Optimization of glycerol-based medium composition for antifungal metabolites production by Bacillus subtilis

Abstract Bacillus species are promising producers of various compounds that have pronounced antimicrobial, antiviral and antitumor activities. Due to its GRAS status, Bacillus subtilis represents an excellent candidate for the usage in plant pathogens biocontrol. In this research, evaluation of antifungal metabolites biosynthesis by Bacillus subtilis ATCC 6633 and optimization of glycerol-based medium composition, using response surface methodology, for the production of compounds effective against Neurospora crassa were investigated. The results of disc-diffusion method indicate that applied Bacillus strain produces compounds with antifungal activity against tested fungus. In order to find optimal cultivation medium composition, the experiments were carried out in accordance with Box-Behnken design, and optimization was performed using the concept of desirability function combined with previously defined mathematical equation, which describes examined bioprocess. The optimization model predicts that maximum inhibition zone diameter against Neurospora crassa of 32.24 mm is achieved when initial content of glycerol, NaNO2 and K2HPO4 were 49.68 g/L, 2.90 g/L and 6.49 g/L, respectively. Additionally, the second optimization set was made to minimize the consumption of medium components and costs of medium preparation. The obtained results are the basis for further research aimed to develop medium appropriate for economically justified production of bioactive compounds at industrial scale.

Genome-wide screening of predicted sugar transporters in Neurospora crassa and the application in hexose fermentation by Saccharomyces cerevisiae / 生物工程学报

The lignocellulolytic filamentous fungus Neurospora crassa is able to assimilate various mono- and oligo-saccharides. However, more than half of predicted sugar transporters in the genome are still waiting for functional elucidation. In this study, system analysis of substrate spectra of predicted sugar transporters in N. crassa was performed at genome-wide level. NCU01868 and NCU08152 have the capability of uptaking various hexose, which are named as NcHXT-1 and NcHXT-2 respectively. Their transport activities for glucose were further confirmed by fluorescence resonance energy transfer analysis. Over-expression of either NcHXT-1 or NcHXT-2 in the null-hexose-transporter yeast EBY.VW4000 restored the growth and ethanol fermentation under submerged fermentation with glucose, galactose, or mannose as the sole carbon source. NcHXT-1/-2 homologues were found in a variety of cellulolytic fungi. Functional identification of two filamentous fungal-conserved hexose transporters NcHXT-1/-2 via genome scanning would represent novel targets for ongoing efforts in engineering cellulolytic fungi and hexose fermentation in yeast.

RNA-Seq, and ye shall find: Sexual-stage-specific A-to-I RNA editing in fungi.

Fusarium graminearum, a pathogen of wheat and barley, is a haploid homothallic ascomycete filamentous fungus (Goswami and Kistler 2004). It overwinters as saprophytic hyphae in plant debris and undergoes the sexual cycle in spring to produce fruiting bodies (perithecia) bearing the progeny ascospores. The genome sequence of the F. graminearum PH-1 strain was reported last year (King et al. 2015). This year, Jin-Rong Xu and colleagues in Northwest A&F University, China, and Purdue University, USA, re-sequenced the PH-1 genome and also performed RNA-Seq analysis on two independent biological replicates each of RNA from conidia, hyphae, and 8-day post-fertilization perithecia (Liu et al. 2016). Alignment of the two replicate perithecial RNA-Seq reads with the reference genome sequence revealed 23,041 and 19,764 single-nucleotide variants (SNVs), of which, respectively, 22,578 and 19,261 corresponded to A (adenosine)- to-G (guanosine) transitions, and 17,613 A-to-G transitions were common to both the replicates. Non- A-to-G variants were far fewer (463 and 503) and only 35.9% were common between the two perithecial replicates, suggesting that the non-A-to-G variants were false-positives. In sum, 26,056 A-to-G variants were identified as putative A-to-I RNA editing sites at which hydrolytic deamination at the C6 position of the purine ring of A produces I (inosine). Since I preferentially base-pairs with C (cytidine), an I within a transcript is read as G by the translation machinery. Also, during reverse transcription, I directs the incorporation of C; thus, it appears as a G in double-stranded cDNA. The conidial and hyphal RNA-Seq data showed only 68 and 112 A-to-G transitions and 335 and 452 non-A-to-G conversions, indicating that the A-to-I RNA editing is specific to the sexual stage. Xu and colleagues had initially set out to do a yeast two-hybrid experiment to identify proteins that interact with a protein kinase named Puk1 (perithecium unique kinase 1). Ascospores from the puk1 mutant have an abnormal morphology. Additionally, qRT-PCR showed that PUK1 transcription is markedly upregulated in perithecia, suggesting that PUK1 expression and function might be restricted to the sexual stage. Therefore, to generate the PUK1 ORF bait, they synthesized cDNA using RNA isolated from perithecial cultures of the PH-1 strain. Sequencing of the construct revealed that two tandem stop codons – UAG UAG – in the PUK1 ORF were changed to UGG UGG in the cDNA, presumably via A-to-I RNA editing. More than 90% of PUK1 reads in perithecial RNA-Seq showed the A-to-I editing, and experiments with site-specific mutant alleles showed that the editing was essential for PUK1 function. This was an unexpected discovery because fungi lack orthologs of the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes that in metazoans converts A to I in double-stranded RNA. Presumably, A-to-I RNA editing in fungi uses different enzymes than animals. This discovery motivated Xu and colleagues to expand the search for RNA editing genome-wide in transcriptomes from vegetative and sexual-stage tissues (conidia, hyphae, and perithecia). The percentage of reads with the A-to-G variant was taken as the RNA editing level at the site. Editing levels varied from 3% to 100%. Strikingly, 47% of genes bearing sites with editing levels >60% tended to be up-regulated or specifically expressed in perithecia compared to conidia and hyphae. A majority of the editing events resulted in amino acid substitutions, which suggested that Ato- I editing might be important for adaptation of protein functions during sexual reproduction. Editing events similar to those in PUK1 were found 69 other genes, including the rid (RIP defective) ortholog and genes important for meiosis (see below). All these genes displayed UAG-to-UGG change in exons that automated annotation had incorrectly predicted as introns.

