Gabriela Frois Duarte

Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis

A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months, respectively. Both singly-inoculated soils and soils that had received mixed inoculants revealed, next to bands resulting from indigenous fungi, the expected bands in the DGGE profiles. The A. oligospora specific amplicon, by virtue of its unique migration in the denaturing gradient, was well detectable, whereas the T. harzianum specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiophene-containing petrol showed the progressive selection of specific fungal bands over time, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and analysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca, N. ochroleuca and Fusarium solani. Fungal isolates obtained from the treated soil on PDA plates were identified as Trichoderma sp., whereas those on Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp).

Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment

As the use of biotechnology products, such as genetically modified microorganisms (GMMs), in the environment might bring about undesirable ecological effects, it is important that the environmental fate of inoculant organisms, as well as any effects of their release, are assessed. Ideally, pilot studies in microcosms or small mesocosms are thus performed prior to a larger-scale (commercial) application, and the results of these studies serve to guide further environmental use of the GMM. In these pilot studies, the methods employed to assess the environmental fate and effects of the GMM will have to be fine-tuned and optimized so as to assess these phenomena in an optimal way. This review examines the methods that are currently available for the assessment of the environmental fate of genetically modified and unmodified microorganisms, as well as the impact following their release. The emphasis will be on monitoring of these phenomena in soil, as a paradigm of assessments in complex environmental matrices. Detection methods based on cultivation, which generally rely on the use of added or intrinsic markers, serve to assess the fate of the culturable fractions of released microorganisms. The specificity of this detection can be enhanced by using a combination of cultivation-based and immunology- and/or DNA-based assessments. Furthermore, specific immunofluorescence or in situ hybridization techniques are suitable to quantify populations of GMMs at the level of microscopy-detectable cells, even though fluorescent in situ hybridization is still plagued by sensitivity problems in oligotrophic environments. Detection methods based on nucleic acids (DNA or RNA) extracted from the environment offer the possibility to monitor the fate of the heterologous genes released, including that following a horizontal gene transfer. Moreover, these methods can also provide a picture of the dynamics of the total numbers of microbial cells released. For a sound assessment of the biosafety of environmental releases, a polyphasic approach to environmental monitoring is recommended, as in most analyses information will be needed on the fate of culturable and nonculturable cells, as well as on that of the specific gene sequences released. Finally, the use of impact analysis via an array of different methods is briefly reviewed, and the merits of novel approaches to assessments of community structure via molecular means (PCR/DGGE, ARDRA or T-RFLP) and metabolic profiling via Biolog are discussed as methods specifically aimed at the detection of shifts in community structure and function (metabolic complement analysis).

Analysis of Bacterial Community Structure in Sulfurous-Oil-Containing Soils and Detection of Species Carrying Dibenzothiophene Desulfurization (dsz) Genes

ABSTRACT The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil. Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed. Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts. However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp.,Arthrobacter sp., and a bacterium of uncertain affiliation.dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment. PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonassp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures. Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively. Most of the strains carrying dszA or dszC were classified asR. erythropolis related, and all revealed the capacity to desulfurize DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8)dszA sequence and a large degree of internal conservation. The 37 sequences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil.

Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community

A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils of different texture. DNA and rRNA yields from these soils were 15-30 and 0.25-1.0 μg g-1 soil, respectively. Following different purification steps, the rRNA as well as genomic DNA extracts obtained were sufficiently pure for either reverse transcription and polymerase chain reaction (PCR) amplification, or direct PCR amplification. Using a set of universal bacterial primers based on conserved regions of the 16S rRNA sequence, both approaches yielded mixed target molecules for subsequent denaturing gradient gel electrophoresis fingerprinting of soil microbial diversity. The amplified rRNA-based bacterial diversity assessment was compared with diversity assessments based on amplified DNA in one selected soil. Results showed similarities as well as differences between the profiles generated on the basis of rRNA and those based on genomic DNA, which suggested that the bacterial communities defined on the basis of their genomic DNA contained variable amounts of rRNA. Copyright (C) 1998 Elsevier Science B.V.

