Wild Strain Research Articles

Transposon-mediated genic rearrangements underlie variation in small RNA pathways

Transposable elements (TEs) can alter host gene structure and expression, whereas host organisms develop mechanisms to repress TE activities. In the nematode Caenorhabditis elegans , a small interfering RNA pathway dependent on the helicase ERI-6/7 primarily silences retrotransposons and recent genes of likely viral origin. By studying gene expression variation among wild C. elegans strains, we found that structural variants and transposon remnants likely underlie expression variation in eri-6/7 and the pathway targets. We further found that multiple insertions of the DNA transposons, Polintons, reshuffled the eri-6/7 locus and induced inversion of eri-6 in some wild strains. In the inverted configuration, gene function was previously shown to be repaired by unusual trans-splicing mediated by direct repeats. We identified that these direct repeats originated from terminal inverted repeats of Polintons . Our findings highlight the role of host-transposon interactions in driving rapid host genome diversification among natural populations and shed light on evolutionary novelty in genes and splicing mechanisms.

Construction and evaluation of a Salmonella Paratyphi A vaccine candidate based on a poxA gene mutation

Salmonella Paratyphi A, the pathogen of paratyphoid A accounts for an obviously growing proportion of cases in many areas. Therefore, development of specific paratyphoid A vaccines is needed. In the present study, the poxA gene of Salmonella Paratyphi A, encoding the aminoacyl-tRNA synthetase, was deleted successfully by the method of lambda Red recombination system, the resulting strain, ΔpoxA was characterized in respect of growth, adhesion and invasion, virulence, immunogenicity and protective efficacy. It was found that the growth of the ΔpoxA strain was significantly delayed compared with the wild type strain, the mutant ΔpoxA was less invasive to Caco-2 BBE epithelioid cells and THP-1 macrophages than the wild type strain, strain ΔpoxA was attenuated at least 1000-fold in mice, significant immune response and efficient protection were provided by the mutant ΔpoxA after oral immunization. It is concluded that the Salmonella Paratyphi A poxA deletion mutant ΔpoxA can be used as a live oral vaccine candidate against paratyphoid A.

The effect of Pseudomonas plecoglossicida vgrG gene on the immune response of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

Pseudomonas plecoglossicida is a pathogen that causes visceral white spot disease that results in significant financial losses. Valine-glycine repeat protein G (VgrG) is one of the core components that make up the spike structure in the type VI secretion system (T6SS), which transports effectors and then contributes to bacterial virulence. However, the specific effect of VgrG on bacterial infection and host response process has not been clearly explained. Herein, we compared the pathogenicity of vgrG gene-deleted (ΔvgrG) strain, vgrG gene-complemented (C-ΔvgrG) strain, and the wild-type (NZBD9) strain of P. plecoglossicida to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Compared to the wild strain, lost vgrG led to a higher survival rate, a lower bacterial load, and several mild pathological changes, such as fewer white nodules on the surface of the spleen and attenuated tissue damage. Moreover, the RNA-Seq of grouper spleen post-challenge indicated 3205 DEGs were involved in the two antibacterial responses, and the antigen presentation and processing pathway and the T-cell receptor signaling pathway were principal significant enrichment items. In summary, our data suggested that deletion of vgrG gene might reduce the pathogenicity of Pseudomonas plecoglossicida on hybrid grouper by making it less able to colonize the host and more easily cleared by the host immune system.

