Minimal Medium Research Articles

Indigo carmine biodegradation by endophytic Microbacterium zeae K5: Enzymatic insights, degradation mechanism, and ecotoxicity analysis

The enormous amount of colored effluent release from dying industries into fresh and marine reservoirs, causing ecotoxicity and serious health problems. Pertaining to this, the current research emphasis on the decolorization and degradation treatment of Indigo carmine (IC). The decolorization of IC with endophytic Microbacteium zeae K5, isolated from the root of the Salix purpurea plant was studied. In a minimal salt medium with shaking, full dye decolorization (400 mg/L) was achieved within a 24 h incubation period. During dye degradation, the activities of enzymes such as laccase (12.02 U/g), manganese peroxidase (4.23 U/g), and quinone dehydrogenase (0.09 U/g) were also observed. The biodegradation of IC into isatin sulfonic acid and isatin was confirmed by UV–VIS spectrophotometry and GC-MS analysis of dye samples extracted with ethyl acetate. Finally, phytotoxicity studies revealed that the IC degraded metabolites toxicity was lower than that of the parent dye compound. The current study demonstrated isolate M. zeae K5 has ability to efficiently break down IC, indicating its potential for future bioremediation uses.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

Non-invasive VOCs detection to monitor the gut microbiota metabolism in-vitro

This work implemented a non-invasive volatile organic compounds (VOCs) monitoring approach to study how food components are metabolised by the gut microbiota in-vitro. The fermentability of a model food matrix rich in dietary fibre (oat bran), and a pure prebiotic (inulin), added to a minimal gut medium was compared by looking at global changes in the volatilome. The substrates were incubated with a stabilised human faecal inoculum over a 24-h period, and VOCs were monitored without interfering with biological processes. The fermentation was performed in nitrogen-filled vials, with controlled temperature, and tracked by automated headspace-solid-phase microextraction coupled with gas chromatography–mass spectrometry. To understand the molecular patterns over time, we applied a multivariate longitudinal statistical framework: repeated measurements—ANOVA simultaneous component analysis. The methodology was able to discriminate the studied groups by looking at VOCs temporal profiles. The volatilome showed a time-dependency that was more distinct after 12 h. Short to medium-chain fatty acids showed increased peak intensities, mainly for oat bran and for inulin, but with different kinetics. At the same time, alcohols, aldehydes, and esters showed distinct trends with discriminatory power. The proposed approach can be applied to study the intertwined pathways of gut microbiota food components interaction in-vitro.

Sexual Education for Children with Special Needs through Learning Media based on Universal Design for Learning

Sexual education is very important to be taught to young children, especially children with special needs. Sexual violence can intentionally occur to someone in a sexual manner. This can even extend to attempted rape, sadism in sexual relationships, other unwanted sexual activities, demeaning and hurting or injuring the victim. One effort to prevent sexual violence is to equip young children with knowledge, skills, and abilities to protect themselves from the threat of sexual violence. The method used is descriptive qualitative research. The subjects in this study are teachers in one Special School. Data collection in this study uses observation, interviews, and documentation techniques. Data analysis techniques use qualitative descriptive analysis techniques. The results of the study show that special teachers need learning media for more structured and systematic inclusive education according to the learning stage process. Until now, there is still minimal learning media used for sexual education, especially for children with special needs. Good learning media must have clear, communicative, concise, and systematic language use. The scope of the content in the learning media must also adjust to the conditions of students who will be given learning.

Growth on Douglas fir media facilitates Cryptococcus virulence factor production and enhances fungal survival against environmental and immune stressors.