Paenibacillus brasilensis sp. nov., a novel nitrogen-fixing species isolated from the maize rhizosphere in Brazil.

Sixteen nitrogen-fixing strains isolated from the rhizosphere of maize planted in Cerrado soil, Brazil, which showed morphological and biochemical characteristics similar to the gas-forming Paenibacillus spp., were phenotypically and genetically characterized. Their identification as members of the genus Paenibacillus was confirmed by using specific primers based on the 16S rRNA gene. SDS-PAGE of whole-cell proteins, API 50CH, morphological and biochemical tests, amplified rDNA-restriction analysis (ARDRA), DNA-relatedness analyses, denaturing-gradient gel electrophoresis (DGGE) and 16S rRNA gene sequence determinations were performed to characterize the novel isolates and to compare them to strains of other nitrogen-fixing Paenibacillus spp. Phenotypic analyses showed that the 16 strains were very homogeneous and shared a high level of relatedness with Paenibacillus polymyxa and Paenibacillus peoriae. However, none of the novel isolates was able to ferment glycerol (positive test for P. polymyxa), L-arabinose or D-xylose (positive tests for P. polymyxa and P. peoriae) or utilize succinate (positive test for P. peoriae). Genetic approaches also indicated a high level of similarity among the novel isolates and P. polymyxa and P. peoriae, but the novel strains clearly could not be assigned to either of these two recognized species. On the basis of the features presented in this study, the 16 novel isolates were considered to represent members of a novel species within the genus Paenibacillus, for which the name Paenibacillus brasilensis is proposed. The type strain is PB1 72(T) (= ATCC BAA-413(T) = DSM 14914(T)).

Brazilian Microbiome Project: revealing the unexplored microbial diversity--challenges and prospects.

The Brazilian Microbiome Project (BMP) aims to assemble a Brazilian Metagenomic Consortium/Database. At present, many metagenomic projects underway in Brazil are widely known. Our goal in this initiative is to co-ordinate and standardize these together with new projects to come. It is estimated that Brazil hosts approximately 20 % of the entire world’s macroorganism biological diversity. It is 1 of the 17 countries that share nearly 70 % of the world’s catalogued animal and plant species, and is recognized as one of the most megadiverse countries. At the end of 2012, Brazil has joined GBIF (Global Biodiversity Information Facility), as associated member, to improve the access to the Brazilian biodiversity data in a free and open way. This was an important step toward increasing international collaboration and clearly shows the commitment of the Brazilian government in directing national policies toward sustainable development. Despite its importance, the Brazilian microbial diversity is still considered to be largely unknown, and it is clear that to maintain ecosystem dynamics and to sustainably manage land use, it is crucial to understand the biological and functional diversity of the system. This is the first attempt to collect and collate information about Brazilian microbial genetic and functional diversity in a systematic and holistic manner. The success of the BMP depends on a massive collaborative effort of both the Brazilian and international scientific communities, and therefore, we invite all colleagues to participate in this project.

Evaluation of mini-VIDAS rapid test for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish

This study was conducted to evaluate the efficacy of the mini-VIDAS Listeria monocytogenes (LMO) system (BioMérieux Vitek, Inc., Missouri, USA) for detection of L. monocytogenes in environmental and fish samples from three Portuguese cold-smoking plants and from their fresh fish suppliers. Mini-VIDAS-LMO is a fully automated system that uses fluorescent ELFA (Enzyme Linked Fluorescent Assay) technology for detection of Listeria monocytogenes antigens in food. It can be a rapid screening method alternative to time consuming classical isolation and identification. Two hundred and ninety five samples were tested in mini-VIDAS-LMO and in parallel by the ISO 11290-1 traditional protocol. The mini-VIDAS-LMO detected 8 of the 11 confirmed positive samples and presented 11 false positive results. The specificity of the mini-VIDAS-LMO found in this experiment was 0.96 and the sensitivity 0.73.

