁺-binding molecular chaperone that facilitates the folding of newly synthesized glycoproteins and regulates the Ca²⁺ homeostasis in the ER lumen. However, functional relevance associated with its localization in other cellular compartments has also been reported. Recent studies suggest that both isoforms of plant CRTs (AtCRT1/2 and AtCRT3) are involved in regulating plant defense against biotrophic pathogens. Here we discuss the cellular functions of CRT and its connection to the emerging functions of AtCRTs in plant immunity.     

Singlet oxygen-mediated and EXECUTER-dependent signalling and acclimation of Arabidopsis thaliana exposed to light stress

Plants respond to environmental changes by acclimation that activates defence mechanisms and enhances the plant''s resistance against a subsequent more severe stress. Chloroplasts play an important role as a sensor of environmental stress factors that interfere with the photosynthetic electron transport and enhance the production of reactive oxygen species (ROS). One of these ROS, singlet oxygen (¹O₂), activates a signalling pathway within chloroplasts that depends on the two plastid-localized proteins EXECUTER 1 and 2. Moderate light stress induces acclimation protecting photosynthetic membranes against a subsequent more severe high light stress and at the same time activates ¹O₂-mediated and EXECUTER-dependent signalling. Pre-treatment of Arabidopsis seedlings with moderate light stress confers cross-protection against a virulent Pseudomonas syringae strain. While non-pre-acclimated seedlings are highly susceptible to the pathogen regardless of whether ¹O₂- and EXECUTER-dependent signalling is active or not, pre-stressed acclimated seedlings without this signalling pathway lose part of their pathogen resistance. These results implicate ¹O₂- and EXECUTER-dependent signalling in inducing acclimation but suggest also a contribution by other yet unknown signalling pathways during this response of plants to light stress.     

Roots, shoots and reproduction: sexual dimorphism in size and costs of reproductive allocation in an annual herb

Females tend to be smaller than males in woody dioecious plant species, but they tend to be larger in herbs. The smaller size of females in woody species has been attributed to higher reproductive costs, yet no satisfactory explanation has been provided for their larger size in herbs. Because herbs have higher nitrogen concentrations in their tissues than woody plants, and because pollen is particularly rich in nitrogen, we predicted that male growth would be more compromised by reproduction than female growth. To test this hypothesis, we conducted three experiments on the annual dioecious herb Mercurialis annua. First, we compared the timing of reproduction between males and females and found that males started flowering earlier than females; early flowering is expected to compromise growth more than later flowering. Second, we compared plants allowed to flower with those prevented from flowering by experimental debudding and found that males incurred a higher reproductive cost than females in terms of both biomass and, particularly, nitrogen. Third, we grew plants under varying levels of nitrogen availability and found that although sexual size dimorphism was unaffected by nitrogen, females, but not males, decreased their relative allocation to both roots and reproduction under high nitrogen availability. We propose that males deal with the high cost of pollen production in terms of nitrogen by allocating biomass to nitrogen-harvesting roots, whereas females pay for carbon-rich seeds and fruits by investing in photosynthetic organs. Sexual dimorphism would thus seem to be the outcome of allocation to above- versus below-ground sinks that supply resources (carbon versus nitrogen) limiting the female and male reproduction differentially.