Evaluating cellular roles and phenotypes associated with trehalose degradation genes in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, 2 types of trehalase activities have been described. Neutral trehalases (Nth1 and Nth2) are considered to be the main proteins that catalyze intracellular trehalose mobilization. In addition to Nth1 and Nth2, studies have shown that acid trehalase Ath1 is required for extracellular trehalose degradation. Although both neutral and acid-type trehalases have been predominantly investigated in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. In this study, we constructed mutants of the trehalose degradation pathway (NTH1, NTH2, and ATH1) in 5 diverse S. cerevisiae strains to examine whether published lab strain phenotypes are also exhibited by wild strains. For each mutant, we assessed a number of phenotypes for comparison to trehalose biosynthesis mutants, including trehalose production, glycogen production, cell size, acute thermotolerance, high-temperature growth, sporulation efficiency, and growth on a variety of carbon sources in rich and minimal medium. We found that all trehalase mutants including single deletion nth1Δ, nth2Δ, and ath1Δ, as well as double deletion nth1nth2Δ, accumulated higher intracellular trehalose levels compared to their isogenic wild-type cells. Also, nth1Δ and nth1Δnth2Δ mutants exhibited mild thermal sensitivity, suggesting a potential minor role for trehalose mobilization when cells recover from stress. In addition, we evaluated phenotypes more directly relevant to trehalose degradation, including both extracellular and intracellular trehalose utilization. We discovered that intracellular trehalose hydrolysis is critical for typical spore germination progression, highlighting a role for trehalose in cell cycle regulation, likely as a storage carbohydrate providing glycolytic fuel. Additionally, our work provides further evidence suggesting Ath1 is indispensable for extracellular trehalose utilization as a carbon source, even in the presence of AGT1.

Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: Emergence of clinically important mutations in sphingolipid biosynthesis.

Invasive fungal infections are associated with high mortality, which is exacerbated by the limited antifungal drug armamentarium and increasing antifungal drug resistance. Echinocandins are a frontline antifungal drug class targeting β-glucan synthase (GS), a fungal cell wall biosynthetic enzyme. Echinocandin resistance is generally low but increasing in species like Candida glabrata, an opportunistic yeast pathogen colonizing human mucosal surfaces. Mutations in GS-encoding genes (FKS1 and FKS2 in C. glabrata) are strongly associated with clinical echinocandin failure, but epidemiological studies show that other, as yet unidentified factors also influence echinocandin susceptibility. Furthermore, although the gut is known to be an important reservoir for emergence of drug-resistant strains, the evolution of resistance is not well understood. Here, we studied the evolutionary dynamics of C. glabrata colonizing the gut of immunocompetent mice during treatment with caspofungin, a widely-used echinocandin. Whole genome and amplicon sequencing revealed rapid genetic diversification of this C. glabrata population during treatment and the emergence of both drug target (FKS2) and non-drug target mutations, the latter predominantly in the FEN1 gene encoding a fatty acid elongase functioning in sphingolipid biosynthesis. The fen1 mutants displayed high fitness in the gut specifically during caspofungin treatment and contained high levels of phytosphingosine, whereas genetic depletion of phytosphingosine by deletion of YPC1 gene hypersensitized the wild type strain to caspofungin and was epistatic to fen1Δ. Furthermore, high resolution imaging and mass spectrometry showed that reduced caspofungin susceptibility in fen1Δ cells was associated with reduced caspofungin binding to the plasma membrane. Finally, we identified several different fen1 mutations in clinical C. glabrata isolates, which phenocopied the fen1Δ mutant, causing reduced caspofungin susceptibility. These studies reveal new genetic and molecular determinants of clinical caspofungin susceptibility and illuminate the dynamic evolution of drug target and non-drug target mutations reducing echinocandin efficacy in patients colonized with C. glabrata.

Tandem mass spectral metabolic profiling of 54 actinobacterial strains and their 459 mutants

Natural products encompass a diverse range of compounds with high impact applications in consumer care, agriculture and most notably, therapeutics. However, despite the expansive chemical repertoire indicated in genomic information of microbes, only a small subset can be obtained under laboratory conditions. To increase accessible chemical space and realize Nature’s full chemical potential, a multi-pronged genetic- and cultivation-based strategy has been employed to activate and upregulate natural product biosyntheses in native and heterologous strains. This data descriptor documents a characterized collection of 2,138 liquid chromatography-tandem mass spectrometry (LC/MS-MS) spectra of fermentation extracts from 54 native actinobacterial strains collected from soil and marine environments in Singapore, and their 459 activated mutants in 3 to 5 media. A total of 743 unique metabolites have been identified, with the activated mutants demonstrating an approximately 2-fold expansion in accessible chemical space over wild type strains. Interrogating this expanded chemical diversity with cheminformatic tools can provide direction for the discovery of novel natural products with desirable functional activity.

