Discrimination of single‐porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra‐species differentiation remains poorly explored. Herein, both transmission Fourier‐transform infrared (FTIR) and attenuated total reflectance (ATR)‐FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR‐spectroscopy techniques is compared with the same collection of unique strains. ATR‐FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Mucopolysaccharidoses in northern Brazil: Targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients' clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers.     

Choosing the best method for stream bioassessment using macrophyte communities: Indices and predictive models

The bioassessment and monitoring of the ecological status of rivers using macrophytes has gained new momentum since macrophytes were recognised as biological quality elements for the implementation of the European Water Framework Directive (WFD; EU/2000/60).Our objectives were to test the suitability of two predictive modelling approaches to macrophyte communities as a tool for water quality assessment, and to compare their performance with other more common approaches—the use of macrophytes as indicators of the trophic status of rivers and multimetric indices. We used floristic and environmental data that were collected in the spring of 2004 and 2005 from around 400 sites on rivers across mainland Portugal, western Iberia.We build two predictive models: MACPACS (MACrophyte Prediction And Classification System) and MAC (Macrophyte Assessment and Classification) based on RIVPACS and the BEAST methods, respectively. Whereas MACPACS is derived from taxa occurrence data, MAC uses a quantitative measure of taxa abundance. Both models showed good performance in predicting reference sites to the correct group and low rate of misclassification errors. However, they performed differently. MAC depicts a reliable response to the overall human-mediated degradation of fluvial systems, as does the multimetric index (RVI, Riparian Vegetation Index), but MACPACS presented only a poor correlation with the Global Human Disturbance Index and with the nutrients input. The incorporation of abundance data in vegetation predictive models appears to be particularly important to the detection of high levels of degradation. The values for correlations with physical–chemical pressure variables were lower than expected for MTR (Mean Trophic Rank) due to an insufficient number of scoring species found in Portuguese fluvial systems. Our results suggest that the most effective methods for bioassessment in Mediterranean-type rivers are either the RVI or the MAC predictive model.     

Temporal and Spatial Patterns in Inputs and Stock of Organic Matter in Savannah Streams of Central Brazil

Coarse particulate organic matter (CPOM) inputs from riparian vegetation into streams and CPOM benthic stock vary naturally in space and time, but most studies in the tropical savannah (Cerrado) have been done over a small temporal scale (<1 year), which does not allow for the determination of inter-annual patterns. We found that CPOM collected over two years differed temporally and spatially, whereas there was no significant variation between years for the benthic stock, which indicates high stability in the energy balance of streams. The largest monthly inputs occurred between August and October, at the end of the dry season and the onset of the rainy season, which was partially explained by precipitation. Other factors such as photoperiod, which was not studied, could also have important roles in this pattern. Spatial differences in CPOM between streams were attributed to topography and channel morphology. The plant density was lowest in the stream with a more irregular topography and a deeper channel, which results in drier riparian soil. The benthic stock was highest in the stream with a flat channel, where the lower water speed facilitates the accumulation of CPOM in the stream bed. Inter-annual differences in CPOM were attributed more to the differences in the beginning of the dry and wet periods between years than to the average values of precipitation. Longer-term studies are needed to clarify this temporal pattern.     

Food quality, feeding preferences, survival and growth of shredders from temperate and tropical streams

1. The importance of leaf quality to the nutritional ecology of lotic shredders is well established for temperate species but virtually unknown for tropical taxa. In the present study, we compared the feeding behaviour and performance of two tropical and two temperate shredders in a series of pair‐wise experiments. 2. Specifically, we tested whether leaf conditioning status (stream‐conditioned versus unconditioned leaves) and geographical origin (temperate Alnus glutinosa versus tropical Hura crepitans leaves) affect the food preference, survivorship, and growth of selected shredders from low and high latitudes in a consistent manner. The animals used in experiments were the caddis‐flies Nectopsyche argentata and Phylloicus priapulus from Venezuela, Sericostoma vittatum from Central Portugal, and the amphipod Gammarus pulex from Northern Germany. 3. In general, all shredders exhibited the same high preference for conditioned over unconditioned leaves, irrespective of the geographical origin of the leaf or shredder species. 4. A corresponding tendency for higher growth was found for sets of animals offered conditioned leaves, with the differences in growth being clearer in the two tropical shredders. Survivorship of the two temperate species was consistently high (> 83%) regardless of the diet offered, whereas the tropical shredders survived better on conditioned (77–90%) as compared with unconditioned (54–87%) leaves, although not significantly so. 5. With the exception of the temperate S. vittatum, shredders did not select or perform better on leaves to which they had previously been exposed, indicating a potential adaptation to native leaf species is over‐ridden by intrinsic leaf properties. 6. Taken together, the results of this study suggest that tropical shredders may exhibit the same basic patterns of food exploitation as their temperate counterparts. Consequently, current concepts relating to the role of shredders in stream detritus dynamics may well be applicable to tropical streams, although essentially derived from temperate systems.     

