Professor of Microbiology at the University of Aveiro. Member of the General Board of the Univ. of Aveiro Director of master studies in Microbiology.
Consulting in environmental microbiology.
Expertise in - environmental microbiology, - microbial diversity, - filamentous fungi in water and plants - gene transfer in water systems - mobile elements and bacterial resistance
Abstract: The diversity of gene cassette promoters in class 1 integrons was investigated in 47 strains isolated from wastewaters. The weak PcW and PcH1 variants predominated, suggesting that, similar to clinical environments, high rates of gene cassette recombination, rather than high expression of gene cassettes, have been preferentially selected in wastewaters.
Abstract: We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular bla(CTX-M). Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a bla(CTX-M) gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcpI was found in the upstream regions of all isolates examined. Culture-independent bla(CTX-M)-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as bla(RAHN-1). The results demonstrate that the occurrence and diversity of blac(CTX-M) genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments.
Abstract: In this study, the occurrence and diversity of integrons were evaluated in 697 isolates belonging to Enterobacteriaceae and Aeromonas spp. isolated from urban wastewaters. Screening of integrons was performed by dot blot hybridization and intI-positive strains were further characterized. The global prevalence of integrons was 3.73%. Three new gene cassettes were identified: a novel aadA variant (aadA17), a gene putatively involved in cell signaling (dcyA) and an open reading frame of unknown function interrupted by a novel insertion sequence (orfER.17::ISAs12). In total, thirteen different gene cassette arrays were detected, 4 representing novel integrons: intI1-dcyA-tniC, intI1-orfER.1.7::ISAs12-aadA13-qacE Delta 1-sul1, intI1-aacA4-catB3-bla(OxA-10)-aadA1-qacE Delta 1-sul1 and intI1-catB8-aadA17-qacE Delta 1-sul1. Approximately 80% of strains were resistant to at least 3 antibiotics of different classes. The presence of novel integron structures in treated effluents suggests that domestic wastewaters may favor the formation of novel combinations of gene cassettes. Moreover, the high prevalence of multiresistant strains highlights the urgent need to employ effective means of effluent disinfection to avoid dissemination of antibiotic-resistant bacteria. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Abstract: In the Azores, the advanced trophic state of the lakes requires a fast intervention to achieve the good ecological status prescribed by the Water Framework Directive. Despite the considerable effort made to describe the phytoplankton growing on the water column, the lack of information regarding the microbial processes in sediments is still high. Thus, for the successful implementation of internal management actions, the present work explored the relationships between geochemical profiles and dominant members of the bacterial community in sediments from eutrophic Azorean lakes. Lake Azul geochemical profiles were quite homogeneous for all parameters, while in lake Furnas the total iron profile presented a peak below the aerobic layer. For lake Verde, the concentrations of all studied parameters (20 +/- 2% loss-on-ignition; 2.10 +/- 0.08 mg g(-1) total phosphorus; 1.31 +/- 0.50 mg g(-1) total nitrogen; 8.06 +/- 0.13 mg g(-1) total iron) in the uppermost sediment layer were approximately two times higher than the ones in sediments from other lakes, decreasing with sediment depth. The higher amounts of phosphorus and organic matter in lake Verde suggested a higher internal contribution of phosphorus to eutrophication. The dominant members of the sediment bacterial community, investigated by denaturing gradient gel electrophoresis, were mostly affiliated to Proteobacteria phylum (Alpha-, Delta-, and Gamma-subclasses), group Bacteroidetes/Chlorobi and phylum Chloroflexi. The Cyanobacteria phylum was solely detected in sediments from lake Verde and lake Furnas that presented the highest amounts of nitrogen and phosphorus both in the water column and sediments, while the other phyla were detected in sediments from the three studied lakes. In conclusion, management measurers to achieve the good ecological status until 2015 should be distinct for the different lakes taking into account the relative magnitude of the nutrient sources and the bacterial diversity in sediments.
