Zwitterionic surface modification of polyethylene via atmospheric plasma-induced polymerization of (vinylbenzyl-)sulfobetaine and evaluation of antifouling properties.

Zwitterionic polymer brushes were grafted from bulk polyethylene (PE) by air plasma activation of the PE surface followed by radical polymerization of the zwitterionic styrene derivative (vinylbenzyl)sulfobetaine (VBSB). Successful formation of dense poly-(VBSB)-brush layers was confirmed by goniometry, IR spectroscopy, XPS and ToF-SIMS analysis. The resulting zwitterionic layers are about 50-100 nm thick and cause extremely low contact angles of 10° (water) on the material. Correspondingly we determined a high density of > 1.0 × 1016 solvent accessible zwitterions/cm2 (corresponding to 2,0 *10-8 mol/cm2) by a UV-based ion-exchange assay with crystal violet. The elemental composition as determined by XPS and characteristic absorption bands in the IR spectra confirmed the presence of zwitterionic sulfobetaine polymer brushes. The antifouling properties of the resulting materials were evaluated in a bacterial adhesion test against gram-positive bacteria (S. aureus). We observed significantly reduced cellular adhesion of the zwitterionic material compared to pristine PE. These microbiological tests were complemented by tests in natural seawater. During a test period of 21 days, confocal microscopy revealed excellent antifouling properties and confirmed the operating antifouling mechanism. The procedure reported herein allows the efficient surface modification of bulk PE with zwitterionic sulfobetaine polymer brushes via a scalable approach. The resulting modified PE retains important properties of the bulk material and has excellent and durable antifouling properties.

Cysteine: an overlooked energy and carbon source.

Biohybrids composed of microorganisms and nanoparticles have emerged as potential systems for bioenergy and high-value compound production from CO2 and light energy, yet the cellular and metabolic processes within the biological component of this system are still elusive. Here we dissect the biohybrid composed of the anaerobic acetogenic bacterium Moorella thermoacetica and cadmium sulphide nanoparticles (CdS) in terms of physiology, metabolism, enzymatics and transcriptomic profiling. Our analyses show that while the organism does not grow on L-cysteine, it is metabolized to acetate in the biohybrid system and this metabolism is independent of CdS or light. CdS cells have higher metabolic activity, despite an inhibitory effect of Cd2+ on key enzymes, because of an intracellular storage compound linked to arginine metabolism. We identify different routes how cysteine and its oxidized form can be innately metabolized by the model acetogen and what intracellular mechanisms are triggered by cysteine, cadmium or blue light.

Different Hydrophobins of Fusarium graminearum Are Involved in Hyphal Growth, Attachment, Water-Air Interface Penetration and Plant Infection.

Hydrophobins (HPs) are small secreted fungal proteins possibly involved in several processes such as formation of fungal aerial structures, attachment to hydrophobic surfaces, interaction with the environment and protection against the host defense system. The genome of the necrotrophic plant pathogen Fusarium graminearum contains five genes encoding for HPs (FgHyd1-5). Single and triple FgHyd mutants were produced and characterized. A reduced growth was observed when the ΔFghyd2 and the three triple mutants including the deletion of FgHyd2 were grown in complete or minimal medium. Surprisingly, the growth of these mutants was similar to wild-type when grown under ionic, osmotic or oxidative stress conditions. All the mutant strains confirmed the ability to develop conidia and perithecia, suggesting that the FgHyds are not involved in normal development of asexual and sexual structures. A reduction in the ability of hyphae to penetrate through the water-air interface was observed for the single mutants ΔFghyd2 and ΔFghyd3 as well as for the triple mutants including the deletion of FgHyd2 and FgHyd3. Besides, ΔFghyd3 and the triple mutant ΔFghyd234 were also affected in the attachment to hydrophobic surface. Indeed, wheat infection experiments showed a reduction of symptomatic spikelets for ΔFghyd2 and ΔFghyd3 and the triple mutants only when spray inoculation was performed. This result could be ascribed to the affected ability of mutants deleted of FgHyd2 and FgHyd3 to penetrate through the water-air interface and to attach to hydrophobic surfaces such as the spike tissue. This hypothesis is strengthened by a histological analysis, performed by fluorescence microscopy, showing no defects in the morphology of infection structures produced by mutant strains. Interestingly, triple hydrophobin mutants were significantly more inhibited than wild-type by the treatment with a systemic triazole fungicide, while no defects at the cell wall level were observed.

Molecular Keys to the Janthinobacterium and Duganella spp. Interaction with the Plant Pathogen Fusarium graminearum.

Janthinobacterium and Duganella are well-known for their antifungal effects. Surprisingly, almost nothing is known on molecular aspects involved in the close bacterium-fungus interaction. To better understand this interaction, we established the genomes of 11 Janthinobacterium and Duganella isolates in combination with phylogenetic and functional analyses of all publicly available genomes. Thereby, we identified a core and pan genome of 1058 and 23,628 genes. All strains encoded secondary metabolite gene clusters and chitinases, both possibly involved in fungal growth suppression. All but one strain carried a single gene cluster involved in the biosynthesis of alpha-hydroxyketone-like autoinducer molecules, designated JAI-1. Genome-wide RNA-seq studies employing the background of two isolates and the corresponding JAI-1 deficient strains identified a set of 45 QS-regulated genes in both isolates. Most regulated genes are characterized by a conserved sequence motif within the promoter region. Among the most strongly regulated genes were secondary metabolite and type VI secretion system gene clusters. Most intriguing, co-incubation studies of J. sp. HH102 or its corresponding JAI-1 synthase deletion mutant with the plant pathogen Fusarium graminearum provided first evidence of a QS-dependent interaction with this pathogen.