Biodegradation of erythrosin B dye by paramorphic Neurospora crassa 74A

The present work used paramorphic forms of Neurospora crassa 74A to remove erythrosine. The fungus culture was grown in medium containing the dye, as only carbon source for 2 and 90 h of interaction. A washing process using distilled water isolated the cellular mass mycelia was dried for 12 h at 105ºC and transformed in fine powder and analyzed in FTIR. The supernatant was analyzed through spectrophotometer UV-Vis and FTIR. Significant differences in the spectrum of UV-VIS and FTIR were observed between the control and the supernatant and between wall control and the walls colored by red, in FTIR for 2 and 90 h. Some significant bands were modified, suggesting the possibility of enzymatic biodegradation in proportion to the time of contact between the dye and fungal biomass.

O presente trabalho utilizou formas paramorfogênicas de Neurospora crassa 74A linhagens, na remoção do corante "Erythrosin B". O fungo, induzido química e fisicamente em forma de "pellets", foi usado no estudo da biodegradação deste corante. A cultura fúngica foi crescida em meio contendo o corante, como única fonte de carbono por 2 e 90h de interação. As paredes celulares foram isoladas por um processo de lavagem em água destilada e o micélio fresco foi secado por 12 h a 105ºC, e transformado num pó fino, e analisado em FTIR. O sobrenadante foi analisado através de espectrofotômetro UV-VIS e FTIR. Diferenças significativas no espectro UV-VIS e no FTIR foram observadas entre o controle e o sobrenadante e entre o controle e as paredes coloridas de vermelho e em FTIR no tempo de 2 e 90 h. Algumas bandas foram modificadas sugerindo a possibilidade de uma biodegradação enzimática em função do tempo de contato entre o corante e a biomassa fúngica.

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents / Alteraciones de crecimiento y ramificación en Neurospora crassa provocadas por concentraciones subinhibitorias de agentes antimicóticos

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 μg/ml inhibited radial growth more than fluconazole at 5.0 μg/ml while amphotericin B at 0.05 μg/ml was more effective than nystatin at 0.05 μg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, α = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment /through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Se investigó el efecto de seis agentes antimicóticos en concentraciones subinhibitorias sobre el crecimiento y la ramificación en Neurospora crassa. El agente azólico ketoconazol a la concentración de 0,5 μg/ml inhibió el crecimiento radial más que el fluconazol a 5,0 μg/ml, y la anfotericina B a 0,05 μg/ ml fue más eficiente que 0,05 μg/ml de nistatina, entre los agentes poliénicos usados. En presencia de griseofulvina, ketoconazol y terbinafina a las concentraciones establecidas se observaron alteraciones morfológicas en las hifas. Los agentes antimicóticos fueron más eficientes sobre el crecimiento vegetativo que sobre la germinación conidial. La terbinafina redujo marcadamente (54,89%) la longitud de la unidad de crecimiento y provocó la hiperramificación del micelio. En todos los casos, existió gran correlación entre la longitud y el número de ápices de las hifas (r > 0,9). Todos los resultados mostraron diferencias altamente significativas de acuerdo con ANOVA (p < 0,001, α = 0,05). Considerando que el ápice de la hifa es la principal interfase entre el hongo y su ambiente, a través de la cual las enzimas y las toxinas son secretadas y los nutrientes son absorbidos, un micelio hiperramificado resultante de dosis ineficientes de agentes antimicóticos sería perjudicial.

