Sofia Costa

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system.

Nematode interactions in nature : Models for sustainable control of nematode pests of crop plants?

van der Putten, W. H., Cook, R., Costa, S., Davies, K. G., Fargette, M., Freitas, H., Hol, W. H. G., Kerry, B. R., Maher, N., Mateille, T., Moens, M., de la Pena, E., Piskiewicz, A., Raeymaekers, A., Rodriguez-Echeverria, S., van der Wurff, A. W. G. (2006). Nematode interactions in nature: models for sustainable control of nematode pests of crop plants? Advances in Agronomy, 89, 227-260.

Soil microorganisms control plant ectoparasitic nematodes in natural coastal foredunes

Belowground herbivores can exert important controls on the composition of natural plant communities. Until now, relatively few studies have investigated which factors may control the abundance of belowground herbivores. In Dutch coastal foredunes, the root-feeding nematode Tylenchorhynchus ventralis is capable of reducing the performance of the dominant grass Ammophila arenaria (Marram grass). However, field surveys show that populations of this nematode usually are controlled to nondamaging densities, but the control mechanism is unknown. In the present study, we first established that T. ventralis populations are top-down controlled by soil biota. Then, selective removal of soil fauna suggested that soil microorganisms play an important role in controlling T. ventralis. This result was confirmed by an experiment where selective inoculation of microarthropods, nematodes and microbes together with T. ventralis into sterilized dune soil resulted in nematode control when microbes were present. Adding nematodes had some effect, whereas microarthropods did not have a significant effect on T. ventralis. Our results have important implications for the appreciation of herbivore controls in natural soils. Soil food web models assume that herbivorous nematodes are controlled by predaceous invertebrates, whereas many biological control studies focus on managing nematode abundance by soil microorganisms. We propose that soil microorganisms play a more important role than do carnivorous soil invertebrates in the top-down control of herbivorous ectoparasitic nematodes in natural ecosystems. This is opposite to many studies on factors controlling root-feeding insects, which are supposed to be controlled by carnivorous invertebrates, parasitoids, or entomopathogenic nematodes. Our conclusion is that the ectoparasitic nematode T. ventralis is potentially able to limit productivity of the dune grass A. arenaria but that soil organisms, mostly microorganisms, usually prevent the development of growth-reducing population densities.

Interactions between nematodes and their microbial enemies in coastal sand dunes

European foredunes are almost exclusively colonised by Ammophila arenaria, and both the natural succession and the die-out of this plant have been linked to populations of plant-parasitic nematodes (PPN). The overarching aim of this study was to investigate top-down control processes of PPN in these natural ecosystems through comparative analyses of the diversity and dynamics of PPN and their microbial enemies. Our specific aims were, first, to identify and quantify PPN microbial enemies in European sand dunes; second, to assess their life history traits, their spatial and temporal variation in these ecosystems, and third, to evaluate their control potential of PPN populations. This was done by seasonal sampling of a range of sites and making observations on both the nematode and the microbial enemy communities in rhizosphere sand. Nine different nematode microbial enemies belonging to different functional groups were detected in European sand dunes. Their high diversity in these low productivity ecosystems could both result from or lead to the lack of dominance of a particular nematode genus. The distribution of microbial enemies was spatially and temporally variable, both among and within sampling sites. Obligate parasites, either with low host-specificity or having the ability to form an environmentally resistant propagule, are favoured in these ecosystems and are more frequent and abundant than facultative parasites. Three microbial enemies correlated, either positively or negatively, with PPN population size: Catenaria spp., Hirsutella rhossiliensis and Pasteuria penetrans. Microbial-enemy supported links in the food-web may be involved in the control of PPN populations through indirect effects. The endospore-forming P. penetrans was the most successful top-down control agent, and was implicated in the direct control of Meloidogyne spp. and indirect facilitation of Pratylenchus spp. Overall, our findings suggest strong and diverse top-down control effects on the nematode community in these natural ecosystems.

Factors affecting cork oak (Quercus suber) regeneration: acorn sowing success and seedling survival under field conditions

Background: The lack of regeneration of cork oak (Quercus suber) is putting at risk the conservation of the montado. Biotic and abiotic factors as well as management are thought to constrain regeneration. Aims: Identify limitations to regeneration during the early stages and relate them to management practices. Methods: Experimental burying of acorns was carried out at three distinctly managed montados. Cache predation, seedling emergence, survival and resprouting were monitored during the first year. Regeneration was evaluated by testing the effect of biotic and abiotic factors in each stage. Results: Regeneration was determined by temperature and relative humidity. Early emerged seedlings showed higher drought tolerance and resprouting ability. Bigger acorns and higher plant cover significantly assisted emergence, survival and resprouting. Mortality causes were drought (38%) and mechanical ploughing (33%), while cache predation and browsing on seedlings were low (1.7%, when excluding tag effect on cache predation, and 0.6%, respectively). Seedling emergence and survival of drought were significantly lower in the most disturbed site than in the other two sites. Conclusions: Abiotic factors restrain regeneration while acorn size and plant cover are key factors determining seedling emergence and survival. The lack of regeneration may be overcome in the short term by preserving the shrub layer. Moreover, reforestation by direct sowing should be also considered.