The isolation of sclerotinin A as an anti-malarial compound by utilization of a global secondary metabolism regulator, laeA gene

Previously, we successfully introduced laeA gene into a fungal strain in order to significantly increase the production of a bioactive compound, allowing use to discover novel biological activity. To demonstrate the universal applicability of the laeA gene introduction strategy for taping the potential of fungal secondary metabolism, in this present study, we created a library of microorganisms which we had the laeA gene inserted, and from that library we aimed to isolate compounds which are produced at significantly greater quantities compared to the respective wild type strains. From this investigation, we were able to isolate sclerotinin A (1) from Pochonia sp. KTF-0504 strain. We revealed that 1 showed anti-malarial activity against Plasmodium falciparum parasite strains. On the other hands, 1 showed no anti-fungal activity against multidrug-sensitive budding yeast. Our study implies that the utilization of the laeA gene in fungi is a versatile method for the discovery of drug candidates.

Prevalence of Aleutian Mink Disease Virus (AMDV) in Free-Ranging American Mink from Biebrza and Narew National Parks (Poland)-An Epidemiological Concern.

Aleutian Mink Disease Virus (AMDV) is the causative agent of Aleutian disease (AD). This progressive and chronic disorder significantly impacts the mink breeding industry, affecting farmed and free-ranging American and European mink. This study investigated AMDV variants isolated from free-ranging American mink in northeastern Poland. Between 2018 and 2019, 26 spleen samples were collected from mink in Narew National Park (NNP) and Biebrza National Park (BNP). DNA was extracted and subjected to PCR to amplify the NS1 gene, followed by sequencing and phylogenetic analysis. The NS1 gene was detected in 50% of samples from NNP minks and in 30% of samples from BNP minks, with an overall prevalence of 42.31%; these findings align with global data and indicate serious ecological and health concerns. Ten closely related AMDV variants and one distinct variant were identified. The grouped variants exhibited high genetic homogeneity, closely related to strains found in mink from the USA, Germany, Greece, Latvia, and Poland; meanwhile, the distinct variant showed similarities to strains found in mink from Finland, Denmark, China, Poland, and Latvia, suggesting multiple infection sources. These findings, consistent with data from Polish mink farms, indicate significant genetic similarity between farmed and wild mink strains, suggesting potential bidirectional transmission. This underscores the importance of a One Health approach, emphasizing the interconnectedness of human, animal, and environmental health. Continuous surveillance and genetic studies are crucial for understanding AMDV dynamics and mitigating their impacts. Measures to reduce transmission between farmed and wild mink populations are vital for maintaining mink health and ecosystem stability.

Citric acid production by Brazilian garlic Aspergillus welwitschiae strains using orange residues

The global production of citrus fruits has expressively grown resulting in large amounts of residue. Orange residues are rich in carbon and could be used as substrates for production of biomolecules such as citric acid. In this study, wild and mutant Aspergillus welwitschiae strains isolated from Brazilian garlic were used to produce citric acid from orange residues, using solid-state fermentation. The results showed that the mutant A. welwitschiae UELAs 15.262/35 produced great amount of citric acid in the fourth day of solid-state fermentation using total and reducing sugars and producing cellulases. On the other hand, there was no production of citric acid by wild A. welwitschiae UELAs 15.262. The use of total and reducing sugars and the production of cellulases by A. welwitschiae UELAs 15.262 were detected, suggesting the use of carbon sources for the production of other metabolites. The great use of reducing sugars and cellulases production by A. welwitschiae UELAs 15.262/35 indicated the consume of carbon sources from the orange residue to produce citric acid at early times of fermentation. These findings lead to the possibility of biotechnological valorization of orange residues for acid organic production by A. welwitschiae strain.

Molecular mechanism of reduced biological fitness of fludioxonil-resistant strains of Botrytis cinerea based on transcriptome analysis.

