Culture-dependent and culture-independent diversity surveys target different bacteria: a case study in a freshwater sample

Compared with culture-independent approaches, traditionally used culture-dependent methods have a limited capacity to characterizewatermicrobiota. Nevertheless, for almost a century the latter have been optimized to detect and quantify relevant bacteria. A pertinent question is if culture-independent diversity surveys give merely an extended perspective of the bacterial diversity or if, even with a higher coverage, focus on a different set of organisms. We compared the diversity and phylogeny of bacteria in a freshwater sample recovered by currently used culture-dependent and culture-independent methods (DGGE and 454 pyrosequencing). The culture-dependent diversity survey presented lower coverage than the other methods. However, it allowed bacterial identifications to the species level, in contrast with the other procedures that rarely produced identifications below the order. Although the predominant bacterial phyla detected by both approaches were the same (Proteobacteria, Actinobacteria, Bacteroidetes), sequence similarity analysis showed that, in general, different operational taxonomical units were targeted by each method. The observation that culture-dependent and independent approaches target different organisms has implications for the use of the latter for studies in which taxonomic identification has a predictive value. In comparison to DGGE, 454 pyrosequencing method had a higher capacity to explore the bacterial richness and to detect cultured organisms, being also less laborious.info:eu-repo/semantics/acceptedVersio

A primer-independent DNA polymerase-based method for competent whole-genome amplification of intermediate to high GC sequences

The dataset (raw sequencing data) that supports the findings of this study are archived in the Universidad Autónoma de Madrid data repository e‐cienciaDatos in DOI: 10.21950/HCNDGMultiple displacement amplification (MDA) has proven to be a useful technique for obtaining large amounts of DNA from tiny samples in genomics and metagenomics. However, MDA has limitations, such as amplification artifacts and biases that can interfere with subsequent quantitative analysis. To overcome these challenges, alternative methods and engineered DNA polymerase variants have been developed. Here, we present new MDA protocols based on the primer-independent DNA polymerase (piPolB), a replicative-like DNA polymerase endowed with DNA priming and proofreading capacities. These new methods were tested on a genomes mixture containing diverse sequences with high-GC content, followed by deep sequencing. Protocols relying on piPolB as a single enzyme cannot achieve competent amplification due to its limited processivity and the presence of ab initio DNA synthesis. However, an alternative method called piMDA, which combines piPolB with Φ29 DNA polymerase, allows proficient and faithful amplification of the genomes. In addition, the prior denaturation step commonly performed in MDA protocols is dispensable, resulting in a more straightforward protocol. In summary, piMDA outperforms commercial methods in the amplification of genomes and metagenomes containing high GC sequences and exhibits similar profiling, error rate and variant determination as the non-amplified samplesMCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe [PGC2018-09723-A-I00 and PID2021-123403NB-I00 to M.R.R.]; C. Egas’ laboratory was funded by the European Union′s Horizon 2020 Research and Innovation Program [685474]; METAFLUIDICS project; C.D.O. and C.M.C. were holder of Fellowships from the Spanish Ministry of University [FPU16/02665] and Spanish Ministry of Science and Innovation [PRE2019-087304], respectivel

Priming of a DNA metabarcoding approach for species identification and inventory in marine macrobenthic communities

In marine and estuarine benthic communities, the inventory and estimation of species richness are often hampered by the need for broad taxonomic expertise across several phyla. The use of DNA metabarcoding has emerged as a powerful tool for the fast assessment of species composition in a diversity of ecological communities. Here, we tested the amplification success of five primer sets targeting different COI-5P regions by 454 pyrosequencing to maximize the recovery of two simulated macrobenthic communities containing 21 species (SimCom1 and SimCom 2). Species identification was first performed against a compiled reference library of macrobenthic species. Reads with similarity results to reference sequences between 70% and 97% were then submitted to GenBank and BOLD to attempt the identification of concealed species in the bulk sample. The combination of at least three primer sets was able to recover more species than any primer set alone, achieving 85% of represented species in SimCom1 and 76% in SimCom2. Our approach was successful to detect low-frequency specimens, as well as concealed species, in the bulk sample, indicating the potential for the application of this approach on marine bioassessment and inventory, including the detection of "hidden" biodiversity that would hardly be possible based on morphology only.This work was supported by FEDER through POFCCOMPETE by national funds from 'Fundacao para a Ciencia e a Tecnologia (FCT)' in the scope of the grant FCOMP-01-0124-FEDER-015429 and also by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER007569). This work was also funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). J.L. was supported by a PhD fellowship (SFRH/BD/69750/2010) from FCT and C.H. by a CAPES Post-doctoral fellowship, under Science Without Borders Program (Ministry of Education, Brazil)info:eu-repo/semantics/publishedVersio