Abstract: The information on bacterial community composition (BCC) in Portuguese water bodies is very scarce. C,rtima River (central western Portugal) is known to have high levels of pollution, namely organic. In the present work, the BCC from a set of 16 water samples collected from C,rtima River Basin and its main tributaries was characterized using 16S rDNA-denaturing gradient gel electrophoresis, a culture-independent molecular approach. Molecular data were related to environmental parameters through multivariate analysis to investigate potential impact of water pollution along the river. Principal component analysis using environmental data showed a water quality gradient from more pristine waters (at the mountain tributaries) to waters with increasingly eutrophic potential (such as Fermentelos Lake). This gradient was mainly defined by factors such as organic and inorganic nutrient sources, electrical conductivity, hydrogen carbonate concentration, and pH. Molecular results showed variations in BCC along C,rtima River Basin but in the main river section, a Bacteroidetes phylotype (Flavobacterium sp.) proved to be dominant throughout the river course. Multivariate analysis suggests that spatial variation of BCC along the C,rtima River Basin depended mainly on parameters such as Chl a, total suspended solid (TSS), total organic carbon, electrical conductivity, and HCO(3)(-) levels. Bacteroidetes phylotypes were all related to higher electrical conductivity and HCO(3)(-) levels although some of these were also correlated with high SO(4)(2-) and others with high soluble reactive phosphorus, nitrate, TN, and Kjeld-N levels. The Gammaproteobacteria occurrence was correlated with high SO(4)(2-) levels. One of the Betaproteobacteria phylotypes showed to correlate with low redox potential (Eh) and high temperature, pH, TSS, and Chl a levels while another one showed a negative correlation with Chl a values.
Abstract: Bacteria from the surface microlayer (bacterioneuston) and underlying waters (bacterioplankton) were isolated upon exposure to UV-B radiation, and their individual UV sensitivity in terms of CFU numbers, activity (leucine and thymidine incorporation), sole-carbon source use profiles, repair potential (light-dependent and independent), and photoadaptation potential, under different physiological conditions, was compared. Colony counts were 11.516.2% more reduced by UV-B exposure in bacterioplankton isolates (P similar to<similar to 0.05). Inhibition of leucine incorporation in bacterioneuston isolates was 10.911.5% higher than in bacterioplankton (P similar to<similar to 0.05). These effects were accompanied by a shift in sole-carbon source use profiles, assessed with Biolog (R) EcoPlates, with a reduction in consumption of amines and amino acids and increased use of polymers, particularly in bacterioneuston isolates. Recovery under starvation was generally enhanced compared with nourished conditions, especially in bacterioneuston isolates. Overall, only insignificant increases in the induction of antibiotic resistant mutant phenotypes (RifR and NalR) were observed. In general, a potential for photoadaptation could not be detected among the tested isolates. These results indicate that UV effects on bacteria are influenced by their physiological condition and are accompanied by a shift in metabolic profiles, more significant in bacterioneuston isolates, suggesting the presence of bacterial strains adapted to high UV levels in the SML.
Abstract: We compare the environmental characteristics and bacterial communities associated with two rushes, Juncus maritimus and Bolboschoenus maritimus, and adjacent unvegetated habitat in a salt marsh subjected to historical mercury pollution. Mercury content was higher in vegetated than unvegetated habitat and increased with sampling depth. There was also a significant relationship between mercury concentration and bacterial composition. Habitat (Juncus, Bolboschoenus or unvegetated), sample depth, and the interaction between both, however, explained most of the variation in composition (similar to 70%). Variation in composition with depth was most prominent for the unvegetated habitat, followed by Juncus, but more constrained for Bolboschoenus habitat. This constraint may be indicative of a strong plant-microbe ecophysiological adaptation. Vegetated habitat contained distinct bacterial communities associated with higher potential activity of aminopeptidase, beta-glucosidase and arylsulphatase and incorporation rates of C-14-glucose and C-14-acetate. Communities in unvegetated habitat were, in contrast, associated with both higher pH and proportion of sulphate reducing bacteria. (C) 2011 Elsevier Ltd. All rights reserved.
