Bacterial functional redundancy along a soil reclamation gradient

A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU), BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site, the number and diversity of bacterial groups responding to four carbon substrates (L-serine, L-threonine, sodium citrate, and or-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning.

Avaliação da agressividade e caracterização genética de linhagens de Ralstonia Solanacearum isoladas de diferentes plantas hospedeiras

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Diagnóstico laboratorial de Mycobacterium spp. em Botucatu e região, utilizando a técnica Reação em Cadeia da Polimerase (PCR) em material biológico e avaliação de condições associadas

Pós-graduação em Saúde Coletiva - FMB

Molecular fingerprinting and amplicon sequencing of the bacterial biofilm on settlement tiles deployed along natural pH gradients in Papua New Guinea

Bacterial biofilms provide cues for the settlement of marine invertebrates such as coral larvae, and are therefore important for the resilience and recovery of coral reefs. This study aimed to better understand how ocean acidification may affect the community composition and diversity of bacterial biofilms on surfaces under naturally reduced pH conditions. Settlement tiles were deployed at coral reefs in Papua New Guinea along pH gradients created by two CO2 seeps, and upper and lower tiles surfaces were sampled 5 and 13 months after deployment. Automated Ribosomal Intergenic Spacer Analysis were used to characterize more than 200 separate bacterial communities, complemented by amplicon sequencing of the bacterial 16S rRNA gene of 16 samples. The bacterial biofilm consisted predominantly of Alpha-, Gamma- and Deltaproteobacteria, as well as Cyanobacteria, Flavobacteriia and Cytophaga, whereas putative settlement-inducing taxa only accounted for a small fraction of the community. Bacterial biofilm composition was heterogeneous with approximately 25% shared operational taxonomic units between samples. Among the observed environmental parameters, pH only had a weak effect on community composition (R² ~ 1%) and did not affect community richness and evenness. In contrast, there were strong differences between upper and lower surfaces (contrasting in light exposure and grazing intensity). There also appeared to be a strong interaction between bacterial biofilm composition and the macroscopic components of the tile community. Our results suggest that on mature settlement surfaces in situ, pH does not have a strong impact on the composition of bacterial biofilms. Other abiotic and biotic factors such as light exposure and interactions with other organisms may be more important in shaping bacterial biofilms than changes in seawater pH.

Small Changes in pH Have Direct Effects on Marine Bacterial Community Composition: A Microcosm Approach

As the atmospheric CO2 concentration rises, more CO2 will dissolve in the oceans, leading to a reduction in pH. Effects of ocean acidification on bacterial communities have mainly been studied in biologically complex systems, in which indirect effects, mediated through food web interactions, come into play. These approaches come close to nature but suffer from low replication and neglect seasonality. To comprehensively investigate direct pH effects, we conducted highly-replicated laboratory acidification experiments with the natural bacterial community from Helgoland Roads (North Sea). Seasonal variability was accounted for by repeating the experiment four times (spring, summer, autumn, winter). Three dilution approaches were used to select for different ecological strategies, i.e. fast-growing or low-nutrient adapted bacteria. The pH levels investigated were in situ seawater pH (8.15-8.22), pH 7.82 and pH 7.67, representing the present-day situation and two acidification scenarios projected for the North Sea for the year 2100. In all seasons, both automated ribosomal intergenic spacer analysis and 16S ribosomal amplicon pyrosequencing revealed pH-dependent community shifts for two of the dilution approaches. Bacteria susceptible to changes in pH were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. Their specific response to reduced pH was often context-dependent. Bacterial abundance was not influenced by pH. Our findings suggest that already moderate changes in pH have the potential to cause compositional shifts, depending on the community assembly and environmental factors. By identifying pH-susceptible groups, this study provides insights for more directed, in-depth community analyses in large-scale and long-term experiments.

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and batch cultures of rumen microorganisms

The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

Influencia del tipo de filtrado y tratamiento con Stomacher del fluido ruminal de ovejas en las poblaciones microbianas del inóculo

Four rumen-fistulated sheep fed a 66:34 alfalfa hay:concentrate diet were used as donors to investigate the effect of rumen contents’ treatment on microbial populations in the resulting fluid. Rumen contents were sampled from each individual sheep and subjected to the following treatments: SQ: squeezed through 4 layers of cheesecloth; FIL: SQ treatment and further filtration through a 100-μm nylon cloth; STO: reated with a Stomacher® for 3 min at 230 rev min-1 and followed by SQ. Microbial populations in the fluid were analysed by real-time PCR and bacterial diversity was assessed by the automated ribosomal intergenic spacer analysis (ARISA) of the 16S ribosomal DNA. Bacterial DNA concentrations and relative abundance of Ruminococcus flavefaciens, arqueal and fungal DNA did not differ (P>0.05) between treatments. In contrast, STO treatment decreased (P<0.05) protozoal DNA concentrations and increased (P<0.05) the relative abundance of Fibrobacter succinogenes compared with SQ method. There were no differences (P>0.05) between treatments either in the Shannon index or in the number of peaks in the ARISA electropherograms, indicating no effect on bacterial diversity. Studies analyzing the influence on the tested methods on fermentation characteristics of different substrates when the fluid is used as inoculum is required.