The yeasts Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that can be isolated from the environment, including the surfaces of many plants. Cryptococcus gattii caused an outbreak on Vancouver Island, British Columbia beginning in 1999 that has since spread to the Pacific Northwest of the United States. Coastal Douglas fir (Pseudotsuga menziesii) is an important lumber species and a major component of the ecosystems in this area. Previous research has explored Cryptococcus survival and mating on Douglas fir plants and plant-derived material, but no studies have been done on the production of cryptococcal virulence factors by cells grown on those media. Here, we investigated the effects of growth on Douglas fir-derived media on the production of the polysaccharide capsule and melanin, two of the most important cryptococcal virulence factors. We found that while the capsule was mostly unchanged by growth in Douglas fir media compared to cells grown in defined minimal media, Cryptococcus spp. can use substrates present in Douglas fir to synthesize functional and protective melanin. These results suggest mechanisms by which Cryptococcus species may survive in the environment and emphasize the need to explore how association with Douglas fir trees could affect its epidemiology for human cryptococcosis.

Phosphate limitation enhances malic acid production on nitrogen-rich molasses with Ustilago trichophora

BackgroundAn important step in replacing petrochemical products with sustainable, cost-effective alternatives is the use of feedstocks other than, e.g., pure glucose in the fermentative production of platform chemicals. Ustilaginaceae offer the advantages of a wide substrate spectrum and naturally produce a versatile range of value-added compounds under nitrogen limitation. A promising candidate is the dicarboxylic acid malic acid, which may be applied as an acidulant in the food industry, a chelating agent in pharmaceuticals, or in biobased polymer production. However, fermentable residue streams from the food and agricultural industry with high nitrogen content, e.g., sugar beet molasses, are unsuited for processes with Ustilaginaceae, as they result in low product yields due to high biomass and low product formation.ResultsThis study uncovers challenges in evaluating complex feedstock applicability for microbial production processes, highlighting the role of secondary substrate limitations, internal storage molecules, and incomplete assimilation of these substrates. A microliter-scale screening method with online monitoring of microbial respiration was developed using malic acid production with Ustilago trichophora on molasses as an application example. Investigation into nitrogen, phosphate, sulphate, and magnesium limitations on a defined minimal medium demonstrated successful malic acid production under nitrogen and phosphate limitation. Furthermore, a reduction of nitrogen and phosphate in the elemental composition of U. trichophora was revealed under the respective secondary substrate limitation. These adaptive changes in combination with the intricate metabolic response hinder mathematical prediction of product formation and make the presented screening methodology for complex feedstocks imperative. In the next step, the screening was transferred to a molasses-based complex medium. It was determined that the organism assimilated only 25% and 50% of the elemental nitrogen and phosphorus present in molasses, respectively. Due to the overall low content of bioavailable phosphorus in molasses, the replacement of the state-of-the-art nitrogen limitation was shown to increase malic acid production by 65%.ConclusionThe identification of phosphate as a superior secondary substrate limitation for enhanced malic acid production opens up new opportunities for the effective utilization of molasses as a more sustainable and cost-effective substrate than, e.g., pure glucose for biobased platform chemical production.

Deciphering the Role of AMPK in Regulating Integrin Profile of Tumor-Derived Exosomes as a Potential Antimetastatic Strategy

Introduction: Cancer cell adapt their metabolic activity to survive in stressful environments with limited nutrients and oxygen. To sense and respond to nutrient cues, cells rely on a “fuel gauge” protein known as AMP-activated protein kinase (AMPK). While previous work has largely focused on AMPK’s role in cell metabolism, its role in metastasis is poorly defined. Interestingly, AMPK also regulates the membrane trafficking of integrins – key proteins in cell adhesion and migration. Recent studies have also shown that circulating integrins in exosomes are predictive of metastasis. Therefore, understanding the biology of AMPK signaling on the exosomal integrin profile not only allow for better cancer treatments but also help developing a potential predictive biomarker. I hypothesize that AMPK activation alters the integrin profile of exosomes in breast and endometrial cancer. Methods: Breast and endometrial cancer cells, MDA-MB-231 and HEC-1 respectively, will be cultured in full nutrient media conditions. 48 hours before exosome harvest, the media will be changed to minimal specialized media for extracellular vesicles (EV) collection. To induce AMPK activation, cells will be treated with limiting concentrations of glucose and glutamine, or with metabolic poisons 2-deoxyglucose (2-DG) and oligomycin. Total Exosome IsolationTM (TEI) solution will be used to enrich released exosomes that will be subjected to mass spectrometry to analyze the exosomal integrin profiles. Anticipated Results: Exosomes secreted from MDA-MB-231 and HEC-1 cells are expected to contain different exosomal integrin profile in both basal and induced AMPK activation conditions. It is anticipated that integrin subunits that promote metastatic and survival signaling such as αvβ5, α6β4, α6β1, will be upregulated in exosomes following AMPK activation. This could suggest tumor cells adapting to nutrient insufficiency by AMPK activation may present a more aggressive exosomal integrin profile to support cancer progression. Discussion: Understanding how metabolic cues such as nutrient insufficiency impact integrin content of tumour-derived exosomes can help identify novel metabolic drug targets to limit pro-survival and pro-metastatic signaling in primary breast tumors. Conclusion: This study investigates how AMPK activation modulates the exosomal integrin profile in breast and endometrial cancer cells, potentially uncovering predictive biomarkers and therapeutic targets for metastatic cancers.

