Strategies and approaches in plasmidome studies, uncovering plasmid diversity disregarding of linear elements?

The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognised not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture dependent or independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e. the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which despite their frequent occurrence in a large number of bacteria are largely neglected in prevalent plasmidome conceptions.Fil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; ArgentinaFil: Wagenknecht, Martin. Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster.; AlemaniaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; ArgentinaFil: Meinhardt, Friedhelm. Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster.; Alemani

Effect of stress factors associated with postharvest citrus conditions on the viability and biocontrol activity of Clavispora lusitaniae strain 146

Only quite recently, we have shown that yeast strains Clavispora lusitaniae 146 and Pichia fermentans 27 can act as efficient biocontrol agents for combating postharvest fungal diseases in lemons. During postharvest and storage conditions, microorganisms are subject to different stress factors that could affect both their survival and their protective capacity. Understanding the tolerance of yeasts to environmental stress factors could support the future development and commercial application of biological control formulations based on such organisms. Thus, the impact of different stressors on the viability and protection efficiency of C. lusitaniae strain 146 and P. fermentans strain 27 was evaluated, and the yeasts were subjected to oxidative stress, thermal treatments, exposure to NaOCl, osmotic stress, and ultraviolet irradiation. Candida oleophila strain O served as the reference control. C. lusitaniae 146 was more resistant to H2O2 in plate assays; however, in liquid media there was no significant difference to the other strains. Strain 146 was less affected by NaOCl, being able to survive with 300 ppm. P. fermentans 27 was the strain most heavily affected by osmotic pressure, while strains 146 and strain O showed a similar adaptation. UV-B irradiation severely affected C. oleophila and P. fermentans, while C. lusitaniae was the most resistant. Strains 146 and 27 were similarly tolerant to thermal shocks, compared to the reference strain, which was less viable. In in vivo tests, exposure to 10 mM H2O2, 45 °C or 200 ppm NaOCl prior to fruit inoculation, reduced the antagonistic activity against the pathogen Penicillium digitatum. However, in no case was the biocontrol efficiency reduced to less than 50%. As C. lusitaniae 146 demonstrated a great potential to combat P. digitatum under a wide range of conditions, the organism is a promising candidate as an effective and valuable alternative to toxic fungicides.Fil: Pereyra, Martina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Diaz, Mariana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Meinhardt, Friedhelm. Münster Universität; AlemaniaFil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán; Argentin

Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions

Complete Genome Sequence of pAP13, a Large Linear Plasmid of a Brevibacterium Strain Isolated from a Saline Lake at 4,200 Meters above Sea Level in Argentina

pAP13 is an 89 kb-linear plasmid hosted by Brevibacterium sp. Ap13, an actinobacterium isolated from the feces of a flamingo from an extremely high-altitude lake in Argentina. Because of the ecological importance of the genus Brevibacterium, the absolute lack of information concerning Brevibacterium linear plasmids, as well as the as the possible ecological significance of this unusual plasmid, pAP13 was completely sequenced, including the inversely oriented termini.Fil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Schuldes, Jörg. Georg August Universität; AlemaniaFil: Thürner, Andrea. Georg August Universität; AlemaniaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Daniel, Rolf. Georg August Universität; AlemaniaFil: Meinhardt, Friedhelm. No especifíca

Killer yeasts for the biological control of postharvest fungal crop diseases

Every year and all over the world the fungal decay of fresh fruit and vegetables frequently generates substantial economic losses. Synthetic fungicides, traditionally used to efficiently combat the putrefactive agents, emerged, however, as the cause of environmental and human health issues. Given the need to seek for alternatives, several biological approaches were followed, among which those with killer yeasts stand out. Here, after the elaboration of the complex of problems, we explain the hitherto known yeast killer mechanisms and present the implementation of yeasts displaying such phenotype in biocontrol strategies for pre-or postharvest treatments to be aimed at combating postharvest fungal decay in numerous agricultural products.Fil: Díaz, Mariana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pereyra, Martina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Picón Montenegro, Ernesto. Universidad Nacional de Tucumán; ArgentinaFil: Meinhardt, Friedhelm. Universidad Nacional de Tucumán; ArgentinaFil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán; Argentin

First Complete Sequence of a Giant Linear Plasmid from a Micrococcus Strain Isolated from an Extremely High-Altitude Lake

Micrococcus sp. V7, an actinobacterial strain adapted to the extreme conditions of the Laguna Vilama, an extremely high-altitude (4,600mabove sea level) lake in the Argentinian Puna, was found to carry the giant linear plasmid pLMV7. We determined its sequence (92,815 bp) as a prerequisite to the investigation of its role for survival in such a harsh environment.Fil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Schuldes, Jörg. Georg August Universität Göttingen; AndorraFil: Thürner, Andrea. Georg August Universität Göttingen; AndorraFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Daniel, Rolf. Georg August Universität Göttingen; AndorraFil: Meinhardt, Friedhelm. No especifíca