RISSC: a novel database for ribosomal 16S–23S RNA genes spacer regions

A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S–23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documen­tation tool for studies on evolution, identification, typing and strain characterization, among others.

Genetic uniformity of international isolates of Leifsonia xyli subsp xyli, causal agent of ratoon stunting disease of sugarcane

An international collection of the sugarcane ratoon stunting disease pathogen, Leifsonia xyli subsp. xyli, was analysed to assess genetic diversity. DNA fingerprinting using BOX primers was performed on 105 isolates, comprising 65 Australian isolates and an additional 40 isolates from Indonesia (n = 8), Japan (n = 1), USA (n = 3), Brazil (n = 2), Mali (n = 2), Zimbabwe (n = 13), South Africa (n = 9) and Reunion (n = 2). Sixty-two of these isolates were also screened using ERIC primers. No variation was found among any of the isolates. The intergenic spacer (IGS) region of the ribosomal RNA genes from 54 isolates was screened for sequence variation using single-stranded conformational polymorphism (SSCP), but none was observed. Direct sequencing of the IGS from a subset of nine isolates, representing all of the countries sampled in this study, confirmed the results of the SSCP analysis. Likewise, no sequence variation was found in the 16S ribosomal RNA genes of the same subset. Four Colombian isolates from sugarcane, morphologically similar to L. xyli subsp. xyli, were putatively shown to be an undescribed Agrococcus species of unknown pathogenicity. The lack of genetic variation among L. xyli subsp. xyli isolates, independent of time of sampling, cultivar of isolation, or country of origin, suggests the worldwide spread of a single pathogenic clone, and further suggests that sugarcane cultivars resistant to ratoon stunting disease in one area should retain this property in other regions.

Isolates of 'Candidatus Nostocoida limicola' Blackall et al. 2000 should be described as three novel species of the genus Tetrasphaera, as Tetrasphaera jenkinsii sp nov., Tetrasphaera vanveenii sp nov and Tetrasphaera veronensis sp nov.

Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (> 94 %) between strains of the filamentous bacterium 'Candidatus Nostocoida limicola' and the cocci Tetrasphaera australiensis and Tetrasphaera japonica and the rod Tetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S-23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the family Intrasporangiaceae in the Actinobacteria. This suggestion receives additional support from DNA-DNA hybridization data and when partial sequences of the rpoC1 gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that 'Candidatus N. limicola' strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the species Tetrasphaera jenkinsii sp. nov. (type strain Ben 74(T) = DSM 17519(T) = NCIMB 14128(T)), 'Candidatus N. limicola' strain Ben 70 into Tetrasphaera vanveenii sp. nov. (type strain Ben 70(T) = DSM 17518(T) = NCIMB 14127(T)) and 'Candidatus N. limicola' strains Ver 1 and Ver 2 into Tetrasphaera veronensis sp. nov. (type strain Ver 1(T) = DSM 17520(T) = NCIMB 14129(T)).

Phylogenetic relationships in Solanaceae and related species based on cpDNA sequence from plastid trnE-trnT region

Intergenic spacers of chloroplast DNA (cpDNA) are very useful in phylogenetic and population genetic studies of plant species, to study their potential integration in phylogenetic analysis. The non-coding trnE-trnT intergenic spacer of cpDNA was analyzed to assess the nucleotide sequence polymorphism of 16 Solanaceae species and to estimate its ability to contribute to the resolution of phylogenetic studies of this group. Multiple alignments of DNA sequences of trnE-trnT intergenic spacer made the identification of nucleotide variability in this region possible and the phylogeny was estimated by maximum parsimony and rooted with Convolvulaceae Ipomoea batalas, the most closely related family. Besides, this intergenic spacer was tested for the phylogenetic ability to differentiate taxonomic levels. For this purpose, species from four other families were analyzed and compared with Solanaceae species. Results confirmed polymorphism in the trnE-trnT region at different taxonomic levels.

