Does a barcoding gap exist in Prokaryotes? Evidences from species delimitation in Cyanobacteria

The amount of information that is available on 16S rRNA sequences for prokaryotes thanks to high-throughput sequencing could allow a better understanding of diversity. Nevertheless, the application of predetermined threshold in genetic distances to identify units of diversity (Operative Taxonomic Units, OTUs) may provide biased results. Here we tests for the existence of a barcoding gap in several groups of Cyanobacteria, defining units of diversity according to clear differences between within-species and among-species genetic distances in 16S rRNA. The application of a tool developed for animal DNA taxonomy, the Automatic Barcode Gap Detector (ABGD), revealed that a barcoding gap could actually be found in almost half of the datasets that we tested. The identification of units of diversity through this method provided results that were not compatible with those obtained with the identification of OTUs with threshold of similarity in genetic distances of 97% or 99%. The main message of our results is a call for caution in the estimate of diversity from 16S sequences only, given that different subjective choices in the method to delimit units could provide different results

Ecology and distribution of Thaumarchaea in the deep hypolimnion of Lake Maggiore

Ammonia-oxidizing Archaea (AOA) play an important role in the oxidation of ammonia in terrestrial, marine, and geothermal habitats, as confirmed by a number of studies specifically focused on those environments. Much less is known about the ecological role of AOA in freshwaters. In order to reach a high resolution at the Thaumarchaea community level, the probe MGI-535 was specifically designed for this study and applied to fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) analysis. We then applied it to a fine analysis of diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence, but showing differences in abundance and distribution within the water column without significant seasonal trends with respect to Bacteria. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from a lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake

Distribution and origin of Thaumarchaea in the deep hypolimnion of Lake Maggiore

Thaumarchaea represents one of the most abundant groups of Archaea on Earth, being found in a variety of environments including soils, oceans, and freshwaters. Ammonia-oxidizing Thaumarchaea (AOA) significantly contribute to the global nitrogen and carbon cycle through chemolithoautotrophic oxidation of reduced nitrogen compounds. Their distribution of in freshwaters is still far less known than in marine and terrestrial environments. In this study, we analyzed the diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence and showing spatial but no seasonal trends with respect to Bacteria. A high resolution at the Thaumarchaea community level was reached with the probe MGI-535 specifically designed in this study and applied for CARD-FISH analysis. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from the lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake

Phylogenetic diversity of freshwater picocyanobacteria

Picocyanobacteria are photosynthetic prokaryotes, coccoid or rod-shaped, with a cell diameter < 2 ?m. They are common in lakes and oceans, and abundant across a wide spectrum of trophic conditions (Callieri et al 2012). The dominant genus of freshwater picocyanobacteria is Synechococcus. Analysis of 16S rRNA gene of freshwater Synechococcus showed its polyphyletic origin, requiring better insights in the present classification of the genus and possibly a revision. We isolated more than 40 picocyanobacterial strains from lakes geographically located in Italy, Mexico and Argentina, and characterized by different origin, trophic conditions and salinity. The strains originated from different water depths and had different pigmentation: phycoerythrin-containing (PE) or phycoerythrin-lacking strains (PC). A fragment of each isolated strain\u27s 16S rRNA gene was amplified (primers 16S5\u27F and B23S5\u27R, Lepere 2000). Sequences of about 750bp were aligned to selected reference sequences belonging to the most representative Synechococcus Groups and used to design a Neighbour-Joining (NJ) phylogenetic tree. Maximal Photosystem II (PSII) quantum yield of dark adapted cultures, Fv/Fm, and photosynthetic parameters from PhytoPAM Rapid Light Curve (RLC) at 14 light intensities (from 4 to 1984 ?E m-2 s-1) were measured for all the strains. The majority of Italian isolates, both PC and PE, assembled within the Cyanobium gracile cluster Group A (Crosbie et al 2003). Lake Maggiore strains convened in Group B together with many of our isolates. Other strains displayed distinct biogeographic differentiation: e.g. those isolated from high altitude tropical athalassohaline lakes in Mexico formed a well distinguished group of halotolerant strains phylogenetically close to the Marine Subcluster 5.2 (Synechococcus WH5701) and to Marine Subcluster 5.3. Notably, Synechococcus strains from tropical lakes had the highest Fv/Fm, typical of ecotypes with high photochemical efficiency of PSII. Synechocccus strains isolated from the ultraoligotrophic, large and deep Lake Nahuel Huapi (North Patagonia, Argentina) resulted as closely related to the Lake Constance Subalpine Group II (Synechococcus BO8805 and BO0014) and to the Marine Subcluster 5.2, with high Fv/Fm. The phylogenetic analysis of our new isolates provides more clear evidence of the non-marine nature of Group A as well as of the phylogenetic relationship between marine and freshwater halotolerant Synechococcus strains

