Null Mutant Strain Research Articles

The Ustilago maydis null mutant strains of the RNA-binding protein UmRrm75 accumulate hydrogen peroxide and melanin

Ustilago maydis is a dimorphic fungus that has emerged as a model organism for the study of fungal phytopathogenicity and RNA biology. In a previous study, we isolated the U. maydis UmRrm75 gene. The deletion of the UmRrm75 gene affected morphogenesis and pathogenicity. UmRrm75 gene encodes a protein containing three RNA recognition motifs. Here we determined that UmRrm75 has chaperone activity in Escherichia coli using the transcription anti-termination assay. Subsequently, we analyzed the growth of ΔUmRrm75 mutants at 15 °C and 37 °C, observing that mutant strains had reduced growth in comparison to parental strains. UmRrm75 gene expression was induced under these non-optimal temperatures. ΔUmRrm75 mutant colonies displayed a dark-brown color at 28 °C, which was confirmed to be melanin based on spectroscopic analysis and spectrometric data. Furthermore, ΔUmRrm75 mutant strains showed the presence of peroxisomes, and increased H2O2 levels, even at 28 °C. The ΔUmRrm75 mutant strains displayed a higher expression of redox-sensor UmYap1 gene and increased catalase activity than the parental strains. Our data show that deletion of the UmRrm75 gene results in higher levels of H2O2, increased melanin content, and abiotic stress sensitivity.

An Ssd1 Homolog Impacts Trehalose and Chitin Biosynthesis and Contributes to Virulence in Aspergillus fumigatus.

Regulation of fungal cell wall biosynthesis is critical to maintain cell wall integrity in dynamic fungal infection microenvironments. Genes involved in this response that impact fungal fitness and host immune responses remain to be fully defined. In this study, we observed that a yeast ssd1 homolog, ssdA, in the filamentous fungus Aspergillus fumigatus is involved in trehalose and cell wall homeostasis. An ssdA null mutant strain exhibited an increase in trehalose levels and a reduction in fungal colony growth rate. In contrast, overexpression of ssdA perturbed trehalose biosynthesis and reduced germination of conidia. The ssdA null mutant strain was more resistant to cell wall-perturbing agents, while overexpression of ssdA increased sensitivity. Overexpression of ssdA significantly increased chitin levels, and both loss and overexpression of ssdA altered subcellular localization of the class V chitin synthase CsmA. Strikingly, overexpression of ssdA abolished adherence to abiotic surfaces and severely attenuated the virulence of A. fumigatus in a murine model of invasive pulmonary aspergillosis. Despite the severe in vitro fitness defects observed upon loss of ssdA, neither surface adherence nor murine survival was impacted. In conclusion, A. fumigatus SsdA plays a critical role in cell wall homeostasis impacting A. fumigatus-host interactions.IMPORTANCE The incidence of life-threatening infections caused by the filamentous fungus Aspergillus fumigatus is increasing along with an increase in the number of fungal strains resistant to contemporary antifungal therapies. The fungal cell wall and the associated carbohydrates required for its synthesis and maintenance are attractive drug targets given that many genes encoding proteins involved in cell wall biosynthesis and integrity are absent in humans. Importantly, genes and associated cell wall biosynthesis and homeostasis regulatory pathways remain to be fully defined in A. fumigatus In this report, we identify SsdA as an important component of trehalose and fungal cell wall biosynthesis in A. fumigatus that consequently impacts the host immune response and fungal virulence in animal models of infection.

The mitochondrial citrate carrier in Yarrowia lipolytica: Its identification, characterization and functional significance for the production of citric acid