Abstract: In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.similar to coli, Enterobacter sp.), FIC (A.similar to salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.similar to veronii, Aeromonas sp., E similar to coli), HI1 (E.similar to coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.
Abstract: In this work the presence of broad-host-plasmids in an estuary in Portugal has been investigated. Pseudomonas putida KT2442 was used as model recipient bacteria in biparental matings with tetracycline and mercury to select for resistance phenotypes. As a result, 7 transconjugants were shown to carry broad-host-plasmids from the IncP-1 group, as seen by PCR amplification of the trfA gene. Sequence analysis confirmed the isolation of 4 plasmids from beta-1 subgroup and 3 assigned to the recently described c subgroup. To our knowledge this is the first report concerning the detection and isolation of IncP-1 beta and epsilon plasmids in estuarine waters. Moreover it is shown that, even though the retrieved plasmids are phylogenetically close to previously characterized plasmids, such as pB10 and pKJK5, respectively, they constitute new molecular variants. (C) 2011 Elsevier Inc. All rights reserved.
Abstract: The effects of UV radiation (UVR) on estuarine bacterioneuston and bacterioplankton were assessed in microcosm experiments. Bacterial abundance and DNA synthesis were more affected in bacterioplankton. Protein synthesis was more inhibited in bacterioneuston. Community analysis indicated that UVR has the potential to select resistant bacteria (e. g., Gammaproteobacteria), particularly abundant in bacterioneuston.
Abstract: Bacterioneuston may play a key role in water-air exchange of gases and in processing organic matter and pollutants that accumulate at the sea-surface microlayer (SML). However, the phylogenetic diversity of bacterioneuston has been poorly characterized. We analyzed 24 samples each from the SML and underlying water (UW) at three sites in the Ria de Aveiro estuary, Portugal. Cultivation and culture-independent techniques were used to compare bacterioneuston and bacterioplankton. Culturable heterotrophic bacteria were enriched in the SML. The culturable community was dominated by Psychrobacter and Acinetobacter. The presence of high numbers of Psychrobacter was a notable result. Differences were confined to a few genera overrepresented in UW samples (Kocuria, Agrococcus and Vibrio). 16S rDNA DGGE profiles were highly stable in terms of number and position of bands between sampling sites and dates but cluster analysis revealed a slight tendency for grouping according to sampled layer. SML-specific DGGE bands affiliated with Actinobacteria, Cyanobacteria, Gammaproteobacteria and Bacteroidetes. Low similarity between nucleotide sequences of DGGE-bands and previously reported sequences suggest the occurrence of SML-specific populations. Enrichment of SML for Pseudomonas and Aeromonas was questioned and the diversity of both communities was analyzed. Consistent differences between SML and UW aeromonads communities were not identified. In terms of Pseudomonas, a culturable operational taxonomic unit was consistently overrepresented within SML samples. Taken together, our results indicate that the similarity between SML and UW communities depends on spatial and temporal factors.
Abstract: Aims: To assess the variability in UV-B (280-320 nm) sensitivity of selected bacterial isolates from the surface microlayer and underlying water of the Ria de Aveiro (Portugal) estuary and their ability to recover from previous UV-induced stress. Methods and Results: Bacterial suspensions were exposed to UV-B radiation (3 center dot 3 W m-2). Effects on culturability and activity were assessed from colony counts and 3H-leucine incorporation rates, respectively. Among the tested isolates, wide variability in UV-B-induced inhibition of culturability (37 center dot 4-99 center dot 3%) and activity (36 center dot 0-98 center dot 0%) was observed. Incubation of UV-B-irradiated suspensions under reactivating regimes (UV-A, 3 center dot 65 W m-2; photosynthetic active radiation, 40 W m-2; dark) also revealed diversity in the extent of recovery from UV-B stress. Trends of enhanced resistance of culturability (up to 15 center dot 0%) and enhanced recovery in activity (up to 52 center dot 0%) were observed in bacterioneuston isolates. Conclusions: Bacterioneuston isolates were less sensitive and recovered more rapidly from UV-B stress than bacterioplankton isolates, showing enhanced reduction in their metabolism during the irradiation period and decreased culturability during the recovery process compared to bacterioplankton. Significance and Impact of the Study: UV exposure can affect the diversity and activity of microbial communities by selecting UV-resistant strains and alter their metabolic activity towards protective strategies.
