Papers by Michele C P Silva
FEMS microbiology ecology, 2014
In the microhabitat that surrounds fungal hyphae in soil, coined the mycosphere, carbonaceous com... more In the microhabitat that surrounds fungal hyphae in soil, coined the mycosphere, carbonaceous compounds that are released from the hyphae stimulate the growth of heterotrophic bacteria, and thus activate organism-to-organism contacts through genetic interactions. Therefore, the mycosphere is postulated to constitute a gene transfer arena, in which a plethora of genes, including locally adaptive ones, are swapped across the resident microbial communities. Such genetic transfers may have plasmids, in particular ones with broad host ranges, as the basis. Indeed, evidence is increasing for the contention that plasmids play crucial roles as accelerators of evolution in the mycosphere, serving as a horizontal gene pool and, therefore, providing competence factors to local bacteria as well as fungi. The evidence so far points at mycosphere roles for two major plasmid classes, the IncP-1 and PromA groups. Moreover, recent data indicate that bacterium-to-fungus gene transfers are detectable ...
IncP-1β Plasmids Are Important Carriers of Fitness Traits for Variovorax Species in the Mycosphere-Two Novel Plasmids, pHB44 and pBS64, with Differential Effects Unveiled
Microbial ecology, Jan 28, 2014
The Laccaria proxima mycosphere strongly selects Variovorax paradoxus cells. Fifteen independent ... more The Laccaria proxima mycosphere strongly selects Variovorax paradoxus cells. Fifteen independent V. paradoxus strains, isolated from mycospheres sampled at two occasions, were investigated with respect to the occurrence of plasmids of sizes <60-100 kb. Two V. paradoxus strains, HB44 and BS64, were found to contain such plasmids, which were coined pHB44 and pBS64. Replicon typing using a suite of plasmid-specific PCR systems indicated that both plasmids belong to the IncP-1β group. Also, both were able to mobilize selectable IncQ group plasmids into Escherichia coli as well as Pseudomonas fluorescens. Moreover, they showed stable replication in these organisms, confirming their broad host range. Strain BS64 was cured of pBS64 and plasmid pHB44 was subsequently moved into this cured strain by making use of the IncQ group tracer plasmid pSUP104, which was then removed at elevated temperature. Thus, both plasmids could be screened for their ability to confer a phenotype upon strain B...
Fluctuations in Ammonia Oxidizing Communities Across Agricultural Soils are Driven by Soil Structure and pH
Frontiers in microbiology, 2012
The milieu in soil in which microorganisms dwell is never constant. Conditions such as temperatur... more The milieu in soil in which microorganisms dwell is never constant. Conditions such as temperature, water availability, pH and nutrients frequently change, impacting the overall functioning of the soil system. To understand the effects of such factors on soil functioning, proxies (indicators) of soil function are needed that, in a sensitive manner, reveal normal amplitude of variation. Thus, the so-called normal operating range (NOR) of soil can be defined. In this study we determined different components of nitrification by analyzing, in eight agricultural soils, how the community structures and sizes of ammonia oxidizing bacteria and archaea (AOB and AOA, respectively), and their activity, fluctuate over spatial and temporal scales. The results indicated that soil pH and soil type are the main factors that influence the size and structure of the AOA and AOB, as well as their function. The nitrification rates varied between 0.11 ± 0.03 μgN h(-1) gdw(-1) and 1.68 ± 0.11 μgN h(-1) gd...