Isolation and Molecular Characterization of Polyester-Degrading Bacterial Strains from Municipal Wastewater and Their Application in Microplastic Degradation in Contaminated Wastewater

Diverse synthetic polymers find their regular application in routine life and are disposed of directly into the environment through various sources. The released microplastic and microfibres from the sources creates contamination in both aquatic and terrestrial environments. When these micropollutants enter the food chain, they have adverse effects on the environment and human health. This study focuses on the application of native bacterial strains isolated for the effective degradation of polyester microfibers. The isolated bacterial strains, namely MFB 6, MFB 8, and MFB 11, tolerant to polyethylene glycol (PEG), were identified as Bacillus sp., Paenibacillus sp., and Aeromonas veronii, respectively, with NCBI gene bank accession numbers ON318306, ON318390, and PP237260. Polyester biodegradation efficiency of 10.5%, 12.6% and 8.4% was achieved by MFB 6, MFB 8 and MFB 11, respectively, after 42 days of investigation in minimal salt medium under shake flask condition. In future the application of these microorganisms will help in sustainable management of synthetic microfiber pollutants and find their suitable application in micro plastic degradation in contaminated wastewater.

A high-throughput assay identifies molecules with antimicrobial activity against persister cells.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Screening of Different Fungi Strains Gongronella sp. WICC F60 and Cordyceps sp. WICC F61 for Degradation of Low-Density Polyethylene

Over the past decade, polyethylene (PE) products have become increasingly important in several industries. Their biggest concerns are plastic material degradation resistance and environmental longevity. Therefore, this study aims to find fungi that are capable of degrading low-density polyethylene (LDPE) efficiently. This study uses Gongronella sp. WICC F60, and Cordyceps sp., WICC F61. The fungi were grown on potato dextrose agar for 30 days. Following that, fungal colonies were placed in potato dextrose broth for incubation. The LDPE sample was then cultured with the fungal strain in a minimum salt medium with a 10% inoculum. After 30 days of biodegradation testing, the analysis was done. These studies measured LDPE sample weight loss, pH, and fungal biomass. It was observed that the LDPE sample weight loss increased with time until the end of inclubation. Gongronella sp. WICC F60 and Cordyceps sp. WICC F61 were able to degrade LDPE plastic. Both fungi show an increase in LDPE weight loss. However, Cordyceps sp. WICC F61 shows more efficiency in LDPE degradation compared to Gongronella sp. WICC F60. The LDPE weight loss using Gongronella sp. WICC F60 was 1.07%, which is lower than Gongronella sp. WICC F60's LDPE weight reduction of 5.56%.