A microbial perspective on biological invasions in aquatic ecosystems

Microorganisms are essential components of all aquatic ecosystems and are primarily responsible for biogeochemical cycles and key environmental processes. Despite their potential to influence the ecological functioning of biological communities and ecosystems at a global scale, the mechanisms of microbial invasions still lack specific and generalizable theories. Here, we review some of the ecological mechanisms, among those relevant under the global change scenario, which may facilitate the spread of microbial invaders, with a specific focus on aquatic prokaryotes (i.e. Bacteria). We summarize a selection of ecological features of vulnerable microbial communities (functional and structural stability, diversity, invasibility, and invasiveness) and environmental stressors for invasions (temperature increase, pollution, nutrients and resources fluctuations, and food web alterations). Owing to the rapid microbial adaptation to laboratory conditions, straightforward experimental approaches appear as suitable and informative tools to explore the invasion mechanisms in artificially assembled communities. We conclude by delineating future research steps required for a better understanding of the potential consequences of microbial invasions in the aquatic environment

Denaturing Gradient Gel Electrophoretic Analysis of Ammonia-Oxidizing Bacterial Community Structure in the Lower Seine River: Impact of Paris Wastewater Effluents

The Seine River is strongly affected by the effluents from the Achères wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study was to investigate the diversity of the ammonia-oxidizing bacterial population by identifying autochthonous bacteria from upstream and/or allochthonous ammonia-oxidizing bacteria from the WWTP effluents. Measurements of potential nitrifying activity, competitive PCR, and denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments specific to ammonia-oxidizing bacteria (AOB) were used to explore the succession and shifts of the ammonia-oxidizing community in the lower Seine River and to analyze the temporal and spatial functioning of the system at several different sampling dates. A major revelation was the stability of the patterns. The CTO primers used in this study (G. A. Kowalchuk, J. R. Stephen, W. D. Boer, J. I. Prosser, T. M. Embley, and J. W. Woldendorp, Appl. Environ. Microbiol. 63:1489-1497, 1997) were shown not to be completely specific to AOB of the β subclass of Proteobacteria. We further demonstrated that when DGGE patterns are interpreted, all the different bands must be sequenced, as one major DGGE band proved to be affiliated with a group of non-AOB in the β subclass of Proteobacteria. The majority of AOB (75 to 90%) present in the lower Seine river downstream of the effluent output belong to lineage 6a, represented by Nitrosomonas oligotropha- and Nitrosomonas ureae-like bacteria. This dominant lineage was represented by three bands on the DGGE gel. The major lineage-6a AOB species, introduced by the WWTP effluents, survived and might have grown in the receiving medium far downstream, in the estuary; it represented about 40% of the whole AOB population. The other two species belonging to lineage 6a seem to be autochthonous bacteria. One of them developed a few kilometers downstream of the WWTP effluent input in an ammonia-enriched environment, and the other appeared in the freshwater part of the estuary and was apparently more adapted to estuarine conditions, i.e., an increase in the amount of suspended matter, a low ammonia concentration, and high turnover of organic matter. The rest of the AOB population was represented in equal proportions by Nitrosospira- and Nitrosococcus mobilis-like species

Intermediate concentrations of synthetic antibiotics drive drastic changes in aquatic bacterial communities

The spread of resistances to commonly used synthetic antibiotics (AB) in natural environments is one of the major threats to human health. A large number of publications investigated the relations between different human pathogens and the spread of AB resistances, but a comparably low number of studies targeted the ecological effect of the presence of synthetic AB on non?pathogenic bacteria in waters. We tested the long?term impact of a cocktail of synthetic antibiotics of commercial use (Imipenem, Levofloaxcin, and Tetracycline) on artificial bacterial communities miming a very simplified natural freshwater community. The experimental setup consisted in a 24 days?long continuous culture system where the bacterial communities were expose to three different AB concentrations: no AB (control), sub?lethal AB concentration (comparable to anthropized European lakes), and in AB concentration 10 folds higher (comparable to heavily contaminated water treatment plants). Bacterial number and morphological distribution were daily monitored (epifluorescence microscopy), while community composition (CARD?FISH) and antibiotic resistance were assessed bi?weekly. Bacterial production, and thus abundance, dropped by 75% in the presence of AB, independently by their concentration, while bacterial community composition resulted highly affected by intermediate AB concentration, with reversal in the proportion of the dominant groups (A. hydrophila and Brevundimonas sp.). These results, for the first time focussing on the ecological consequences of AB on a experimental bacterial community, highlight the potential risk posed by low AB concentrations in waters. Their effect is not only limited to the spread of potentially pathogenic strains, but it extends to modification of interspecific ecological interactions, weakening the natural resistance of the aquatic communities to the success of allochtonous, and potentially dangerous, strains

