Friedhelm Meinhardt

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.

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.

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.

Restriction modification systems (R-M systems), consisting of a restriction endonuclease and a cognate methyltransferase, constitute an effective means of a cell to protect itself from foreign DNA. Identification, characterization, and deletion of the restriction modification system BliMSI, a putative isoschizomer of ClaI from Caryophanon latum, were performed in the wild isolate Bacillus licheniformis MS1. BliMSI was produced as recombinant protein in Escherichia coli, purified, and in vitro analysis demonstrated identical restriction endonuclease activity as for ClaI. A recombinant E. coli strain, expressing the heterologous bliMSIM gene, was constructed and used as the host for in vivo methylation of plasmids prior to their introduction into B. licheniformis to improve transformation efficiencies. The establishment of suicide plasmids in the latter was rendered possible. The subsequent deletion of the restriction endonuclease encoding gene, bliMSIR, caused doubled transformation efficiencies in the respective mutant B. licheniformis MS2 (∆bliMSIR). Along with above in vivo methylation, the establishment of further gene deletions (∆upp, ∆yqfD) was performed. The constructed triple mutant (∆bliMSIR, ∆upp, ∆yqfD) enables rapid genome manipulation, a requirement for genetic engineering of industrially important strains.

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.

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.

Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA(Gln) and tRNA(Glu) respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S-phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate-limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage.

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.

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. The rather wide-spread and extremely A/T rich yeast virus like elements (VLEs, also termed linear plasmids) which encode toxic anticodon nucleases (ACNases) ensure autoselection in the cytoplasm by preventing functional nuclear capture of the cognate immunity genes, but how? When expressed in the nucleus, the mRNA of the VLE immunity genes is split into fragments to which poly(A) tails are added. Consistently, lowering the A/T content by gene synthesis prevented transcript cleavage and permitted functional nuclear expression providing full immunity against the respective ACNase toxin. Thus, internal poly(A) cleavage is likely to prevent functional nuclear immunity gene expression.