Fludioxonil is a fungicide used to control gray mold. However, the frequency of resistance in the field is low, and highly resistant strains are rarely isolated. The biological fitness of the resistant strain is lower than that of the wild strain. Therefore, the molecular mechanism underlying the decrease in the fitness of the fludioxonil-resistant strain of Botrytis cinerea was explored to provide a theoretical basis for resistance monitoring and management. Transcriptome analysis was performed on five different-point mutant resistant strains of fludioxonil, focusing on mining and screening candidate genes that lead to reduced fitness of the resistant strains and the functional verification of these genes. The differentially expressed genes (DEGs) of the five point-mutation resistant strains intersected with 1869 DEGs. Enrichment analysis showed that three downregulated genes (Bcin05g07030, Bcgad1, and Bcin03g05840) were enriched in multiple metabolic pathways and were downregulated in both domesticated strains. Bcin05g07030 and Bcin03g05840 were involved in mycelial growth and development, pathogenicity, and conidial yield, and negatively regulated oxidative stress and cell wall synthesis. Bcgad1 was involved in mycelial growth and development, conidial yield, oxidative stress, and cell wall synthesis. Furthermore, Bcin05g07030 was involved in osmotic stress and spore germination, whereas Bcin03g05840 and Bcgad1 negatively regulated osmotic stress and cell wall integrity. These results enable us to further understand the molecular mechanism underlying the decrease in the biological fitness of B. cinerea fludioxonil-resistant strains. © 2024 Society of Chemical Industry.

Detection of Brucella S19 Vaccine Strain DNA in Domestic and Wild Ungulates from Brazilian Pantanal.

The Pantanal region, the largest floodplain in the world, has a huge biodiversity and is an important livestock center. Bovine brucellosis has been reported in the region over the last three decades, posing implications for cattle industry as well as for the maintenance of biodiversity. We aimed to investigate the presence of B. abortus S19 vaccine strain DNA in unvaccinated domestic and wild ungulates from the Brazilian Pantanal. Fifty-two heifers, 63 ovine, 24 domestic pigs, 28 feral pigs, and three Pampas deer were sampled. Brucella spp. was detected through bcsp31 PCR of blood samples in 45.3% (77/170) of the sampled animals, of which 36.4% (28/77) showed positivity in ery PCR corresponding to B. abortus S19 strain. Feral pigs presented the highest occurrence of positive samples in bcsp31 PCR (75%), followed by ovine (47.6%), domestic pigs (41.7%), and unvaccinated heifers (30.8%). We did not observe positivity in Pampas deer. Our results strongly suggest that vaccination against bovine brucellosis may promote spill-over of B. abortus S19 strain in the Pantanal region. Moreover, our data indicate that wild strains of Brucella circulates in the Pantanal Biome.

Superior osmotic stress tolerance in oilseed rape transformed with wild-type Rhizobium rhizogenes

Key messageNatural transformation with R. rhizogenes enhances osmotic stress tolerance in oilseed rape through increasing osmoregulation capacity, enhancing maintenance of hydraulic integrity and total antioxidant capacity.Transformation of plants using wild strains of agrobacteria is termed natural transformation and is not covered by GMO legislation in, e.g., European Union and Japan. In this study, offspring lines of Rhizobium rhizogenes naturally transformed oilseed rape (Brassica napus), i.e., A11 and B3 (termed root-inducing (Ri) lines), were investigated for osmotic stress resilience. Under polyethylene glycol 6000 (PEG) 10% (w/v)-induced osmotic stress, the Ri lines, particularly A11, had less severe leaf wilting, higher stomatal conductance (8.2 times more than WT), and a stable leaf transpiration rate (about 2.9 mmol m−2 s−1). Although the leaf relative water content and leaf water potential responded similarly to PEG treatment between the Ri lines and WT, a significant reduction of the turgid weight to dry weight ratio in A11 and B3 indicated a greater capacity of osmoregulation in the Ri lines. Moreover, the upregulation of plasma membrane intrinsic proteins genes (PIPs) in roots and downregulation of these genes in leaves of the Ri lines implied a better maintenance of hydraulic integrity in relation to the WT. Furthermore, the Ri lines had greater total antioxidant capacity (TAC) than the WT under PEG stress. Collectively, the enhanced tolerance of the Ri lines to PEG-induced osmotic stress could be attributed to the greater osmoregulation capacity, better maintenance of hydraulic integrity, and greater TAC than the WT. In addition, Ri-genes (particularly rolA and rolD) play roles in response to osmotic stress in Ri oilseed rape. This study reveals the potential of R. rhizogenes transformation for application in plant drought resilience.

