Susana S. Santos

Metagenomes provide valuable comparative information on soil microeukaryotes

Despite the critical ecological roles of microeukaryotes in terrestrial ecosystems, most descriptive studies of soil microbes published so far focused only on specific groups. Meanwhile, the fast development of metagenome sequencing leads to considerable data accumulation in public repositories, providing microbiologists with substantial amounts of accessible information. We took advantage of public metagenomes in order to investigate microeukaryote communities in a well characterized grassland soil. The data gathered allowed the evaluation of several factors impacting the community structure, including the DNA extraction method, the database choice and also the annotation procedure. While most studies on soil microeukaryotes are based on sequencing of PCR-amplified taxonomic markers (18S rRNA genes, ITS regions), this work represents, to our knowledge, the first report based solely on metagenomic microeukaryote DNA. Choosing the correct annotation procedure and reference database has proven to be crucial, as it considerably limits the risk of wrong assignments. In addition, a significant and pronounced effect of the DNA extraction method on the taxonomical structure of soil microeukaryotes has been identified. Our analyses suggest that publicly available metagenome data can provide valuable information on soil microeukaryotes for comparative purposes when handled appropriately, complementing the current view provided by ribosomal amplicon sequencing methods.

Natural Hot Spots for Gain of Multiple Resistances: Arsenic and Antibiotic Resistances in Heterotrophic, Aerobic Bacteria from Marine Hydrothermal Vent Fields

ABSTRACT Microorganisms are responsible for multiple antibiotic resistances that have been associated with resistance/tolerance to heavy metals, with consequences to public health. Many genes conferring these resistances are located on mobile genetic elements, easily exchanged among phylogenetically distant bacteria. The objective of the present work was to isolate arsenic-, antimonite-, and antibiotic-resistant strains and to determine the existence of plasmids harboring antibiotic/arsenic/antimonite resistance traits in phenotypically resistant strains, in a nonanthropogenically impacted environment. The hydrothermal Lucky Strike field in the Azores archipelago (North Atlantic, between 11°N and 38°N), at the Mid-Atlantic Ridge, protected under the OSPAR Convention, was sampled as a metal-rich pristine environment. A total of 35 strains from 8 different species were isolated in the presence of arsenate, arsenite, and antimonite. ACR3 and arsB genes were amplified from the sediments total DNA, and 4 isolates also carried ACR3 genes. Phenotypic multiple resistances were found in all strains, and 7 strains had recoverable plasmids. Purified plasmids were sequenced by Illumina and assembled by EDENA V3, and contig annotation was performed using the “Rapid Annotation using the Subsystems Technology” server. Determinants of resistance to copper, zinc, cadmium, cobalt, and chromium as well as to the antibiotics β-lactams and fluoroquinolones were found in the 3 sequenced plasmids. Genes coding for heavy metal resistance and antibiotic resistance in the same mobile element were found, suggesting the possibility of horizontal gene transfer and distribution of theses resistances in the bacterial population.

Comparison of three DNA extraction methods for recovery of soil protist DNA.

The use of molecular methods to investigate protist communities in soil is in rapid development this decade. Molecular analysis of soil protist communities is usually dependant on direct genomic DNA extraction from soil and inefficient or differential DNA extraction of protist DNA can lead to bias in downstream community analysis. Three commonly used soil DNA extraction methods have been tested on soil samples from three European Long-Term Observatories (LTOs) with different land-use and three protist cultures belonging to different phylogenetic groups in different growth stages. The methods tested were: ISOm-11063 (a version of the ISO-11063 method modified to include a FastPrep ®-24 mechanical lysis step), GnS-GII (developed by the GenoSol platform to extract soil DNA in large-scale soil surveys) and a commercial DNA extraction kit - Power Lyzer™ PowerSoil® DNA Isolation Kit (MoBio). DNA yield and quality were evaluated along with DNA suitability for amplification of 18S rDNA fragments by PCR. On soil samples, ISOm-11063 yields significantly higher DNA for two of the three soil samples, however, MoBio extraction favors DNA quality. This method was also more effective to recover copies of 18S rDNA numbers from all soil types. In addition and despite the lower yields, higher DNA quality was observed with DNA extracted from protist cultures with the MoBio method. Likewise, a bead-beating step shows to be a good solution for DNA extraction of soil protists, since the recovery of DNA from protist cultures and from the different soil samples with the ISOm method proved to be efficient in recovering PCR-amplifiable DNA. This study showed that soil DNA extraction methods provide biased results towards the cyst stages of protist organism.

Soil DNA Extraction Procedure Influences Protist 18S rRNA Gene Community Profiling Outcome

Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location. The commercial DNA extraction kit was associated with the highest diversity estimates and with a corresponding higher retrieval of Excavata, Cercozoa and Amoebozoa-related taxa. Overall, our findings indicate that this extraction offers a compromise between rare and dominant taxa representation, while providing high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays.

Effects of Bacillus cereus Endospores on Free-Living Protist Growth

We studied the predator–prey interactions between heterotrophic protists and endospores of Bacillus cereus group bacteria, in order to gain insight on survival and dispersal of B. cereus endospores in the environment. It has been hypothesised that the spore stage protects against digestion by predating protists. Therefore, experiments were carried out to investigate the impact of B. cereus endospores and vegetative cells, as the only food source, on individual amoeboid, flagellated and ciliated protists. The presence of fluorescent-labelled intracellular bacteria confirmed that B. cereus endospores as well as vegetative cells were ingested by protists and appeared intact in the food vacuoles when observed by epifluorescence microscopy. Furthermore, protist growth and bacterial predation were followed by qPCR. Protists were able to grow on vegetative cells as well as endospores of B. cereus, despite the lower cell division rates observed for some protists when feeding on bacterial endospores. Survival and proliferation of ingested bacteria inside protists cells was also observed. Finally, B. cereus spore germination and growth was observed within all protists with higher abundance in the amoeboid protist after antibiotic treatment of the protist surface. These observations support that protists can act as a potential breeding ground for B. cereus endospores.