Predictive Models for Freshwater Biological Assessment: Statistical Approaches,Biological Elements and the Iberian Peninsula Experience: A Review

In the last three decades, predictive models have been developed and applied worldwide for freshwater bioassessment. They consist of statistical tools that follow the concept of the Reference Condition Approach. Composed of several sequential steps, these assessment tools assess the deviation of given site assemblages from the expected biological condition in the absence of human disturbance. The most common approaches (RIVPACS/AUSRIVAS and BEAST) are based on a posteriori classifications that use the biological composition of a community to classify reference sites in groups, and afterwards to establish which environmental features best discriminate the biological groups obtained. Here, we review the predictive modeling procedures used in freshwaters bioassessment (RIVPACS/AUSRIVAS, BEAST, ANNA, Artificial Neural Networks, Bayesian Belief Networks and others) as well as the biological elements to which they have been applied. We also review the Spanish and Portuguese experiences in the development and application of predictive models, with particular attention to regional environmental conditions, the different modeling approaches, and the available implementation tools. Moreover, and considering the natural continuity within the Iberian Peninsula (which include several transnational rivers), we discuss the possibilities of the development of common predictive models across the region, considering all factors that may influence their performance, such as the target scale used to develop the models (regional or peninsular); common reference criteria; sampling and sorting procedures; the taxonomic resolution used in the models; the temporal variability (mainly in the Iberian Mediterranean region); and the biological elements to consider. We concluded that there are good technical conditions for the implementations of a common predictive approach throughout the Iberian Peninsula, which should allow a global biological assessment of streams with different biological elements and seasons that could be used by water managers in the context of the Water Framework Directive. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Thermal unfolding of plastocyanin from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 and comparison with its thermophilic counterpart from Phormidium laminosum

The thermal unfolding of plastocyanin from the mesophilic cyanobacterium Synechocystis is described herein, and the results are compared with those obtained for the homologous thermophilic protein from Phormidium laminosum. The thermal unfolding is irreversible under all the conditions that were investigated. Plastocyanin from the thermophilic organism, both in the native state and in the apoprotein form, proved to be more thermostable than its mesophilic counterpart under all experimental conditions. Synechocystis reduced plastocyanin has been shown to be more stable than the oxidized species, both with respect to the required temperature for protein unfolding and with respect to the kinetics of the process. This behavior contrasts with that observed for Phormidium plastocyanin, in which the oxidized form is the more stable one. The unfolding pH dependence and kinetic studies indicate that around physiological pH, the most kinetically stable form is also the one more resistant to temperature variations, suggesting a close compromise between function and stability. Molecular dynamics simulations suggest that Phormidium and Synechocystis plastocyanins follow different unfolding pathways that affect different protein areas and which could be responsible for the observed dissimilar thermal resistance.     

Identification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus

The glutamic acid residues of the peptidoglycan of Staphylococcus aureus and many other bacteria become amidated by an as yet unknown mechanism. In this communication we describe the identification, in the genome of S. aureus strain COL, of two co-transcribed genes, murT and gatD, which are responsible for peptidoglycan amidation. MurT and GatD have sequence similarity to substrate-binding domains in Mur ligases (MurT) and to the catalytic domain in CobB/CobQ-like glutamine amidotransferases (GatD). The amidation of glutamate residues in the stem peptide of S. aureus peptidoglycan takes place in a later step than the cytoplasmic phase – presumably the lipid phase - of the biosynthesis of the S. aureus cell wall precursor. Inhibition of amidation caused reduced growth rate, reduced resistance to beta-lactam antibiotics and increased sensitivity to lysozyme which inhibited culture growth and caused degradation of the peptidoglycan.     

Chironomidae of the Holarctic region: a comparison of ecological and functional traits between North America and Europe

Chironomidae (Diptera) are widespread, abundant, diverse and ubiquitous, and include genera and species that are distributed across the Holarctic region. However, the geographical barriers between continents should have resulted in intraspecific population differentiation with reflection on individual biological and ecological traits. Our aim was to test for potential differences in Chironomidae species/genus and traits between the Nearctic and Palearctic regions. We compared the Chironomidae trait information gathered in two databases; one database was developed in Europe and the other in North America. Common genera and species of both databases were selected, and the common traits were adjusted into the same trait categories. Data were transformed into presence/absence and divided into Eltonian (biological/functional) and Grinnellian (ecological) traits. Common genera and common species were analyzed using Fuzzy correspondence analysis (FCA). Differences between databases occur for all trait domains. Yet, Eltonian traits showed lower level of concordance than Grinnellian traits at the species level. Different biological characteristics in the Nearctic and Palearctic regions may indicate that Chironomidae have different adaptions to similar ecological environments due to intraspecific variability or even trait plasticity.