Abstract: Studies of cyanobacterial nostocacean taxa are important to the global scientific community, mainly because a significant number of beneficial strains that belong to the order Nostocales fix atmospheric nitrogen, thus contributing to the fertility of agricultural soils worldwide, while others behave as nuisance microorganisms in aquatic ecosystems due to their involvement in toxic bloom events. However, in spite of their ecological importance and environmental concerns, their identification and taxonomy are still problematic and doubtful, often being based on current morphological and physiological studies, which generate confusing classification systems and usually vary under different conditions. Therefore, the present research aimed to investigate through a polyphasic approach differences in morphological, biochemical and genotypic features of three nostocacean cyanobacterial strains isolated from central-western Portuguese shallow freshwater bodies. Morphometric, genetic (16S rRNA, nifH and hetR fragments) and biochemical (fatty acid methyl ester; FAME profiles) data were used to characterize the strains. Morphological analysis and sequencing of 16S rRNA fragments showed that the strains belonged to Anabaena cylindrica (UTAD_A212), Aphanizomenon gracile (UADFA16) and Nostoc muscorum (UTAD_N213) species. These strains showed clear distinct morphological and genetic features, allowing easy allocation to their respective genera. The same happened by using partial sequences of hetR and nifH genes, in spite of the scarcity of deposited sequences. Biochemical characterization showed that the FAME profiles obtained were consistent with both morphological and molecular analyses. It was suggested that the ratio of monounsaturated to polyunsaturated FAMEs, together with the unsaturation index, could be used as genus-specific chemotaxonomic biomarkers.
Abstract: The effects of ultraviolet-B (0.4 W m(-2)) radiation on the abundance, diversity and heterotrophic metabolism of bacterioneuston and bacterioplankton communities from Ria de Aveiro (Portugal) were assessed and compared to those of freshwater communities from Lake Vela (Portugal) in microcosm experiments. Exposure to 9 h of artificial ultraviolet radiation (UVR) led to 24-33% reduction in bacterial abundance and up to a 70% decrease in bacterial diversity. Maximum extracellular enzyme activity and monomer incorporation rates were reduced by 16-90% and 80-100%, respectively. Recovery of bacterial activity during post-UV dark incubations ranged from 10 to 100% for extracellular enzyme activity and 40% for monomer incorporation rates. In general, the heterotrophic activity of bacterioneuston was more inhibited by UVR than that of bacterioplankton. However, DGGE profiles revealed greater UVR-induced reductions in the diversity of bacterioplankton compared to bacterioneuston. The similarity between bacterioneuston and bacterioplankton communities in samples collected at early morning was lower than at noon (pre-exposed communities) and increased upon experimental irradiation, possibly indicating selection for UV-resistant bacteria. The observation that UV exposure resulted in enhanced reduction of bacterioneuston activity, but a lower reduction in bacterial diversity accompanied by enhanced dark recovery potential compared to bacterioplankton, indicates re-directioning of bacterioneuston metabolism towards stress defence/recovery strategies rather than the sustained heterotrophic metabolism. Our results indicate that UVR can significantly decrease the abundance, diversity and activity of bacteria inhabiting the surface and sub-surface layers of freshwater and estuarine systems with potentially important impacts on the biogeochemical cycles in these environments.