Temporal Dynamics of Abundance and Composition of Nitrogen-Fixing Communities across Agricultural Soils
PLoS ONE, 2013
IncP-1 and PromA Group Plasmids Are Major Providers of Horizontal Gene Transfer Capacities Across Bacteria in the Mycosphere of Different Soil Fungi
Microbial Ecology, 2014
Soil Biology and Biochemistry, 2013
Soil Biology and Biochemistry, 2012
The effects of plants on the microbiota involved in the oxidation of ammonia in soils have been c... more The effects of plants on the microbiota involved in the oxidation of ammonia in soils have been controversial. Here, we investigated the dynamics in the abundances and community structures of the bacterial and archaeal ammonia oxidizers (AOB and AOA, respectively) in two fields that were cropped with potato. Six different potato cultivars were used, including a genetically-modified one, in a fourfold replicated experimental setup. On the basis of bulk and rhizosphere soil extracted microbial community DNA, AOB and AOA quantitative PCR as well as PCR-DGGE were performed. In addition, samples were used for the production and analysis of amoA gene fragment based clone libraries. Regardless of sample type (bulk versus rhizosphere soil) and across soils, the population sizes of AOA (of the order 10 4 e10 8 amoA gene copies g À1 dry soil), were generally higher than those of AOB in the same samples (about 10 4 e10 5 g À1 dry soil), resulting in ratio's of log-transformed values > 1.0. Whereas the AOB numbers were generally raised in the rhizosphere versus bulk soils in both soils, the opposite was true for the AOA numbers. Moreover, significant effects of cultivar type on both the AOB and AOA community structures were found in both soils, and these extended to beyond the rhizospheres. The effects were found across the whole growth season. Soil type did not significantly affect the community structures of AOA, but had a small effect on the community structure of AOB. Analysis of the structures of the AOB communities revealed a prevalence of AOB subgroups 2, 3a, 3b and 4 in one field soil and of 2 and 4 in the other one. With respect to the AOA, soil/sediment clusters (SS) I, II, III and IV were found to prevail.
Soil Biology and Biochemistry, 2012
Soil microbial communities play a major role in organic matter decomposition, however the importa... more Soil microbial communities play a major role in organic matter decomposition, however the importance of the individual species involved is still unclear. To identify the dynamics and identity of bacterial species involved in decomposition of potato tissue as well as the assimilation of carbon from fresh plant material, 13 C-labeled green potato tissues (13 C 99.2%) were incorporated in soil microcosm for 39 days at the level of 2.5% (w/dry weight soil). The DNA was extracted from the soil after 1, 6, 15, 25 and 39 days. The heavy (13 C) and light (12 C) fractions of DNA were separated by ultracentrifugation and the structures of the bacterial and fungal communities were characterized by DGGE. Primary and secondary 13 C-sequestrators were identified by sequencing DGGE bands that had appeared only in the heavy DNA fractions. Over the course of the experiment, the most dominant 13 C-labeled phylogenetic group (class or phylum) was g-Proteobacteria (51.4%), followed by Actinobacteria (27%), b-Proteobacteria (8.1%) and a-Proteobacteria (5.4%). Two taxa, namely Firmicutes and Verrucomicrobia, were represented by just one sequence type. These bacterial taxa were differentiated into primary (Arthrobacter, Pseudomonas) and secondary sequestrators (Actinobacteria, Dyella, Mesorhizobium and Sphingomonas). The latter were possibly involved in either the redistribution of previously consumed carbon or in a possible degradation of the more complex plant compounds. On the basis of this analysis, only 5 to 8 bacterial taxa were involved in carbon sequestration at any one measured time point. Our results show the importance of specific microbial taxa in the decomposition and mineralization of plant residues in soil, which will allow us to better understand the role of such communities in carbon cycling.
PLoS ONE, 2012
Background: Soil microbial communities are in constant change at many different temporal and spat... more Background: Soil microbial communities are in constant change at many different temporal and spatial scales. However, the importance of these changes to the turnover of the soil microbial communities has been rarely studied simultaneously in space and time. Methodology/Principal Findings: In this study, we explored the temporal and spatial responses of soil bacterial, archaeal and fungal b-diversities to abiotic parameters. Taking into account data from a 3-year sampling period, we analyzed the abundances and community structures of Archaea, Bacteria and Fungi along with key soil chemical parameters. We questioned how these abiotic variables influence the turnover of bacterial, archaeal and fungal communities and how they impact the long-term patterns of changes of the aforementioned soil communities. Interestingly, we found that the bacterial and fungal b-diversities are quite stable over time, whereas archaeal diversity showed significantly higher fluctuations. These fluctuations were reflected in temporal turnover caused by soil management through addition of Nfertilizers. Conclusions: Our study showed that management practices applied to agricultural soils might not significantly affect the bacterial and fungal communities, but cause slow and long-term changes in the abundance and structure of the archaeal community. Moreover, the results suggest that, to different extents, abiotic and biotic factors determine the community assembly of archaeal, bacterial and fungal communities.