Mitochondrial citrate carrier plays a central role in exporting acetyl-CoA in the form of citrate from mitochondria to cytosol thereby connecting carbohydrate catabolism and lipogenesis. In this study, Yarrowia lipolytica mitochondrial citrate carrier was functionally defined and characterized. Firstly, deletion of Y. lipolytica YlCTP1 and YlYHM2 genes coding putative tricarboxylate mitochondrial carriers were performed. ΔYlctp1 strain did not differ significantly from wild type strain in terms of growth rate, organic acids and lipid production. In contrast, ΔYlyhm2 strain did not grow in liquid citrate-containing minimal medium. Moreover, in glucose-containing lipogenic medium YlYHM2 null mutant strain did not produce citric acid; the production of isocitric acid and lipids were decreased. Reintroduction of YlYHM2 gene as well as heterologous expression of Aspergillus niger gene AnYHM2 into ΔYlyhm2 strain restored the growth in minimal citrate medium and even enhanced citric acid production by 45% in both variants compared with wild type strain during test tube cultivation. Mitochondrial extracts isolated from YlYHM2 null mutant and wild type strain were incorporated into liposomes; citrate/citrate and α-ketoglutarate/α-ketoglutarate homoexchange activities were reduced by 87% and 40% in ΔYlyhm2 strain, respectively, compared with the wild type, whereas citratein/α-ketoglutarateout and α-ketoglutaratein/citrateout heteroexchanges were decreased by 87% and 95%, respectively. YlYhm2p was expressed in Escherichia coli, purified and reconstituted into liposomes. Besides high efficiency to citrate and α-ketoglutarate transport, YlYhm2p also transported oxaloacetate, succinate, fumarate, and to a much lesser extent, aconitate, malate, isocitrate, oxoadipate, and glutamate. The activity of reconstituted YlYhm2p was inhibited strongly by SH-blocking reagents, pyridoxal-5′-phosphate, and partly by N-ethylmaleimide. Co-expression of YlYHM2 and adenosine monophosphate deaminase YlAMPD genes resulted in the production of 49.7 g/L of citric acid during test tube cultivation, whereas wild type strain accumulated 30.1 g/L of citric acid. Large-scale cultivation in bioreactor of the engineered strain resulted in 97.1 g/L of citric acid production with a process selectivity of 94.2% and an overall citric acid yield of 0.5 g/g. The maximal specific rate of citric acid synthesis was 0.93 g/L/h. Therefore, the physiological role of YlYhm2p in glucose-containing medium is to catalyze both import of citrate into mitochondria for catabolic reactions and export of citrate as a source of acetyl-CoA from mitochondria. Possible shuttles for citrate exporting are discussed. Moreover, for the first time evidence has been given for the improvement of TCA cycle intermediate production by manipulation of a gene coding a mitochondrial carrier.

Interplay between small RNA pathways shapes chromatin landscapes in C. elegans.

The nematode Caenorhabditis elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both ‘silencing’ siRNAs bound by Worm-specific Argonautes (WAGO) and ‘activating’ siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here, we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partially rescued null mutant strain (WM193), this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements and an increased number of small RNAs that match enhancers in both drh-3 and csr-1 mutants. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to sites with increased silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

Epimedium flavonoids mitigate proteotoxicity and extend healthspan via DAF-16 inC. elegans

Objective: Epimedium flavonoids (EF), the raw extract of medicinal herb Epimedium, have been shown to have broad beneficial effects in the elderly including those with neurodegeneration. The goal of this study is to investigate whether EF is protective against proteotoxicity and whether it extends healthspan in C. elegans.Methods: Animals were treated with EF supplemented in the growth medium. Two C. elegans models of human proteotoxic disease, CL4176 expressing an aggregated amyloid-[Formula: see text] (1–42) peptide (A[Formula: see text]1–42) and AM140 expressing a polyglutamine (polyQ) protein, were exploited to test the anti-proteotoxicity of EF. Proteotoxicity-induced paralysis in CL4176 and AM140 was evaluated. Lifespan, stress resistance, and locomotion were tested in wild-type N2 C. elegans. Lifespan assays were also performed in CF1038, a daf-16 null mutant strain. DAF-16 nuclear translocation was analyzed in TJ356, a strain expressing a functional DAF-16::GFP fusion protein. The mRNA levels of downstream targets of DAF-16 were measured by qPCR.Results: EF significantly reduced A[Formula: see text]1–42- and polyQ-induced paralysis in CL4176 and AM140, indicating the anti-proteotoxic potency of EF. EF significantly extended the lifespan and promoted stress resistance and locomotion in N2, demonstrating a healthspan extension effect of EF. DAF-16 nuclear localization and its downstream targets, sod-3 and hsp-16.2 mRNA levels, were significantly elevated with EF. EF did not increase the lifespan of daf-16 null mutant CF1038, revealing a DAF-16-dependent mechanism of EF effect on lifespan.Conclusion: We found that EF, a natural extract from a widely used medicinal herb Epimedium, protects against proteotoxicity and extends the healthspan via DAF-16 in C. elegans. Our work may provide molecular insights into the clinical benefits of EF and Epimedium.

Length doesn't matter-telomere damage triggers cellular senescence in the ageing heart.

Telomere shortening induces cellular senescence in proliferative cells. Yet, it is presently unclear how it is triggered in post‐mitotic cells such as cardiac myocytes. A new study by Anderson et al (2019) reports that during ageing of the heart, cellular senescence develops independently of telomere length, but is evoked by DNA damage, which preferentially accumulates at the telomere. Removal of senescent cells using senolytic drugs ameliorated cardiac hypertrophy and fibrosis and may inform novel approaches to improve the conditions for the ageing heart.