Abstract: Metallo-p,-lactamases (MBLs) or class B beta-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of beta-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for beta-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for p-lactam hydrolysis by these enzymes. (C) 2011 Elsevier Ltd. All rights reserved.
Abstract: The subclass B2 metallo-beta-lactamase (MBL) Sfh-I from Serratia fonticola UTAD54 was cloned and overexpressed in Escherichia coli. The recombinant protein binds one equivalent of zinc, as shown by mass spectrometry, and preferentially hydrolyzes carbapenem substrates. However, compared to other B2 MBLs, Sfh-I also shows limited hydrolytic activity against some additional substrates and is not inhibited by a second equivalent of zinc. These data confirm Sfh-I to be a subclass B2 metallo-beta-lactamase with some distinctive properties.
Abstract: Cyanobacterial blooms of Aphanizomenon spp. occur worldwide, with deleterious effects on aquatic communities. The present work aimed to investigate the effects of different levels of phosphorus and nitrogen on the growth of Aphanizomenon bloom-forming strains, belonging to the species A. issatschenkoi, A. aphanizomenoides and A. gracile. Algal inhibition tests were also performed to assess the potential allelopathic effects of the strainsâ filtrates on the growth of four green algae (Pseudokirchneriella subcapitata, Chlorella vulgaris, Pandorina morum and Coelastrum astroideum). Phosphorus had a significant effect on the growth of all the strains tested; orthophosphate concentrations <= 0.3 mg PO(4)(3-) L(-1) led to a decrease of more than 50% in the cyanobacterial growth of most strains. The growth of A. gracile strains was unaffected by variation in nitrate levels, but strains of A. issatschenkoi (UADFA1) and A. aphanizomenoides (UADFA6, UADFA7 and UADFA13) were moderately to extremely sensitive to nitrate depletion in the medium. These results strengthen the idea that intra-specific variation may play an important role in bloom dynamics; however, culture conditions and mutations may explain the morphological and physiological changes observed. Allelopathic assays revealed significant inhibition of microalgal growth by filtrates of strains from the three species, but also indicated intra-specific differences in allelopathic activity.
Abstract: At the present time, there is still a lack of information about environmental parameters modulating variations on bacterial diversity in temperate lakes, particularly from Portugal. Fermentelos Lake (Central Portugal) is a shallow water body that sustains an important wetland area. The strong nutrient inputs from agriculture and industrial runoffs have led to its current eutrophic status. The present work aimed to understand which factors modulate the seasonal bacterioplankton diversity at this lake using 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) and multivariate analysis. Environmental data demonstrated eutrophic features throughout all samples with nitrate concentrations reaching 12.0 mg N (NO(3) (-)) l(-1) in March 2006, while the highest conductivity (609 mu S cm(-1)), soluble reactive phosphorus (0.37 mg l(-1)), total suspended solids (87.2 mg l(-1)) and chlorophyll a (286.6 mu g l(-1)) levels were recorded in August 2007. Over the past two decades there was a general increase in nitrate, pH and conductivity levels at this lake, suggesting the eutrophication process is still in progress. Multivariate analysis showed that summer versus winter DGGE patterns could be established for bacterial assemblages and were mainly defined by water temperature and chlorophyll a. Actinobacteria were dominant throughout the study period although a general preference for higher temperature, pH, total suspended solids, conductivity, soluble reactive phosphorus (SRP) and chlorophyll a levels was observed. The highest concentrations of nitrogen sources were related to Bacteroidetes and phototrophic eukaryote (cryptophycean) dominance. The expansion of Betaproteobacteria, Alphaproteobacteria and Cyanobacteria phylotypes was generally associated to high temperature, pH, conductivity and SRP values.