Investigation on uneven flow distribution in triply periodic minimal surface heat exchangers

The development in manufacturing engineering has made it possible to adopt the unique porous structure known as the triply periodic minimal surface to increase energy efficiency in a variety of energy-related sectors. With the unique morphological characteristics, triply periodic minimal surface exhibits remarkable mechanical strength and thermal performance, which makes it a potential candidate for compact, high-performance heat exchanger constructions. In this study, a theoretical model based on triply periodic minimal surface porous media is developed. Based on the hypothesis that the triply periodic minimal surface is isotropic and the heat transfer intensity of fluid and solid is uniform throughout the core of the heat exchanger, the simulation of the full-size heat exchanger is simplified by using a porous media model. The flow distribution characteristics of triply periodic minimal surface heat exchangers, as well as the effect mechanism of key factors, including the channel porosity, header configuration, the type of triply periodic minimal surface, the core length and Reynolds number on the flow distribution performance, are investigated. A dual-header configuration is proposed to improve the flow uniformity of triply periodic minimal surface heat exchangers. The results indicate that the flow distribution performance of triply periodic minimal surface based heat exchangers can be enhanced by the reduction of channel porosity. When the cold-side porosity is 0.7, the flow rate deviates from the mean value by 220% at its maximum for a Primitive heat exchanger. A dual-header configuration eliminates the columnar extreme high velocity space in the core and greatly reduces the “weak zone” of flow distribution, which improves the flow non-uniformity by 45–90% over the single-inlet headers. This paper provides data and theoretical support for the optimal design and practical application of the complex, high-performance triply periodic minimal surface based heat exchangers.

Robust, fully quantifiable and scalable bioprocess utilizing spent sulfite liquor with Corynebacterium glutamicum

In this study, a bioprocessing strategy was designed to valorize ultra-filtered spent sulfite liquor (UF-SSL) without prior detoxification steps as well as using it purely as a carbon source supplement to defined or complex media. Hence, a minimal medium for the bioconversion of UF-SSL with Corynebacterium glutamicum was developed and process robustness and reproducibility were validated. Process quantifiability was ensured by development of a biomass measurement technique for matrices with high water-insoluble solids and verified using elemental balancing. Mechanistic modeling based on Monod equations was used to identify batch kinetics. In a final step, scale-up of the developed process was performed to showcase process maturity towards commercialisation.

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named “LAbrini”, exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain “LAbrini” to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

Modes of action of Trichoderma virens Gl006 and Bacillus velezensis Bs006: decoding the arsenal of the microbial consortium

The consortium based on Trichoderma virens Gl006 and Bacillus velezensis Bs006 was selected in a previous study because the high effectiveness against Fusarium wilt of cape gooseberry (Fusarium oxysporum f. sp. physali—Foph). The compatibility among the strains was determined previously, but the modes of action exerted by the consortium remained unknown. In this study, the modes of action displayed by the Gl006 and Bs006 consortium against the Foph strain Map5 were determined and compared with the modes of action of the single microorganisms. Production of cell wall degrading enzymes (CWDE), cyclic lipopeptides (CLPs) by Bs006 and gliotoxin by Gl006, and fusaric acid (FA) production by Foph were evaluated in the co-culture of the strains in several culture media. Furthermore, the survival of the strains in the soil, the colonization of plant tissues and the induction of systemic responses through a split root model bioassay were evaluated. While Bs006 showed protease, β-1,3-glucanase, and chitobiosidase enzymatic activities, and production of CLPs, Gl006 showed β-N-acetylglucosaminidase, chitobiosidase, total chitinase, β-1,3-glucanase, and protease activities. All the metabolites produced by the single strains were also found in the consortium, but in a culture medium dependent manner. The FA was found in the experiment on the interaction of the consortium with Foph Map5 in the minimal basal medium and in potato dextrose broth. The population of Foph was reduced by the consortium in vitro, under the synthetic media that favored the production of all the CWDE tested. In soil conditions, the consortium reduced the population of Foph by 63% in a synergistic way. Bs006 was recovered from the inner tissues of the plant, where Gl006 prevented the entry of Foph to the roots and reduced the incidence of the disease under the split root model. These results suggest that the consortium of Gl006 and Bs006 displays modes of action through CWDE and CLP´s and indirectly through induction of systemic resistance, which could act complementary since neither of them were enhanced in consortium. Here we characterized a consortium capable of reducing the population of Foph in soil and control the disease in a synergistic way.