Transcriptome analysis reveals the high ribosomal inhibitory action of 1,4-naphthoquinone on Meloidogyne luci infective second-stage juveniles

The root-knot nematode (RKN) Meloidogyne luci presents a threat to the production of several important crops. This nematode species was added to the European Plant Protection Organization Alert list in 2017. The scarce availability of efficient nematicides to control RKN and the phasing out of nematicides from the market have intensified the search for alternatives, such as phytochemicals with bionematicidal properties. The nematicidal activity of 1,4-naphthoquinone (1,4-NTQ) against M. luci has been demonstrated; however, knowledge of the potential mode(s) of action of this compound is still scarce. In this study, the transcriptome profile of M. luci second-stage juveniles (J2), the infective stage, in response to 1,4-NTQ exposure was determined by RNA-seq to identify genes and pathways that might be involved in 1,4-NTQ’s mode(s) of action. Control treatments, consisting of nematodes exposed to Tween® 80 (1,4-NTQ solvent) and to water, were included in the analysis. A large set of differentially expressed genes (DEGs) was found among the three tested conditions, and a high number of downregulated genes were found between 1,4-NTQ treatment and water control, reflecting the inhibitory effect of this compound on M. luci, with a great impact on processes related to translation (ribosome pathway). Several other nematode gene networks and metabolic pathways affected by 1,4-NTQ were also identified, clarifying the possible mode of action of this promising bionematicide

Understanding the Role of PIN Auxin Carrier Genes under Biotic and Abiotic Stresses in Olea europaea L..

The PIN-FORMED (PIN) proteins represent the most important polar auxin transporters in plants. Here, we characterized the PIN gene family in two olive genotypes, the Olea europaea subsp. europaea var. sylvestris and the var. europaea (cv. ‘Farga’). Twelve and 17 PIN genes were identified for vars. sylvestris and europaea, respectively, being distributed across 6 subfamilies. Genes encoding canonical OePINs consist of six exons, while genes encoding non-canonical OePINs are composed of five exons, with implications at protein specificities and functionality. A copia-LTR retrotransposon located in intron 4 of OePIN2b of var. europaea and the exaptation of partial sequences of that element as exons of the OePIN2b of var. sylvestris reveals such kind of event as a driving force in the olive PIN evolution. RNA-seq data showed that members from the subfamilies 1, 2, and 3 responded to abiotic and biotic stress factors. Co-expression of OePINs with genes involved in stress signaling and oxidative stress homeostasis were identified. This study highlights the importance of PIN genes on stress responses, contributing for a holistic understanding of the role of auxins in plants

Bacterial communities in Serpa cheese by culture dependent techniques, 16S rRNA gene sequencing and high-throughput sequencing analysis

Serpa cheese is one of the traditional regional Portuguese cheeses having the Protected Denomination of Origin (PDO) designation. This study investigated the bacterial community in the traditional Portuguese Serpa cheese. The microorganisms identified at the end of ripening (30 days) mainly were lactic acid bacteria (LAB). Lactobacillus paracasei/Lactobacillus casei was the main species in cheese from PDO registered industries, whereas in non-PDO registered industries Lactobacillus brevis was highlighted, among other LAB. Enterobacteriaceae species were detected at 20% to 40% of the total isolates. The results obtained by high-throughput sequencing analysis confirmed that LAB was the main microbial group, with Lactococcus genus contributing to approximately 40% to 60% of the population, followed by Leuconostoc and Lactobacillus. The Enterobacteriaceae family was also important. The differences between bacterial communities from PDO and non-PDO registered industries suggest that the lack of regulation of the cheese-making practices may influence unfavorably. The new knowledge about bacterial diversity in Serpa cheese could be useful to set up new ripening conditions, which favor the development of desirable microorganisms