Strategies to Maintain Redox Homeostasis in Yeast Cells with Impaired Fermentation-Dependent NADPH Generation.

Redox homeostasis is the balance between oxidation and reduction reactions. Its maintenance depends on glutathione, including its reduced and oxidized form, GSH/GSSG, which is the main intracellular redox buffer, but also on the nicotinamide adenine dinucleotide phosphate, including its reduced and oxidized form, NADPH/NADP+. Under conditions that enable yeast cells to undergo fermentative metabolism, the main source of NADPH is the pentose phosphate pathway. The lack of enzymes responsible for the production of NADPH has a significant impact on yeast cells. However, cells may compensate in different ways for impairments in NADPH synthesis, and the choice of compensation strategy has several consequences for cell functioning. The present study of this issue was based on isogenic mutants: Δzwf1, Δgnd1, Δald6, and the wild strain, as well as a comprehensive panel of molecular analyses such as the level of gene expression, protein content, and enzyme activity. The obtained results indicate that yeast cells compensate for the lack of enzymes responsible for the production of cytosolic NADPH by changing the content of selected proteins and/or their enzymatic activity. In turn, the cellular strategy used to compensate for them may affect cellular efficiency, and thus, the ability to grow or sensitivity to environmental acidification.

Synthesis, breast cancer activity, molecular docking and dynamic simulation of 1,4-Dihydropyridine derivatives

Breast cancer (BC) is the most prevalent cancer diagnosed in women, accounting for more than 1 in 10 new cancer diagnoses each year. Even though chemotherapy is successful in BC treatments, resistance to spreading these medications requires a new therapy. A new 1,4-Dihydropyridine (ZL1-6) series was synthesized and characterized using spectroscopic techniques such as FT-IR, 1H-NMR, 13C-NMR, and mass spectroscopy. The efficacy of these compounds against MFC7 breast cancer cells was assessed through a combination of in vitro, molecular docking, molecular dynamics, and MM-GBSA analyses. Notably, compound ZL4 exhibited promising activity against MFC7 cells (IC50 = 93.09 μg/ml), with a docking core of -6.728 kcal/mol and an MM-GBSA of -47.82 kcal/mol potentially involving the inhibition of EGFR as a mechanism of action. It is worth mentioning that these compounds were inactive against both E. Coli and S. Aureus wild strains. These findings provide a foundation for the development of innovative treatments for breast cancer.

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Performance and yield estimation of sugarcane varieties for pot still cachaça production

Cachaça is an alcoholic beverage obtained through the distillation of fermented sugarcane juice. It is the sugarcane distillate most consumed in Brazil. Despite the traditional nature and economic importance of cachaça, the production chain in Brazil is not technologically homogeneous; low-yielding varieties of sugarcane with susceptibility to various diseases are common. The pot still cachaças undergo delicate distillation processes in copper pot stills using wild yeast strains for fermentation, and the most exceptional and expensive cachaças can be found on the market in Brazil. The objective of this study was to assess modern sugarcane varieties for the production of pot still cachaça and to propose a new methodology to obtain the expected yield in liters of cachaça per hectare. A randomized block experimental design was used in a 4 (sugarcane varieties) × 3 (harvest times) factorial arrangement, with five replications. At the end of three crop seasons after harvest, the yield components, soluble solids content, and expected cachaça yield were evaluated. The highest levels of soluble solids (ºBrix) were for CTC4 and RB966928. Based on data from three crop seasons, the CTC9002 variety showed the highest stalk yield per hectare (SYH). The estimated yield methodology allowed for comprehensive assessment of the main sugarcane production characteristics, providing reliability and practicality in estimating cachaça yield. Based on analysis of yield components and mean cachaça yield (liters/ha) over three crop seasons, CTC9002 is recommended for inclusion in the panel of varieties for cachaça production.

Recent genetic drift in the co-diversified gut bacterial symbionts of laboratory mice.