Different Selective Effects on Rhizosphere Bacteria Exerted by Genetically Modified versus Conventional Potato Lines
PLoS ONE, 2013
Ecological Indicators, 2014
Soil microorganisms are the most important determinants of soil functioning. In order to understa... more Soil microorganisms are the most important determinants of soil functioning. In order to understand the relevance of stress-induced changes (e.g. as promoted by genetically modified plants), the natural variation (or normal operating range, NOR) of soil function has to be better understood. Quantitative assessment of the NOR, taking into account the relevant and most sensitive microbial groups, may lead to the first quantitative characterization of the NOR of an entire soil system. Thus, the focus of this work was on quantitative measurements of key genes involved in the nitrogen cycle, next to broader taxonomic assessment of the microbial groups, by real-time PCR as well as by PCR-DGGE and potential activities. In total, a robust dataset of more than 2000 measurements was obtained, and a NOR was developed based upon this dataset. The NOR can be considered as a space containing n dimensions, where n is the number of variables measured. When a soil (at field capacity) is not disturbed, all combinations of the variables fall inside the NOR. The distance between an investigated state and the center of the NOR represents a quantitative measurement that summarizes the state of the soil, taking into account the multivariate nature of the data. Parameterization of the model was done using microcosm experiments as well as via sampling of selected field soils during 3-years period. One advantage of the proposed approach is that the data itself show which variables are of concern and contribute most to the NOR, next to which ones produce noise. The method will assist in distinguishing the critical parameters in soil which are outside of the NOR as well as in the prevention of unnecessary actions.
Brazilian Journal of Microbiology, 2013
The petroleum-derived degrading Dietzia cinnamea strain P4 recently had its genome sequenced and ... more The petroleum-derived degrading Dietzia cinnamea strain P4 recently had its genome sequenced and annotated. This allowed employing the data on genes that are involved in the degradation of n-alkanes. To examine the physiological behavior of strain P4 in the presence of n-alkanes, the strain was grown under varying conditions of pH and temperature. D. cinnamea P4 was able to grow at pH 7.0-9.0 and at temperatures ranging from 35°C to 45°C. Experiments of gene expression by real-time quantitative RT-PCR throughout the complete growth cycle clearly indicated the induction of the regulatory gene alkU (TetR family) during early growth. During the logarithmic phase, a large increase in transcriptional levels of a lipid transporter gene was noted. Also, the expression of a gene that encodes the protein fused rubredoxin-alkane monooxygenase was enhanced. Both genes are probably under the influence of the AlkU regulator.
FEMS Microbiology Ecology, 2011
The nitrogen (N)-fixing community is a key functional community in soil, as it replenishes the po... more The nitrogen (N)-fixing community is a key functional community in soil, as it replenishes the pool of biologically available N that is lost to the atmosphere via anaerobic ammonium oxidation and denitrification. We characterized the structure and dynamic changes in diazotrophic communities, based on the nifH gene, across eight different representative Dutch soils during one complete growing season, to evaluate the amplitude of the natural variation in abundance and diversity, and identify possible relationships with abiotic factors. Overall, our results indicate that soil type is the main factor influencing the N-fixing communities, which were more abundant and diverse in the clay soils (n = 4) than in the sandy soils (n = 4). On average, the amplitude of variation in community size as well as the range-weighted richness were also found to be higher in the clay soils. These results indicate that N-fixing communities associated with sandy and clay soil show a distinct amplitude of variation under field conditions, and suggest that the diazotrophic communities associated with clay soil might be more sensitive to fluctuations associated with the season and agricultural practices. Moreover, soil characteristics such as ammonium content, pH and texture most strongly correlated with the variations observed in the diversity, size and structure of N-fixing communities, whose relative importance was determined across a temporal and spatial scale.
Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop
Plant and Soil, 2013
Dynamics of bacterial community succession in a salt marsh chronosequence: evidences for temporal niche partitioning
The ISME Journal, 2014
Applied and Environmental Microbiology, 2012
ABSTRACTAlthough mangroves represent ecosystems of global importance, the genetic diversity and a... more ABSTRACTAlthough mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on thenifHgene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. ThenifHgene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, thenifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in thenifHgene diversities were noted along the land-sea transect within the previously oiled mangrove. ThenifHgene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of...
Although mangroves represent ecosystems of global importance, the genetic diversity and abundance... more Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies.