Neurospora crassa mat A-2 and mat A-3 proteins weakly interact in the yeast two-hybrid system and affect yeast growth

Mating-type genes control the entry into the sexual cycle, mating identity and sexual development in fungi. The mat A-2 and mat A-3 genes, present in the mat A idiomorph of the filamentous fungus Neurospora crassa, are required for post-fertilization functions but are not essential for mating identity. Their putative roles as transcription factors are based on the similarity of mat A-2 with the Podospora anserina SMR1 gene and an HMG motif present in the mat A-3 gene. In this work the yeast two-hybrid system was used to identify transcriptional activity and protein-protein interaction of N. crassa mat A-2 and mat A-3 genes. We observed that the mat A-3 protein alone is capable of weakly activating transcription of yeast reporter genes; it also binds with low specificity to the GAL1 promoter sequence, possibly due to its HMG domain. Our results also indicate that mat A-3 is capable to form homodimers, and interact with mat A-2. Interference on yeast growth was observed on some transformants suggesting a toxic action of the mat A-2 protein. Our data on pattern of interactions of mat proteins contributes towards understanding the control of vegetative and sexual cycles in filamentous fungi.

SCREENING OF STRAINS PRODUCING EXTRACELLULAR MELANIN / 微生物学通报

The growth and pigment production of 47 strains on casein medium were studied comparatively.T4 and Neurospora crassa AS 3.1602 were selected for their capacity of producing melanin on five different culture media. Melanin produced by T4 was investigated, and T4 was identified as Proteus mirabilis primarily.

An enzymic method for the determination of chitin and chitosan in fungal cell walls.

The free and N-acetyl glucosamine contents, serving as a measure of the amounts of chitosan and chitin respectively, were determined in the chitinase hydrolysates of the cell wall of a wild strain of Neurospora crassa. Chitinase, obtained from cultures of Serratia marcescens, could hydrolyse the cell wall completely apart from being capable of hydrolysing preparations of chitin and chitosan. The free and N-acetyl glucosamines, released by chitinase hydrolysis, were determined by a modified Morgan-Elson reaction carried out in the presence and absence of acetic anhydride. The method is capable of estimating chitin and chitosan contents in as little as 100 μg of cell wall material.

Carbon starvation mediated changes in carbohydrate metabolism in Neurospora crassa.

Carbon starvation conditions were found to increase the activities of gluconeogenic enzymes such as malic enzyme, cytosolic malate dehydrogenase and isocitrate lyase along with proteases and inhibition in glucose catabolic enzymes such as G6P dehydrogenase and FDP aldolase in Neurospora crassa.

Excretion of pyruvate in nickel toxicity in wild type and Ni2 + resistant mutants of Neurospora crassa.

The parent wild strain Neurospora crassa Em 5297a and three Ni2+ resistant Neurospora crassa mutants have been shown to excrete pyruvate into the culture medium in Ni2+ and Co2 + toxicities. Ni2+ has a more pronounced effect in this regard. The excretion is progressive with growth inhibition and is abolished by Mg2+ in all strains and by Fe3+ partially in the Em strain but not in Neurospora crassa NiR1. Pyruvate, citrate and malate supplementation reverse growth inhibition caused by excess Ni2+, but with concomitant suppression of Ni2+ accumulation. It is suggested that one of the features of Ni2+ toxicity in Neurospora crassa is a derangement in carbohydrate metabolism at step(s) beyond pyruvate and that this is possibly due to decreased in vivo activity of Mg2+ dependent processes.

A new repressible alkaline phosphatase in Neurospora crassa EM 5297a.

Neurospora crassa Em 5297a can utilize sodium β-glycerophosphate as a sole phosphorous source (in the place of KH2PO4). Under these conditions a repressible alkaline phosphatase is elaborated which has different pH optimum towards β-glycerophosphate (10.2) and pyrophosphate (9.0) as substrates. This enzyme does not require any metal ion for its activity and could be assayed in the presence of EDTA. However, under conditions of cobalt toxicity, the activity of this enzyme is high and is decreased in copper and nickel toxicities.

Antifungal activity of bacitracin and its interaction with metals.

Bacitracin was more growth-inhibitory to Neurospora crassa on a minimal magnesium medium than on a normal magnesium-medium. Both magnesium and manganese were able to counteract the growth inhibition. The antifungal activity of bacitracin was potentiated by zinc. Potassium could not counteract the growth inhibition by this antibiotic. The mycelial magnesium levels were low in bacitracin-inhibited cultures.

Cobalt transport in a cobalt-resistant strain of Neurospora crassa.

Uptake of Co2+ by cobalt-resistant strain is dependent on Co2+ concentration in the medium and is linear with time. The uptake is unaffected by metabolic inhibitors and decreased at low pH values. The uptake is independent of temperature in the range 0–40° C. The transport system is a passive diffusion process, unlike in the parent wild type strain where it is energy-dependent. It is possible that Mg2+ transport system is not involved in Co2+ transport in this strain, since the Co2+ uptake is not suppressed by Mg2+ as in the parent strain.

The effect of copper on histidine biosynthesis in Neurospora crassa.

The inhibition of growth of a wild strain of Neurospora crassa by Cu2+ is counteracted by histidine, histidine methyl ester, histidinol and Mn2+. In the presence of Cu2+, the total free amino acid content decreased by 30%. The decreased free amino acid pools of arginine, histidine and tyrosine were restored on the addition of Mn2+. Histidinol phosphate phosphatase showed a decrease in activity in the presence of Cu2+. This inhibition was reversed on the addition of excess Mn2+.. The data suggest that copper toxicity in the mould is due to suppression of histidine biosynthesis.