Laboratory mice (Mus musculus domesticus) harbor gut bacterial strains that are distinct from those of wild mice1 but whose evolutionary histories are poorly understood. Understanding the divergence of laboratory-mouse gut microbiota (LGM) from wild-mouse gut microbiota (WGM) is critical, because LGM and WGM have been previously shown to differentially affect mouse immune-cell proliferation2,3, infection resistance4, cancer progression2, and ability to model drug outcomes for humans5. Here, we show that laboratory mice have retained gut bacterial symbiont lineages that diversified in parallel (co-diversified) with rodent species for > 25 million years, but that LGM strains of these ancestral symbionts have experienced accelerated accumulation of genetic load during the past ~ 120 years of captivity. Compared to closely related WGM strains, co-diversified LGM strains displayed significantly faster genome-wide rates of fixation of nonsynonymous mutations, indicating elevated genetic drift, a difference that was absent in non-co-diversified symbiont clades. Competition experiments in germ-free mice further indicated that LGM strains within co-diversified clades displayed significantly reduced fitness in vivo compared to WGM relatives to an extent not observed within non-co-diversified clades. Thus, stochastic processes (e.g., bottlenecks), not natural selection in the laboratory, have been the predominant evolutionary forces underlying divergence of co-diversified symbiont strains between laboratory and wild house mice. Our results show that gut bacterial lineages conserved in diverse rodent species have acquired novel mutational burdens in laboratory mice, providing an evolutionary rationale for restoring laboratory mice with wild gut bacterial strain diversity.

The relationship between resistance evolution and carbon metabolism in Staphylococcus xylosus under ceftiofur sodium stress.

Staphylococcus xylosus has emerged as a bovine mastitis pathogen with increasing drug resistance, resulting in substantial economic impacts. This study utilized iTRAQ analysis to investigate the mechanisms driving resistance evolution in S. xylosus under ceftiofur sodium stress. Findings revealed notable variations in the expression of 143 proteins, particularly glycolysis-related proteins (TpiA, Eno, GlpD, Ldh) and peptidoglycan (PG) hydrolase Atl. Following the induction of ceftiofur sodium resistance in S. xylosus, the emergence of resistant strains displaying characteristics of small colony variants (SCVs) was observed. The transcript levels of TpiA, Eno, GlpD and Ldh were up-regulated, TCA cycle proteins (ICDH, MDH) and Atl were down-regulated, lactate content was increased, and NADH concentration was decreased in SCV compared to the wild strain. That indicates a potential role of carbon metabolism, specifically PG hydrolysis, glycolysis, and the TCA cycle, in the development of resistance to ceftiofur sodium in S. xylosus.

Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas 'cutting' combined with homologous recombination of a 'repair' template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains, and rely on relatively low efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an E. coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo , which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic ( HIS3, URA3 ) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide for how to use this system.

A New Convenient Method to Assess Antibiotic Resistance and Antimicrobial Efficacy against Pathogenic Clostridioides difficile Biofilms.

Clostridioides difficile is a widely distributed anaerobic pathogen. C. difficile infection is a serious problem in healthcare. Its biofilms have been found to exhibit biocorrosivity, albeit very little, but sufficient for it to correlate with biofilm growth/health. This work demonstrated the use of a disposable electrochemical biofilm test kit using two solid-state electrodes (a 304 stainless steel working electrode, and a graphite counter electrode, which also served as the reference electrode) in a 10 mL serum vial. It was found that the C. difficile 630∆erm Adp-4 mutant had a minimum inhibitory concentration (MIC) for vancomycin twice that of the 630∆erm wild type strain in biofilm prevention (2 ppm vs. 1 ppm by mass) on 304 stainless steel. Glutaraldehyde, a commonly used hospital disinfectant, was found ineffective at 2% (w/w) for the prevention of C. difficile 630∆erm wild type biofilm formation, while tetrakis(hydroxymethyl)phosphonium sulfate (THPS) disinfectant was very effective at 100 ppm for both biofilm prevention and biofilm killing. These antimicrobial efficacy data were consistent with sessile cell count and biofilm imaging results. Furthermore, the test kit provided additional transient biocide treatment information. It showed that vancomycin killed C. difficile 630∆erm wild type biofilms in 2 d, while THPS only required minutes.