13C pulse-labeling assessment of the community structure of active fungi in the rhizosphere of a genetically starch-modified potato (Solanum tuberosum) cultivar and its parental isoline
The aim of this study was to gain understanding of the carbon flow from the roots of a
geneticall... more The aim of this study was to gain understanding of the carbon flow from the roots of a
genetically modified (GM) amylopectin-accumulating potato (Solanum tuberosum) cultivar
and its parental isoline to the soil fungal community using stable isotope probing (SIP).
• The microbes receiving 13C from the plant were assessed through RNA⁄ phospholipid fatty
acid analysis with stable isotope probing (PLFA-SIP) at three time-points (1, 5 and 12 d after
the start of labeling). The communities of Ascomycota, Basidiomycota and Glomeromycota
were analysed separately with RT-qPCR and terminal restriction fragment length polymorphism
(T-RFLP).
• Ascomycetes and glomeromycetes received carbon from the plant as early as 1 and 5 d
after labeling, while basidiomycetes were slower in accumulating the labeled carbon. The rate
of carbon allocation in theGMvariety differed from that in its parental variety, thereby affecting
soil fungal communities.
• We conclude that both saprotrophic and mycorrhizal fungi rapidly metabolize organic substrates
flowing from the root into the rhizosphere, that there are large differences in utilization
of root-derived compounds at a lower phylogenetic level within investigated fungal phyla,
and that active communities in the rhizosphere differ between the GM plant and its parental
cultivar through effects of differential carbon flow from the plant.
Transcriptional profiling of genes involved in n-hexadecane compounds assimilation in the hydrocarbon degrading Dietzia cinnamea P4 strain
The petroleum-derived degrading Dietzia cinnamea strain P4 recently had its genome sequenced and
... more The petroleum-derived degrading Dietzia cinnamea strain P4 recently had its genome sequenced and
annotated. This allowed employing the data on genes that are involved in the degradation of n-alkanes.
To examine the physiological behavior of strain P4 in the presence of n-alkanes, the strain was
grown under varying conditions of pH and temperature. D. cinnamea P4 was able to grow at pH
7.0-9.0 and at temperatures ranging from 35 °C to 45 °C. Experiments of gene expression by
real-time quantitative RT-PCR throughout the complete growth cycle clearly indicated the induction
of the regulatory gene alkU (TetR family) during early growth. During the logarithmic phase, a large
increase in transcriptional levels of a lipid transporter gene was noted. Also, the expression of a gene
that encodes the protein fused rubredoxin-alkane monooxygenase was enhanced. Both genes are
probably under the influence of the AlkU regulator.
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Papers by Michele C P Silva
genetically modified (GM) amylopectin-accumulating potato (Solanum tuberosum) cultivar
and its parental isoline to the soil fungal community using stable isotope probing (SIP).
• The microbes receiving 13C from the plant were assessed through RNA⁄ phospholipid fatty
acid analysis with stable isotope probing (PLFA-SIP) at three time-points (1, 5 and 12 d after
the start of labeling). The communities of Ascomycota, Basidiomycota and Glomeromycota
were analysed separately with RT-qPCR and terminal restriction fragment length polymorphism
(T-RFLP).
• Ascomycetes and glomeromycetes received carbon from the plant as early as 1 and 5 d
after labeling, while basidiomycetes were slower in accumulating the labeled carbon. The rate
of carbon allocation in theGMvariety differed from that in its parental variety, thereby affecting
soil fungal communities.
• We conclude that both saprotrophic and mycorrhizal fungi rapidly metabolize organic substrates
flowing from the root into the rhizosphere, that there are large differences in utilization
of root-derived compounds at a lower phylogenetic level within investigated fungal phyla,
and that active communities in the rhizosphere differ between the GM plant and its parental
cultivar through effects of differential carbon flow from the plant.
annotated. This allowed employing the data on genes that are involved in the degradation of n-alkanes.
To examine the physiological behavior of strain P4 in the presence of n-alkanes, the strain was
grown under varying conditions of pH and temperature. D. cinnamea P4 was able to grow at pH
7.0-9.0 and at temperatures ranging from 35 °C to 45 °C. Experiments of gene expression by
real-time quantitative RT-PCR throughout the complete growth cycle clearly indicated the induction
of the regulatory gene alkU (TetR family) during early growth. During the logarithmic phase, a large
increase in transcriptional levels of a lipid transporter gene was noted. Also, the expression of a gene
that encodes the protein fused rubredoxin-alkane monooxygenase was enhanced. Both genes are
probably under the influence of the AlkU regulator.