Iron homeostasis in Mycobacterium tuberculosis is essential for persistence

Tuberculosis, caused by the obligate intracellular pathogen Mycobacterium tuberculosis (Mtb), is responsible for 2–3 million deaths annually worldwide. Intracellular adaptability, which is critical for long-term persistence, requires the pathogen to neutralize host-mediated insults. The iron–sulphur (Fe–S) cofactor is essential for many enzymes critical for such ‘adaptation’. The Mtb genome harbors only one putative iron–sulphur cluster (ISC) operon (rv1460-66) predicted to be involved in the generation of the Fe–S cofactor. Except for rv1460, all other genes in this operon are anticipated to be essential. The current study investigated the role of rv1460, an sufR homologue of Mtb (sufRTB), in maintaining intracellular Fe homeostasis and its implications on mycobacterial pathogenesis. We found that Mtb ISC locus (rv1461–66) was transcribed as a single multigene transcript. We successfully generated the sufRTB null mutant strain (ΔsufRTB) of Mtb, suggesting nonessentiality of the gene under normal growth conditions. The mutant strain demonstrated enhanced biofilm generation and failed to grow under a low-Fe condition. Growth characterization studies indicated that SufRTB-mediated intracellular Fe homeostasis is essential for Mtb to persist in the host. Targeting mycobacterial persistence by inhibiting SufRTB protein activity may be a novel intervention strategy in tuberculosis treatment.

NanI Sialidase Is an Important Contributor to Clostridium perfringens Type F Strain F4969 Intestinal Colonization in Mice.

Clostridium perfringens type F (formerly enterotoxigenic C. perfringens type A) strains produce an enterotoxin (CPE) to cause acute cases of food poisoning and chronic nonfoodborne human gastrointestinal diseases (NFD), e.g., antibiotic-associated diarrhea (AAD). NFD strains also produce NanI sialidase, an extracellular enzyme that releases sialic acids from sialyated host macromolecules. Recent in vitro studies suggested that NanI may contribute to NFD strain intestinal colonization by enhancing the adherence of such strains to intestinal cells and promoting their bacterial growth using generated sialic acid as an energy source. The current study tested this hypothesis by developing a mouse intestinal colonization model involving clindamycin pretreatment to produce conditions mimicking those during AAD. In this model, the type F NFD strain F4969 persisted for at least 4 days in the small intestine, cecum, and colon. When clindamycin-pretreated mice were challenged by oral gavage with equivalent numbers of F4969 bacteria or its isogenic nanI null mutant, significantly lower numbers of the nanI mutant were recovered from all intestinal segments, and it was completely cleared from the small intestine by day 4. Complementation of the mutant to restore NanI production also promoted colonization. When the same nanI null mutant strain was coinoculated into the mouse model together with a nanI-producing strain, the numbers of this mutant were restored to wild-type F4969 levels in all intestinal segments. This result suggests that sialidases produced by other bacteria might also provide some support for C. perfringens intestinal colonization. Collectively, these in vivo findings identify NanI to be the first known significant contributor to chronic intestinal colonization by NFD strains.

CORT0C04210 is required for Candida orthopsilosis adhesion to human buccal cells

Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis sensu lato species complex. C. orthopsilosis annotated genome harbors 3 putative agglutinin-like sequence (ALS) genes named CORT0B00800, CORT0C04210 and CORT0C04220. The aim of this study was to investigate the role played by CORT0C04210 (CoALS4210) in the virulence and pathogenicity of this opportunistic yeast. Heterozygous and null mutant strains lacking one or both copies of CoALS4210 were obtained using the SAT1-flipper cassette strategy and were characterized in in vitro, ex vivo and in vivo models. While no differences between the mutant and the wild-type strains were observed in in vitro growth or in the ability to undergo morphogenesis, the CoALS4210 null mutant showed an impaired adhesion to human buccal epithelial cells compared to heterozygous and wild type strains. When the pathogenicity of CoALS4210 mutant and wild type strains was evaluated in a murine model of systemic candidiasis, no statistically significant differences were observed in fungal burden of target organs. Since gene disruption could alter chromatin structure and influence transcriptional regulation of other genes, two independent CRISPR/Cas9 edited mutant strains were generated in the same genetic background used to create the deleted strains. CoALS4210-edited strains were tested for their in vitro growing ability, and compared with the deleted strain for adhesion ability to human buccal epithelial cells. The results obtained confirmed a reduction in the adhesion ability of C. orthopsilosis edited strains to buccal cells. These findings provide the first evidence that CRISPR/Cas9 can be successfully used in C. orthopsilosis and demonstrate that CoALS4210 plays a direct role in the adhesion of C. orthopsilosis to human buccal cells but is not primarily involved in the onset of disseminated candidiasis.