Abstract: Four Azorean volcanic muds were submitted to maturation (ageing process) with mineral water for 60 days under different abiotic conditions. This study intends to assess the geochemistry of these natural resources before and after maturation and to evaluate the ability of materials to be colonized by microorganisms under different mixing and light maturation procedures. Besides the geochemical and mineralogical analyses, bacterial quantification and diatom community identification and relative quantification were also provided. Principal component analysis was carried out on geochemical data. Our data reveal that muds have potentiality for thermal application and the modifications induced by maturation are affected by raw materials properties. Slightly geochemical modifications were verified probably due to the weathering of primary minerals. The bacteria and mainly the algae development on samples under light and no mixing maturation conditions were recorded rendering to muds potentialities for therapeutic uses.
Abstract: Endocrine disrupting compounds (EDCs) have been studied due to their impact on human health and increasing awareness of their impact on wildlife species. Studies concerning the organ-specific molecular effects of EDC in invertebrates are important to understand the mechanisms of action of this class of toxicants but are scarce in the literature. We have used a dose/response approach to unravel the protein expression in different organs of isopods exposed to bisphenol A (BPA) and vinclozolin (Vz) and assess their potential use as surrogate species. Male isopods were exposed to a range of Vz or of BPA concentrations. After animal dissection, proteins were extracted from gut, hepatopancreas and testes. Protein profiles were analysed by electrophoresis and differentially expressed proteins were identified by MALDI mass spectrometry. EDCs affected proteins involved in the energy metabolism (arginine kinase), proteins of the heat shock protein family (Hsp70 and GRP78) and most likely microtubule dynamics (tubulin). Different proteins expressed at different concentrations in different organs are indicative of the organ-specific effects of BPA and Vz. Additionally, several proteins were up-regulated at lower but not higher BPA or Vz concentrations, bringing new data to the non-monotonic response curve controversy. Furthermore, our findings suggest that some common responses to EDCs in both vertebrates and invertebrates may exist. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.
Abstract: Aims: Aeromonas is ubiquitous in aquatic environments and may cause infectious diseases in fish and humans. However, reliable and specific methods to evaluate the diversity and dynamics of Aeromonas populations are currently unavailable. This study aimed to develop PCR-DGGE methodologies for culture-independent analysis of Aeromonas populations in water systems. Methods and Results: Three primer sets were designed to amplify selected sections of genes gyrB, rpoD and sodB from Aeromonas. Their specificity was confirmed by in silico analysis and by PCR on DNA from pure cultures. Estuarine water samples were analyzed by PCR-DGGE using those primers. DGGE patterns clearly clustered according to seasonal factors, and Aeromonas communities were surprisingly stable along a salinity gradient. Sequences of cloned amplicons affiliated to sequences belonging to seven Aeromonas species previously isolated from the same environment. Conclusions: The three systems used showed to be useful to describe the diversity of Aeromonas communities. However, the combined use of more than one primer set is advisable. Significance and Impact of the Study: The methods presented here can be applied to understand the natural pool of Aeromonas and also to monitor and control these bacteria in aquatic reservoirs.
Abstract: To investigate the mobile gene pool present in wastewater environments, total community DNA was obtained from two distinct raw effluents: urban and slaughterhouse wastewaters. Bacterial community structure was evaluated by DGGE analysis of 16S rRNA gene fragments. Detection of broad-host-range plasmid sequences and integrase encoding genes was carried out through PCR and Southern hybridization. Gene cassette libraries were obtained using primers targeting consensus sequences that flank gene cassettes. Analysis of DGGE profiles revealed a complex and distinct bacterial community among effluents (similarity < 25%). Despite that, All plasmid-specific sequences searched (rep for IncN, trfA for IncP-1 and oriV for IncQ and IncW) and integrase genes were present in both effluents. Gene cassettes recovered from clone libraries showed low homology with genes encoding putative enzymes involved in the metabolism of amino sugars, cell wall synthesis, motility, gene regulation, intercellular signalling and secretion pathways and in the synthesis of cellulose, folic acid and antibiotics. Additionally, in the majority of clones, no identifiable open reading frames homologues were found in the databases. According to our results, wastewater environments promote the development of bacterial communities that support and bring together different types of molecular elements that, in association, play a major role in bacterial adaptation and evolution. (C) 2009 Elsevier Masson SAS. All rights reserved.