Taking Root: Enduring Effect of Rhizosphere Bacterial Colonization in Mangroves

Mangrove forests are of global ecological and economic importance, but are also one of the world's most threatened ecosystems. Here we present a case study examining the influence of the rhizosphere on the structural composition and diversity of mangrove bacterial communities and the implications for mangrove reforestation approaches using nursery-raised plants.A barcoded pyrosequencing approach was used to assess bacterial diversity in the rhizosphere of plants in a nursery setting, nursery-raised transplants and native (non-transplanted) plants in the same mangrove habitat. In addition to this, we also assessed bacterial composition in the bulk sediment in order to ascertain if the roots of mangrove plants affect sediment bacterial composition. We found that mangrove roots appear to influence bacterial abundance and composition in the rhizosphere. Due to the sheer abundance of roots in mangrove habitat, such an effect can have an important impact on the maintenance of bacterial guilds involved in nutrient cycling and other key ecosystem functions. Surprisingly, we also noted a marked impact of initial nursery conditions on the rhizosphere bacterial composition of replanted mangrove trees. This result is intriguing because mangroves are periodically inundated with seawater and represent a highly dynamic environment compared to the more controlled nursery environment.In as far as microbial diversity and composition influences plant growth and health, this study indicates that nursery conditions and early microbial colonization patterns of the replants are key factors that should be considered during reforestation projects. In addition to this, our results provide information on the role of the mangrove rhizosphere as a habitat for bacteria from estuarine sediments

An Insightful Model to Study Innate Immunity and Stress Response in Deep‐Sea Vent Animals: Profiling the Mussel Bathymodiolus azoricus

Deep‐sea environments are, in some cases, largely unexplored ecosystems, where life thrives driven by the geochemical features of each location. Among these environments, chemosynthesis‐based ecosystems, in the Mid Atlantic Ridge, have an exclusive combination of high depth, high sulfur, and high methane concentrations. This is believed to modulate the biological composition of vent communities and influence the overall vent animal transcriptional activity of genes involved in adaptation processes to extreme environments. This opens, thus, the possibility of finding gene expression signatures specific to a given hydrothermal vent field. Regardless of the extreme physicochemical conditions that characterize deep‐sea hydrothermal vents, the animals dwelling around the vent sites exhibit high productivity and thus must cope with toxic nature of vent surrounding, seemingly deleterious to the animals, while developing surprisingly successful strategies to withstand adverse environmental conditions, including environmental microbes and mechanical stress whether ensuing from animal predation or venting activity. The deep‐sea vent mussel Bathymodiolus azoricus has adapted well to deep‐sea extreme environments and represents the dominating faunal community from hydrothermal vent sites in the Mid‐Atlantic Ridge, owing its successful adaptation and high biomasses to specialized exploitation of methane and sulfide sources from venting activity. Its extraordinary capabilities of adapting and thriving in chemosynthesis‐based environments, largely devoid of photosynthetic primary production and characterized by rapid geochemical regime changes are due to symbiotic associations with chemosynthetic bacteria within its large gills. In an attempt to understand physiological reactions in animals normally set to endure extreme deep‐sea environments, our laboratory has undertaken, for the last few years, a series of investigations, aimed at characterizing molecular indicators of adaptation processes of which components of the host defense system has received most attention. This study reviews recent advances on the characterization of molecules and genes participating in immune reactions, using in vivo and ex vivo models, to elucidate cellular and humoral defense mechanisms in vent mussels and the strategies they have adopted to survive under extreme environments