Posttranslational hypusination of the eukaryotic translation initiation factor-5A regulates Fusarium graminearum virulence.

Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A.

Disruption of the GABA shunt affects mitochondrial respiration and virulence in the cereal pathogen Fusarium graminearum.

The cereal pathogen Fusarium graminearum threatens food and feed production worldwide. It reduces the yield and poisons the remaining kernels with mycotoxins, notably deoxynivalenol (DON). We analyzed the importance of gamma-aminobutanoic acid (GABA) metabolism for the life cycle of this fungal pathogen. GABA metabolism in F. graminearum is partially regulated by the global nitrogen regulator AreA. Genetic disruption of the GABA shunt by deletion of two GABA transaminases renders the pathogen unable to utilize the plant stress metabolites GABA and putrescine. The mutants showed increased sensitivity against oxidative stress, GABA accumulation in the mycelium, downregulation of two key enzymes of the TCA cycle, disturbed potential gradient in the mitochondrial membrane and lower mitochondrial oxygen consumption. In contrast, addition of GABA to the wild type resulted in its rapid turnover and increased mitochondrial steady state oxygen consumption. GABA concentrations are highly upregulated in infected wheat tissues. We conclude that GABA is metabolized by the pathogen during infection increasing its energy production, whereas the mutants accumulate GABA intracellularly resulting in decreased energy production. Consequently, the GABA mutants are strongly reduced in virulence but, because of their DON production, are able to cross the rachis node.

The ATF/CREB transcription factor Atf1 is essential for full virulence, deoxynivalenol production, and stress tolerance in the cereal pathogen Fusarium graminearum.

Fusarium graminearum is a necrotrophic plant pathogen of cereals that produces mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA) in grains. The stress-activated mitogen-activated protein kinase FgOS-2 is a central regulator in F. graminearum and controls, among others, virulence and DON and ZEA production. Here, we characterized the ATF/CREB-activating transcription factor FgAtf1, a regulator that functions downstream of FgOS-2. We created deletion and overexpression mutants of Fgatf1, the latter being also in an FgOS-2 deletion mutant. FgAtf1 localizes to the nucleus and appears to interact with FgOS-2 under osmotic stress conditions. Deletion mutants in Fgatf1 (ΔFgatf1) are more sensitive to osmotic stress and less sensitive to oxidative stress compared with the wild type. Furthermore, sexual reproduction is delayed. ΔFgatf1 strains produced higher amounts of DON under in vitro induction conditions than that of the wild type. However, during wheat infection, DON production by ΔFgatf1 is strongly reduced. The ΔFgatf1 strains displayed strongly reduced virulence to wheat and maize. Interestingly, constitutive expression of Fgatf1 in the wild type led to hypervirulence on wheat, maize, and Brachypodium distachyon. Moreover, constitutive expression of Fgatf1 in the ΔFgOS-2 mutant background almost complements ΔFgOS-2-phenotypes. These data suggest that FgAtf1 may be the most important transcription factor regulated by FgOS-2.

CbCTB2, an O-methyltransferase is essential for biosynthesis of the phytotoxin cercosporin and infection of sugar beet by Cercospora beticola.

BACKGROUND: Cercospora leaf spot disease, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beets (Beta vulgaris) worldwide. Cercosporin, a light-inducible toxin, is essential for necrosis of the leaf tissue and development of the typical leaf spots on sugar beet leaves. RESULTS: In this study we show that the O-methyltransferase gene CTB2 is essential for cercosporin production and pathogenicity in two C. beticola isolates. We established a transformation system for C. beticola protoplasts, disrupted CTB2, and transformed the Δctb2 strains as well as a wild type strain with the DsRed reporter gene. The Δctb2 strains had lost their pigmentation and toxin measurements demonstrated that the Δctb2 strains were defective in cercosporin production. Infection of sugar beets with the wild type and Δctb2 DsRed strains showed that the deletion strain was severely impaired in plant infection. Histological analysis revealed that the CTB2-deficient isolate cannot enter the leaf tissue through stomata like the wild type. CONCLUSIONS: Taken together, these observations indicate that cercosporin has a dual function in sugar beet infection: in addition to the well-known role in tissue necrosis, the toxin is required for the early phase of sugar beet infection.

Lipase 8 affects the pathogenesis of Candida albicans.

The production of lipases can affect microbial fitness and virulence. We examined the role of the lipase 8 (LIP8) gene in the virulence of Candida albicans by constructing Deltalip8 strains by the URA-blaster disruption method. Reverse transcription-PCR experiments demonstrated the absence of LIP8 expression in the homozygous knockout mutants. Reconstituted strains and overexpression mutants were generated by introducing a LIP8 open reading frame under control of a constitutive actin promoter. Knockout mutants produced more mycelium, particularly at higher temperatures and pH >or=7. Diminished LIP8 expression resulted in reduced growth in lipid-containing media. Mutants deficient in the LIP8 gene were significantly less virulent in a murine intravenous infection model. The results clearly indicate that Lip8p is an important virulence factor of C. albicans.