Glyphosate-Induced Phosphonatase Operons in Soil Bacteria of the Genus Achromobacter.

Achromobacter insolitus and Achromobacter aegrifaciens, bacterial degraders of the herbicide glyphosate, were found to induce phosphonatase (phosphonoacetaldehyde hydrolase, EC 3.11.1.1) when grown on minimal media with glyphosate as the sole source of phosphorus. The phosphonatases of the strains were purified to an electrophoretically homogeneous state and characterized. The enzymes differed in their kinetic characteristics and some other parameters from the previously described phosphonatases. The phosphonatase of A. insolitus was first revealed to separate into two stable forms, which had similar kinetic characteristics but interacted differently with affinity and ion-exchange resins. The genomes of the investigated bacteria were sequenced. The phosphonatase genes were identified, and their context was determined: the bacteria were shown to have gene clusters, which, besides the phosphonatase operon, included genes for LysR-type transcription activator (substrate sensor) and putative iron-containing oxygenase PhnHD homologous to monooxygenases PhnY and TmpB of marine organophosphonate degraders. Genes of 2-aminoethylphosphonate aminotransferase (PhnW, EC 2.6.1.37) were absent in the achromobacterial phosphonatase operons; instead, we revealed the presence of genes encoding the putative flavin oxidase HpnW. In silico simulation showed 1-hydroxy-2-aminoethylphosphonate to be the most likely substrate of the new monooxygenase, and a number of glycine derivatives structurally similar to glyphosate to be substrates of flavin oxidase.

Evaluating lignin degradation under limited oxygen conditions by bacterial isolates from forest soil

Lignin, a heterogeneous aromatic polymer present in plant biomass, is intertwined with cellulose and hemicellulose fibrils, posing challenges to its effective utilization due to its phenolic nature and recalcitrance to degradation. In this study, three lignin utilizing bacteria, Klebsiella sp. LEA1, Pseudomonas sp. LEA2, and Burkholderia sp. LEA3, were isolated from deciduous forest soil samples in Nan province, Thailand. These isolates were capable of growing on alkali lignin and various lignin-associated monomers at 40 °C under microaerobic conditions. The presence of Cu2+ significantly enhanced guaiacol oxidation in Klebsiella sp. LEA1 and Pseudomonas sp. LEA2. Lignin-related monomers and intermediates such as 2,6-dimethoxyphenol, 4-vinyl guaiacol, 4-hydroxybenzoic acid, benzoic acid, catechol, and succinic acid were detected mostly during the late stage of incubation of Klebsiella sp. LEA1 and Pseudomonas sp. LEA2 in lignin minimal salt media via GC–MS analysis. The intermediates identified from Klebsiella sp. LEA1 degradation suggested that conversion and utilization occurred through the β-ketoadipate (ortho-cleavage) pathway under limited oxygen conditions. The ability of these bacteria to thrive on alkaline lignin and produce various lignin-related intermediates under limited oxygen conditions suggests their potential utility in oxygen-limited processes and the production of renewable chemicals from plant biomass.

Regulation of nitrogen utilization and mycotoxin biosynthesis by the GATA transcription factor AaAreA in Alternaria alternata.