Microbial Ecology and Nematode Control in Natural Ecosystems

Plant-parasitic nematodes have traditionally been studied in agricultural systems, where they can be pests of importance on a wide range of crops. Nevertheless, nematode ecology in natural ecosystems is receiving increasing interest because of the role of nematodes in soil food webs, nutrient cycling, influences on vegetation composition, and because of their indicator value. In natural ecosystems, plant-parasitic nematode populations can be controlled by bottom-up, horizontal and top-down mechanisms, with more than one mechanism acting upon a given population. Moreover, in natural ecosystems soil nematodes inhabit probably more heterogeneous environment than in agricultural soils. New breakthroughs are to be expected when new molecular-based methods can be used for nematode research in natural ecosystems. Thus far, nematode ecology has strongly relied on coupling conventional abundance and diversity measurements with conceptual population ecology. Biochemical and molecular methods are changing our understanding of naturally co-evolved multitrophic plant-nematode-antagonist interactions in nature, the inter-connections within the soil food web and the extent to which nematodes are involved in many, disparate, soil processes. We foresee that finer nematode interactions that lead to their management and control can only be fully understood through the joint effort of different research disciplines that investigate such interactions from the molecular to the ecosystem level.

Laimaphelenchus suberensis sp. nov. associated with Quercus suber in Portugal

Laimaphelenchus suberensis sp. nov. obtained from declining Quercus suber trees of Herdade da Gouveia de Baixo, Alentejo, Portugal, is described and illustrated based on morphological, biometrical and molecular characters. The diagnosis of Laimaphelenchus species has been commonly based on the presence or absence of a vulval flap and on the shape structure of the tail tip. The species described here has been included in the Laimaphelenchus group without vulval flap, and can be distinguished from morphologically similar species by its tail tip shape structure that has a stalk-like terminus and three diffuse tubercles with 4–6 finger-like protrusions. For the molecular analyses, the mitochondrial DNA region from the cytochrome oxidase subunit I (mtCOI), the D2-D3 expansion segments of the large subunit (LSU) and small subunit (SSU) of rRNA gene were amplified and sequenced. Sequences of L. suberensis sp. nov. clustered separately from all Laimaphelenchus spp. with available sequences in Genbank, confirming its identification as a new species. This is the second report of the genus Laimaphelenchus in Portugal, associated with Q. suber: L. heidelbergi and L. suberensis sp. nov.

Plant sex and phenological stage affect interactions with rhizosphere nematode communities

ABSTRACT Background: In dioecious plants, females and males associate differently with mycorrhizal fungi, but interactions with other rhizosphere organisms are less well studied. Aims: We investigated the effect of plant sex on rhizosphere nematode communities associated with Corema album, a dioecious shrub occurring in coastal habitats. Methods: Rhizosphere samples were collected from males and females in three populations (150 plants), during fruiting and flowering. Nematode communities were characterised and compared between plant sexes through statistical analyses of the abundance of trophic groups, plant parasitic nematode (PPN) genera and ecological indices. Results: Free-living nematodes showed no statistically significant differences owing to plant sex. Conversely, PPN community composition was significantly different between plant sexes during fruiting but not flowering, suggesting that physiological requirements over the annual phenological cycle of the plant influence ecological interactions with the rhizosphere. Of the 13 PPN genera identified, the ectoparasitic Criconema and Hemicriconemellawere more abundant in the rhizosphere of males during fruiting, whereas the endoparasitic Meloidogyne associated more frequently with females, suggesting that plant host suitability is related to PPN feeding strategy. Conclusions: It appears that interactions of individuals of different sexes of C. album with the rhizosphere nematode community vary with phenological stage, especially for PPN.

Leaf decomposition of cork oak under three different land uses within a montado of southern Portugal

This study compared litter decomposition dynamics of cork oak at three sites under different land-uses (grassland, shrubland and woodland), in a montado ecosystem in Southern Portugal. The montado is a protected habitat within the EU Habitats Directive, but the long-term persistence of cork oak is endangered in these ecosystems, with health of poor cork oak and low natural regeneration rates being the main causes of degradation. Moreover, human management has resulted in the conversion of woodlands to grasslands and may have long-term effects on soil nutrient availability, eventually modifying soil nutrient budgets. Knowledge of the ecological processes is therefore relevant for ecosystem management and species conservation. In the study, the estimated amount of leaf fall from cork oak showed no significant differences between land uses, despite the positive influence of tree crown size on leaf fall. Decomposition was affected by season, vegetation cover, leaf thickness and litter quality. Differences in land use that exposed soil to harsh climate conditions negatively affected soil microbial dynamics, resulting in lower decomposition rates in the more disturbed sites with lower canopy cover.

Temporal effects dominate land use as factors affecting soil nematode communities in Mediterranean oak woodlands

We analysed the soil nematode community within three different montados (agricultural, pastoral and forestry uses), focusing on temporal variation. Nematodes were classified into trophic groups (bacterivores, fungivores, omnivores, predators and plant-parasitic nematodes (PPN)) and we calculated the maturity index for free-living taxa (MI), maturity index for plant-parasitic taxa (PPI) and the nematode channel ratio (NCR). Temporal variations were most evident during winter when there was a rise in the abundance of the five functional groups. Concordantly, there was a simultaneous increase of soil moisture and organic matter, due to litter decomposition. Fungivore abundance was highest in the forest and the temporal occurrence and abundance of many PPN genera was largely determined by land use. Land management was responsible for differences in plant community structure and composition, thus plant diversity increased from the agricultural to the forestry use. Because the montado shows great temporal variability in vegetation structure our results of MI, PPI and PPI/MI ratio explain the significant changes in the nutritional status over time, with the highest values recorded in the spring. NCR is a good indicator of energy efficiency in the soil decomposition process. Its lowest values were attained in the forest, where the slow-growing plant species favoured a lower activity of the bacterial energy channel and a prevalence of the fungal-based decomposition energy channel. Nematode composition reflected plant succession, changes in decomposition in the soil food web and temporal variations in the structure of soil.