Persistence of antibiotic resistance genes in large subalpine lakes: the role of anthropogenic pollution and ecological interactions

The spread of antibiotic-resistant bacteria is a threatening risk for human health at a global scale. Improved knowledge on the cycle of antibiotic resistance spread between human and the environment is a major requirement for the management of the current crisis. Compared to the well-studied cycle in clinical settings much less is known about the factor allowing their persistence in the environment. In fact, lakes and rivers exposed to high anthropogenic impact seem to become long-term reservoirs for resistance genes. The presence of antibiotic resistance genes (ARGs) within the resident microbiome of large subalpine lakes (i.e. Lake Geneva, Lake Maggiore) has recently been investigated in both the water column and the sediment. These studies suggest a correlation between the abundance of some ARGs and the anthropogenic impact. Within the system, however, abiotic factors and the food-web structure determine the survival of specific bacterial genotypes and thus the resistance genes they harbour. Thus, a growing body of work suggests an important role of ecological interactions in the persistence or elimination of such genes from the environment. This article reviews the current literature regarding the presence of ARGs in subalpine lakes, the impact of anthropogenic pollution on their appearance and the potential role of various ecological interactions on their persistence in the system

Archaea and Bacteria in deep lake hypolimnion: In situ dark inorganic carbon uptake

The interest for microorganisms inhabiting the hypolimnion and for their role in biogeochemical cycles of lakes is considerable, but knowledge is far from complete. The presence of chemolithoautotrophic Bacteria and mesophilic Archaea (e.g., Thaumarchaeota) assimilating inorganic carbon in the deep hypolimnion of lakes has been ascertained. We measured, for the first time at 350 m in Lake Maggiore (Northern Italy), the prokaryotic in situ dark [14C]HCO3 incorporation with a new custom-made apparatus, which takes samples and adds tracers in situ. Thereby stress factors affecting prokaryotes during sample recovery from the depth were avoided. We tested the new instrument at different depths and conditions, performing parallel conventional on board incubations. We found that dark [14C]HCO3 incorporations had lower standard deviation in in situ incubations with respect to the on board ones, but their means were not statistically different. At 350 m we estimated an uptake of 187.7±15 μg C m–3 d–1, which is in line with the published uptake rates in aquatic systems. By inhibiting the bacterial metabolism, we found that Archaea were responsible for 28% of the total CO2 uptake. At the same depth, Thaumarchaeota, on average, constituted 11% of total DAPI counts. Dark [14C]HCO3 incorporation integrated along the aphotic water column was 65.8±5.2 mg C m–2 d–1 which corresponds to 87% of picophytoplanktonic autotrophic fixation in the euphotic layer. This study provides the first evidence of Bacteria and Archaea dark CO2 fixation in the deep hypolimnion of a subalpine lake and indicates a potentially significant prokaryotic CO2 sink

Phylogenetic diversity of non-marine picocyanobacteria

We studied the phylogenetic diversity of nonmarine picocyanobacteria broadening the sequence data set with 43 new sequences of the 16S rRNA gene. The sequences were derived from monoclonal strains isolated from four volcanic high-altitude athalassohaline lakes in Mexico, five glacial ultraoligotrophic North Patagonian lakes and six Italian lakes of glacial, volcanic and morenic origin. The new sequences fall into a number of both novel and previously described clades within the phylogenetic tree of 16S rRNA gene. The new cluster of Lake Nahuel Huapi (North Patagonia) forms a sister clade to the subalpine cluster II and the marine Synechococcus subcluster 5.2. Our finding of the novel clade of ‘halotolerants’ close to the marine subcluster 5.3 (Synechococcus RCC307) constitutes an important demonstration that euryhaline and marine strains affiliate closely. The intriguing results obtained shed new light on the importance of the nonmarine halotolerants in the phylogenesis of picocyanobacteria.Fil: Callieri, Cristiana. Consiglio Nazionale delle Ricerche. Istituto per lo Studio degli Ecosistemi; ItaliaFil: Coci, Manuela. Consiglio Nazionale delle Ricerche. Istituto per lo Studio degli Ecosistemi; ItaliaFil: Corno, Gianluca. Consiglio Nazionale delle Ricerche. Istituto per lo Studio degli Ecosistemi; ItaliaFil: Macek, Miroslav. Universidad Nacional Autónoma de México; MéxicoFil: Modenutti, Beatriz Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; ArgentinaFil: Balseiro, Esteban Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; ArgentinaFil: Bertoni, Roberto. Consiglio Nazionale delle Ricerche. Istituto per lo Studio degli Ecosistemi; Itali