Autoselection of Cytoplasmic Yeast Virus Like Elements Encoding Toxin/Antitoxin Systems Involves a Nuclear Barrier for Immunity Gene Expression

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle

Screening methods for isolation of biocontrol epiphytic yeasts against penicillium digitatum in lemons

Worldwide, the green rot caused by Penicillium digitatum is one of the most aggressive postharvest diseases of lemons. Searching for sustainable alternatives to chemical fungicides, epi-phytic yeasts as potential biocontrol agents were isolated from citrus fruits using a tailor-made selective medium. For disclosing their antagonistic potential against P. digitatum, obtained isolates were subjected to direct screening methods, both in vitro and in vivo. In the course of the primary in vitro screening that comprised dual culture assays, 43 yeast strains displaying antagonistic activities against the pathogen were selected. Subsequently, such strains were subjected to an in vivo screening that consisted of a microscale test, allowing the selection of six yeast strains for further analysis. In the final screening using macroscale in vivo tests, three strains (AcL2, AgL21, and AgL2) displaying the highest efficiencies to control P. digitatum were identified. The protection efficiencies in lemons were 80 (AcL2), 76.7 (AgL21), and 75% (AgL2). Based on sequence analysis of the PCR amplified D1/D2 domains of the 26S rRNA genes, they were identified as representatives of the species Clavispora lusitaniae. Interestingly, the strains exhibited a broad action spectrum among citrus fruits as they were also able to combat the green mold disease in grapefruit and two orange varieties. The direct screening methods applied in this study favored the recovery of effi-cient candidates for application as biological control agents to combat fungal infestations of citrus fruits.Fil: Pereyra, Martina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Diaz, Mariana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Soliz Santander, Fabricio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Poehlein, Anja. Universität Göttingen; AlemaniaFil: Meinhardt, Friedhelm. Westfälische Wilhelms Universität; AlemaniaFil: Daniel, Rolf. Universität Göttingen; AlemaniaFil: Dib, Julian Rafael. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentin

Assessment of the plasmidome of an extremophilic microbial community from the Diamante Lake, Argentina

Diamante Lake located at 4589 m.a.s.l. in the Andean Puna constitutes an extreme environment. It is exposed to multiple extreme conditions such as an unusually high concentration of arsenic (over 300 mg L−1) and low oxygen pressure. Microorganisms thriving in the lake display specific genotypes that facilitate survival, which include at least a multitude of plasmid-encoded resistance traits. Hence, the genetic information provided by the plasmids essentially contributes to understand adaptation to different stressors. Though plasmids from cultivable organisms have already been analyzed to the sequence level, the impact of the entire plasmid-borne genetic information on such microbial ecosystem is not known. This study aims at assessing the plasmidome from Diamante Lake, which facilitates the identification of potential hosts and prediction of gene functions as well as the ecological impact of mobile genetic elements. The deep-sequencing analysis revealed a large fraction of previously unknown DNA sequences of which the majority encoded putative proteins of unknown function. Remarkably, functions related to the oxidative stress response, DNA repair, as well as arsenic- and antibiotic resistances were annotated. Additionally, all necessary capacities related to plasmid replication, mobilization and maintenance were detected. Sequences characteristic for megaplasmids and other already known plasmid-associated genes were identified as well. The study highlights the potential of the deep-sequencing approach specifically targeting plasmid populations as it allows to evaluate the ecological impact of plasmids from (cultivable and non-cultivable) microorganisms, thereby contributing to the understanding of the distribution of resistance factors within an extremophilic microbial community.Fil: Perez, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Saona Acuña, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Poehlein, Anja. Universität Göttingen; AlemaniaFil: Meinhardt, Friedhelm. Münster Universität; AlemaniaFil: Daniel, Rolf. Universität Göttingen; AlemaniaFil: Dib, Julian Rafael. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentin

Complete Genome Sequence of the Linear Plasmid pJD12 Hosted by Micrococcus sp. D12, Isolated from a High-Altitude Volcanic Lake in Argentina

The linear plasmid pDJ12 from Micrococcus D12, isolated from the high-altitude volcanic Diamante Lake in the northwest of Argentina, was completely sequenced and annotated. It is noteworthy that the element is probably conjugative and harbors genes potentially instrumental in coping with stress conditions that prevail in such an extreme environmentFil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina. Universidad Nacional de Tucumán; ArgentinaFil: Angelov, Angel. Technische Universitat Munchen; AlemaniaFil: Liebl, Wolfang. Technische Universitat Munchen; AlemaniaFil: Döbber, Johannes. Westfalische Wilhelms Universitat; AlemaniaFil: Voget, Sonja. Georg-August University; AlemaniaFil: Schuldes, Jörg. Georg-August University; AlemaniaFil: Gorriti, Marta Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); ArgentinaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); ArgentinaFil: Meinhardt, Friedhelm. Westfalische Wilhelms Universitat; AlemaniaFil: Daniele, Rolf. Georg-August University; Alemani