GEITLERINEMA SPECIES (OSCILLATORIALES, CYANOBACTERIA) REVEALED BY CELLULAR MORPHOLOGY, ULTRASTRUCTURE, AND DNA SEQUENCING

Geitlerinema amphibium (C. Agardh ex Gomont) Anagn. and G. unigranulatum (Rama N. Singh) Komarek et M. T. P. Azevedo are morphologically close species with characteristics frequently overlapping. Ten strains of Geitlerinema (six of G. amphibium and four of G. unigranulatum) were analyzed by DNA sequencing and transmission electronic and optical microscopy. Among the investigated strains, the two species were not separated with respect to cellular dimensions, and cellular width was the most varying characteristic. The number and localization of granules, as well as other ultrastructural characteristics, did not provide a means to discriminate between the two species. The two species were not separated either by geography or environment. These results were further corroborated by the analysis of the cpcB-cpcA intergenic spacer (PC-IGS) sequences. Given the fact that morphology is very uniform, plus the coexistence of these populations in the same habitat, it would be nearly impossible to distinguish between them in nature. On the other hand, two of the analyzed strains were distinct from all others based on the PC-IGS sequences, in spite of their morphological similarity. PC-IGS sequences indicate that these two strains could be a different species of Geitlerinema. Using morphology, cell ultrastructure, and PC-IGS sequences, it is not possible to distinguish G. amphibium and G. unigranulatum. Therefore, they should be treated as one species, G. unigranulatum as a synonym of G. amphibium.

Characterization of fungal soil communities by F-RISA and arbuscular mycorrhizal fungi from Araucaria angustifolia forest soils after replanting and wildfire disturbances

The Fungal Ribosomal Intergenic Spacer Analysis (F-RISA) was used to characterize soil fungal communities from three ecosystems of Araucaria angustifolia from Brazil: a native forest and two replanted forest ecosystems, one of them with a past history of wildfire. The arbuscular mycorrhizal fungi (AMF) infection was evaluated in Araucaria roots of 18-month-old axenic plants previously inoculated with soils collected from those areas in a greenhouse experiment. The principal component analysis of F-RISA profiles showed different soil fungal community between the three studied areas. Sixty three percent of F-RISA fragments amplified in the soil and the substrate samples presented lengths between 500 and 700 bp. The number of Operational Taxonomic Units (OTUs) was 34 for soil and 38 for substrate, however, more fragments were detected in soil (214) than in substrate (163). An in silico F-RISA analysis to compare our data with ITS1-5.8S-ITS2 sequences from NCBI database showed the presence of Ascomycota, Basidiomycota and Glomeromycota among the soil and substrate fungal communities. AMF infection was higher in plants inoculated with soil from the native forest and the replanted forest with wildfire, both presenting similar chemical characteristics but with different disturbance levels. These results indicate that soil chemical composition may influence the soil fungal community structures rather than the anthropogenic or fire disturbances.

Mycobacterium chelonae valve endocarditis resulting from contaminated biological prostheses

Objectives: A rapid-growing mycobacteria biological prosthetic valve (BPV) endocarditis related to prosthetic manufacturing process is described in Brazil. Methods: From 1999 to 2008, thirty-nine patients underwent BPV replacement due to culture-negative suspected endocarditis. All these cases had histological sections stained by Ziehl-Neelsen method. Clinical and microbiological data were reviewed in all acid-fast bacilli (AFB) positive cases. The 16S-23S internal transcribed sequence (ITS) was amplified using DNA extracted from paraffin-embedded samples, digested with restrictions enzymes and/or sequenced. Results: Eighteen AFB positive BPV (18/39)(46%) were implanted in 13 patients and were from the same manufacturer. Four of them were implanted in other hospitals. Thirteen BPV were histologically proven endocarditis and five showed a colonization pattern. The examination of six non-implanted ""sterile"" BPV from this manufacturer resulted in 5 AFB positive. Mycobacterium chelonae was the AFB identified by ITS restriction analysis and sequencing. Conclusions: Rapid-growing mycobacteria infections must be suspected and Ziehl-Neelsen stain always performed on histology of either early or late BPV endocarditis, particularly when blood cultures are negative. (C) 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Molecular rates parallel diversification contrasts between carnivorous plant sister lineages

In the carnivorous plant family Lentibulariaceae, the bladderwort lineage (Utricularia and Genlisea) is substantially more species-rich and morphologically divergent than its sister lineage, the butterworts (Pinguicula). Bladderworts have a relaxed body plan that has permitted the evolution of terrestrial, epiphytic, and aquatic forms that capture prey in intricately designed suction bladders or corkscrew-shaped lobster-pot traps. In contrast, the flypaper-trapping butterworts maintain vegetative structures typical of angiosperms. We found that bladderwort genomes evolve significantly faster across seven loci (the trnL intron, the second trnL exon, the trnL-F intergenic spacer, the rps16 intron, rbcL, coxI, and 5.8S rDNA) representing all three genomic compartments. Generation time differences did not show a significant association. We relate these findings to the contested speciation rate hypothesis, which postulates a relationship between increased nucleotide substitution and increased cladogenesis. (C) 2002 The Willi Hennig Society.