The role of mycorrhization helper bacteria in the establishment and action of ectomycorrhizae associations

More than 95 % short roots of most terrestrial plants are colonized by mycorrhizal fungi as soon as they emerge in the upper soil profiles. The establishment of mycorrhizal association involves profound morphological and physiological changes in root and fungus. It is affected by other rhizospheric microorganisms, specifically by the bacteria. Bacteria may have developed mechanisms of selective interaction with surrounding microorganisms, with neutral or positive effects on mycorrhizal associations, but negative effect on root pathogens in general. Because of the beneficial effect of bacteria on mycorrhizae, the concept of Mycorrhization Helper Bacteria (MHB) was created. Five main actions of MHB on mycorrhizae were proposed: in the receptivity of root to the mycobiont, in root-fungus recognition, in fungal growth, in the modification of rhizospheric soil and in the germination of fungal propagules. MHB appear to develop a gradation of specificity for the mycobiont, but little or no specificity for the host plant in symbiosis. One of the main groups of MHB is the fluorescent Pseudomonas, well represented in diversity and cell density studies of mycorrhizal associations. This review covers the activity of MHB in the establishment of ectomycorrhizae, taking as model the effects of Pseudomonas sp. described in scientific literature.

Optimization of electroporation procedure to transform B. polymyxa SCE2 and other nitrogen-fixing Bacillus

Abstract An efficient method for genetic transformation of different nitrogen-fixing Bacillus polymyxa strains by electroporation is presented. Various parameters on plasmid transformation of b. polymyxa SCE2 with plasmid pC194 were investigated to enhance transformation efficiency: voltage, buffer strength and type of electroporation buffer, plasmid DNA concentration, purity of plasmid preparation and plasmid source. Electroporation of B. polymyxa SCE2 resulted in a transformation efficiency as high as 3.2 × 10 5 transformants/μg of pC194 when the optimized procedure was used. Stability of different plasmids (pE194, pBD64, pBC16 and pFT30) transformed to B. polymyxa SCE2-43 and/or SCE2-43 was analysed. pE194 showed high frequency of curing while pFT30 and pBC16 were stable after 10 transfers in non-selective medium. Plasmid DNA isolated from transformants had not undergone detectable deletions. Although the electroporation protocol presented here can be used to transform B. azotofixans , lower efficiencies of transformations were achieved. No transformants were detected in other nitrogen-fixing strains belonging to B. macerans .

Characterization of bacterial strains capable of desulphurisation in soil and sediment samples from Antarctica

The presence of sulphur in fossil fuels and the natural environment justifies the study of sulphur-utilising bacterial species and genes involved in the biodesulphurisation process. Technology has been developed based on the natural ability of microorganisms to remove sulphur from polycyclic aromatic hydrocarbon chains. This biotechnology aims to minimise the emission of sulphur oxides into the atmosphere during combustion and prevent the formation of acid rain. In this study, the isolation and characterization of desulphurising microorganisms in rhizosphere and bulk soil samples from Antarctica that were either contaminated with oil or uncontaminated was described. The growth of selected isolates and their capacity to utilise sulphur based on the formation of the terminal product of desulphurisation via the 4S pathway, 2-hydroxybiphenyl, was analysed. DNA was extracted from the isolates and BOX-PCR and DNA sequencing were performed to obtain a genomic diversity profile of cultivable desulphurising bacterial species. Fifty isolates were obtained showing the ability of utilising dibenzothiophene as a substrate and sulphur source for maintenance and growth when plated on selective media. However, only seven genetically diverse isolates tested positive for sulphur removal using the Gibbs assay. DNA sequencing revealed that these isolates were related to the genera Acinetobacter and Pseudomonas.