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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.

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 H.sub.2 O.sub.2 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 H.sub.2 O.sub.2, 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.

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.

Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity.

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.

Blueberry production is affected by fungal postharvest pathogens, including Botrytis cinerea and Alternaria alternata, the causative agents of gray mold disease and Alternaria rot, respectively. Biocontrol agents adapted to blueberries and local environments are not known to date. Here, we report on the search for and the identification of cultivable blueberry epiphytic bacteria with the potential to combat the aforementioned fungi. Native, blueberry-borne bacterial strains were isolated from a plantation in Tucumán, Argentina and classified based on 16S rRNA gene sequences. Antagonistic activities directed at B. cinerea and A. alternata were studied in vitro and in vivo. The 22 bacterial strains obtained could be attributed to eleven different genera: Rosenbergiella, Fictibacillus, Bacillus, Pseudomonas, Microbacterium, Asaia, Acinetobacter, Curtobacterium, Serratia, Sphingomonas and Xylophilus. Three strains displaying antagonistic impacts on the fungal pathogens were identified as Bacillus velezensis (BA3 and BA4) and Asaia spathodeae (BMEF1). These strains are candidates for biological control agents of local blueberry production and might provide a basis for the development of eco-friendly, sustainable alternatives to synthetic pesticides.

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, epiphytic 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 efficient candidates for application as biological control agents to combat fungal infestations of citrus fruits.

We functionally analysed the two-component regulatory system DegSU (historically SacU) in Bacillus megaterium DSM319 by generating a genetic knock out as well as a sacU32 mutation. The latter--known to cause a hypersecretion phenotype in Bacillus subtilis--had no influence on extracellular protease and amylase activity in B. megaterium. Since the B. megaterium DegU complemented a Bacillus licheniformis [increment]degSU mutant, functionality of the protein was proven. Expression of the sacB encoded levansucrase was found to be dependent on DegSU in B. megaterium. Consistently, the fusion of the sacB promoter to gfp revealed a strong increase in GFP-expression in the sacU32 strain. On 2 D-gels of the secretome, a large number of intracellular proteins was seen. The culture medium contained only 42 secreted proteins which can be assigned to polypeptides involved in the metabolism of the cell wall, polypeptides with proteolytic activities and those with unknown functions. Though overall protease activity matches with the wild type, two proteolytic enzymes (Vpr and YwaD) are missing in the secretome of the [increment]degSU strain, while other degradative enzymes are not affected. In line with such findings, no increase of proteolytic or other degradative enzymes was seen in the sacU32 mutant. Thus, compared to B. subtilis and B. licheniformis, the number of extracellular proteins influenced by DegSU is surprisingly low in B. megaterium, a feature, probably advantageous as to the use of the sacU32 mutant for production of secreted proteins.[PUBLICATION ABSTRACT]

Amadori products (fructosamines)—ubiquitously occurring in nature—are precursors of the toxic and cell damaging ‘advanced glycation endproducts’; thus, it is not surprising that numerous organisms have developed systems to degrade such compounds. The deglycating enzymes differ with respect to their mechanisms as well as to their substrate specificities. Furthermore, different physiological functions are proposed for the different enzymes. The fructosamine 3-kinases of mammals and homologous proteins (fructosamine 3-kinase related proteins), which are common to all taxa, are thought to focus on intracellular repair functions. In contrast, in Bacillus subtilis and Escherichia coli, the cooperative action of a kinase and a deglycase facilitates Amadori degradation. As genes encoding these enzymes are co-transcribed with ABC transporter genes, it is thought that these genes facilitate the utilisation of extracellular Amadori products. Indeed, it has been shown that fructosamines can serve as the sole carbon and nitrogen sources. Here, we provide an overview of known deglycating systems with the emphasis on Amadori product degradation in bacteria.

Natural genetic competence renders bacteria able to take up and, in case there is sufficient homology to the recipient’s chromosome, integrate exogenously supplied DNA. Well studied in Bacillus subtilis , genetic competence is—in several aspects—known to be differently regulated in Bacillus licheniformis . We now report on the identification of a novel, chromosomally encoded homolog of a competence inhibitor in B. licheniformis (ComI) that has hitherto only been described as a plasmid borne trait in the ancestral B. subtilis NCIB3610. Bioinformatical analysis that included 80 Bacillus strains covering 20 different species revealed a ComI encoding gene in all of the examined B. licheniformis representatives, and was identified in few among the other species investigated. The predicted ComI of B.   licheniformis is a highly conserved peptide consisting of 28 amino acids. Since deletion of comI in B.   licheniformis DSM13 resulted in twofold increased transformation efficiency by genetic competence and overexpression resulted in threefold decreased transformability, the function as a competence inhibitor became evident.