Alternaria alternata is a prevalent postharvest pathogen that generates diverse mycotoxins, notably alternariol (AOH) and alternariol monomethyl ether (AME), which are recurrent severe contaminants. Nitrogen sources modulate fungal growth, development, and secondary metabolism, including mycotoxin production. The GATA transcription factor AreA regulates nitrogen source utilization. However, little is known about its involvement in the regulation of nitrogen utilization in A. alternata. To examine the regulatory mechanism of AaAreA on AOH and AME biosynthesis in A. alternata, we analyzed the impact of diverse nitrogen sources on the fungal growth, conidiation and mycotoxin production. The use of a secondary nitrogen source (NaNO3) enhanced mycelial elongation and sporulation more than the use of a primary source (NH4Cl). NaNO3 favored greater mycotoxin accumulation than did NH4Cl. The regulatory roles of AaAreA were further clarified through gene knockout. The absence of AaAreA led to an overall reduction in growth in minimal media containing any nitrogen source except NH4Cl. AaAreA positively regulates mycotoxin biosynthesis when both NH4Cl and NaNO3 are used as nitrogen sources. Subcellular localization analysis revealed abundant nuclear transport when NaNO3 was the sole nitrogen source. The regulatory pathway of AaAreA was systematically revealed through comprehensive transcriptomic analyses. The deletion of AaAreA significantly impedes the transcription of mycotoxin biosynthetic genes, including aohR, pksI and omtI. The interaction between AaAreA and aohR, a pathway-specific transcription factor gene, demonstrated that AaAreA binds to the aohR promoter sequence (5'-GGCTATGGAAA-3'), activating its transcription. The expressed AohR regulates the expression of downstream synthase genes in the cluster, ultimately impacting mycotoxin production. This study provides valuable information to further understand how AreA regulates AOH and AME biosynthesis in A. alternata, thereby enabling the effective design of control measures for mycotoxin contamination.

Optimizing in vitro spherulation cues in the fungal pathogen Coccidioides.

Coccidioides spp . are part of a group of thermally dimorphic fungal pathogens, which grow as filamentous cells (hyphae) in the soil and transform to a different morphology upon inhalation into the host. The Coccidioides host form, the spherule, is unique and highly under characterized due to both technical and biocontainment challenges. Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremost technical challenges has been reliably growing spherules in culture without the formation of contaminating hyphae, and consistently inducing endospore release from spherules. Here, we present optimization of in vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, using freshly-harvested arthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal media to test spherule growth on various carbon and nitrogen sources. We defined a critical role for the dispersant Tamol in both early spherule formation and prevention of the accumulation of a visible film around spherules. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization of this virulence-relevant morphology.

Laboratory horror stories: Poison in the agars.

The fission yeast Schizosaccharomyces pombe is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our two laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar, from different suppliers, were toxic. Interestingly, the inhibitory effect was attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.

Improvement in Salt Tolerance Ability of Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is a popular platform for bioremediation due to its robust tolerance to environmental stress and strong biodegradation capacity. Limited research on the salt tolerance of P. putida KT2440 has hindered its application. In this study, the strain KT2440 was tested to tolerate a maximum of 4% w/v NaCl cultured with minimal salts medium. Transcriptomic data in a high-salinity environment showed significant expression changes in genes in membrane components, redox processes, chemotaxis, and cellular catabolic processes. betB-encoding betaine-aldehyde dehydrogenase was identified from the transcriptome data to overexpress and enhance growth profile of the strain KT2440 in minimal salts medium containing 4% w/v NaCl. Meanwhile, screening for exogenous salt-tolerant genes revealed that the Na+/H+ antiporter EcnhaA from Escherichia coli significantly increased the growth of the strain KT2440 in 4% w/v NaCl. Then, co-expression of EcnhaA and betB (KT2440-EcnhaA-betB) increased the maximum salt tolerance of strain KT2440 to 5% w/v NaCl. Further addition of betaine and proline improved the salt tolerance of the engineered strain to 6% w/v NaCl. Finally, the engineered strain KT2440-EcnhaA-betB was able to degrade 56.70% of benzoic acid and 95.64% of protocatechuic acid in minimal salt medium containing 4% w/v NaCl in 48 h, while no biodegradation was observed in the normal strain KT2440 in the same conditions. However, the strain KT2440-EcnhaA-betB failed to degrade catechol in minimal salt medium containing 3% w/v NaCl. This study illustrated the improvement in the salt tolerance performance of Pseudomonas putida KT2440 and the feasibility of engineered strain KT2440 as a potential salt-tolerant bioremediation platform.