Abstract: Although a number of studies have indicated that microbes are key players in nutrient cycling, limitations on how to accurately assess their diversity have constrained further knowledge on the role of microbial diversity in organic matter decomposition in streams. Microbial diversity on leaf litter of Alnus glutinosa was assessed by microscopic analysis of bacterial cells and released fungal conidia, and by the number of operational taxonomic units (OTUs) from denaturing gradient gel electrophoresis using two different primer pairs targeting the rDNA of fungi (ITS2 and 5â end of the 18S region) and bacteria (V3 region and V6-V8 regions). Fingerprints of fungal and bacterial DNA showed a higher diverse microbial community on decomposing leaves than that assessed by microscopy-based techniques. Higher number of OTUs was obtained with primers targeting the ITS2 region of fungi, but the selected primers for bacteria showed similar number of OTUs. A succession of fungal or bacterial taxa throughout leaf decomposition was found, regardless of the chosen primer. These microbial communities ensured a rapid decomposition of submerged leaf litter (k = -0.045 day(-1)). Fungal biomass (up to 58 mg g(-1) AFDM) contributed with more than 98% to the total microbial biomass, supporting a greater role of fungi than bacteria in leaf-litter decomposition in streams. (C) 2009 Elsevier GmbH. All rights reserved.
Abstract: The diversity within the genus Aphanizomenon (Nostocales, Cyanobacteria) is still controversial and more studies are needed to clarify its heterogeneity. Aphanizomenon strains isolated from the eutrophic shallow Vela Lake (Centralwestern Portugal) during bloom formation in summer months from 2001 to 2007 were characterized combining phenotypic and molecular approaches. The molecular affiliation of the strains was established through sequencing of the 16S rDNA, as well as nifH and hetR genes fragments, and inter-specific genetic variability was checked through repetitive sequences fingerprinting. The morphological characters were characteristic of the descriptions of Aph. issatschenkoi (Cuspidothrix issatschenkoi), Aphanizomenon gracile and Aph. aphanizomenoides. Molecular phylogenetic results corroborated the morphological identification and fingerprinting showed a persistent occurrence of Aph. gracile and Aph. aphanizomenoides strains over the years but also indicated that Aph. aphanizomenoides strains are not clonal within a same bloom. Partial nifH and hetR gene sequences of the cyanobacterial strains showed a high phylogenetic differentiation capacity, even at the strain level.
Abstract: The growing interest in the application of proteomic technologies to solve toxicology issues and its relevance in ecotoxicology research has resulted in the emergence of âecotox-icoproteomicsâ. There is a general consensus that ecotoxicoproteomics is a powerful tool to spot early molecular events involved in toxicant responses, which are responsible for the adverse effects observed at higher levels of biological organization, thus contributing to elucidate the mode of action of stressors and to identify specific biomarkers. Ultimately, early-warning indicators can then be developed and deployed in âin situâ bioassays and in environmental risk assessment. The number of field experiments or laboratory trials using ecologically relevant test-species and involving proteomics has been, until recently, insufficient to allow a critical analysis of the real benefits of the application of this approach to ecotoxicology. This article intends to present an overview on the applications of proteomics in the context of ecotoxicology, focusing mainly on the prospective research to be done in invertebrates. Although these represent around 95% of all animal species and in spite of the key structural and functional roles they play in ecosystems, proteomic research in invertebrates is still in an incipient stage. We will review applications of ecotoxicoproteomics by evaluating the technical methods employed, the organisms and the contexts studied, the advances achieved until now and lastly the limitations yet to overcome will be discussed.
Notes: Fonseca, Fatima Sarmento, Ana Cristina Henriques, Isabel Samyn, Bart van Beeumen, Jozef Domingues, Pedro Domingues, Maria Rosario Saavedra, Maria Jose Correia, Antonio