Cor interacts with outer membrane proteins to exclude FhuA-dependent phages

Superinfection exclusion (Sie) of FhuA-dependent phages is carried out by Cor in the Escherichia coli mEp167 prophage lysogenic strain. In this work, we present evidence that Cor is an outer membrane (OM) lipoprotein that requires the participation of additional outer membrane proteins (OMPs) to exclude FhuA-dependent phages. Two Cor species of ~13 and ~8.5kDa, corresponding to the preprolipoprotein/prolipoprotein and lipoprotein, were observed by Western blot. Cell mutants for CorC17F, CorA18D and CorA57E lost the Sie phenotype for FhuA-dependent phages. A copurification affinity binding assay combined with LC_ESI_MS/MS showed that Cor bound to OMPs: OmpA, OmpC, OmpF, OmpW, LamB, and Slp. Interestingly, Sie for FhuA-dependent phages was reduced on Cor overexpressing FhuA+ mutant strains, where ompA, ompC, ompF, ompW, lamB, fhuE, genes were knocked out. The exclusion was restored when these strains were supplemented with plasmids expressing these genes. Sie was not lost in other Cor overexpressing FhuA+ null mutant strains JW3938(btuB-), JW5100(tolB-), JW3474(slp-). These results indicate that Cor interacts and requires some OMPs to exclude FhuA-dependent phages.

The F1Fo-ATP Synthase β Subunit Is Required for Candida albicans Pathogenicity Due to Its Role in Carbon Flexibility.

Previous work has explored link between mitochondrial biology and fungal pathogenicity in F1Fo-ATP synthase in Candida albicans. In this work we have detailed the more specific roles of the F1Fo-ATP synthase β subunit, a key protein subunit of F1Fo-ATP synthase. The ability to assimilate alternative carbons in glucose-limited host niches is known to be a critical factor for infection caused by opportunistic pathogens including C. albicans. The function of the F1Fo-ATP synthase β subunit was characterized through the construction of an ATP2 gene null mutant (atp2Δ/Δ) and the gene-reconstituted strain (atp2Δ/ATP2) in order to understand the link between carbon metabolism and C. albicans pathogenesis. Cell growth, viability, cellular ATP content, mitochondrial membrane potential (ΔΨm), and intracellular ROS were compared between null mutant and control strain. Results showed that growth of the atp2Δ/Δ mutant in synthetic medium was slower than in complex medium. However, the synthetic medium delayed the onset of reduced cell viability and kept cellular ATP content from becoming fully depleted. Consistent with these observations, we identified transcriptional changes in metabolic response that activated other ATP-generating pathways, thereby improving cell viability during the initial phase. Unlike glucose effects, the atp2Δ/Δ mutant exhibited an immediate and sharp reduction in cell viability on non-fermentable carbon sources, consistent with an immediate depletion of cellular ATP content. Along with a reduced viability in non-fermentable carbon sources, the atp2Δ/Δ mutant displayed avirulence in a murine model of disseminated candidiasis as well as lower fungal loads in mouse organs. Regardless of the medium, however, a decrease in mitochondrial membrane potential (ΔΨm) was found in the atp2Δ/Δ mutant but ROS levels remained in the normal range. These results suggest that the F1Fo-ATP synthase β subunit is required for C. albicans pathogenicity and operates by affecting metabolic flexibility in carbon consumption.

Impact of veA on the development, aggressiveness, dissemination and secondary metabolism of Penicillium expansum.

Penicillium expansum, the causal agent of blue mould disease, produces the mycotoxins patulin and citrinin amongst other secondary metabolites. Secondary metabolism is associated with fungal development, which responds to numerous biotic and abiotic external triggers. The global transcription factor VeA plays a key role in the coordination of secondary metabolism and differentiation processes in many fungal species. The specific role of VeA in P.expansum remains unknown. A null mutant PeΔveA strain and a complemented PeΔveA:veA strain were generated in P.expansum and their pathogenicity on apples was studied. Like the wild-type and the complemented strains, the null mutant PeΔveA strain was still able to sporulate and to colonize apples, but at a lower rate. However, it could not form coremia either invitro or invivo, thus limiting its dissemination from natural substrates. The impact of veA on the expression of genes encoding proteins involved in the production of patulin, citrinin and other secondary metabolites was evaluated. The disruption of veA drastically reduced the production of patulin and citrinin on synthetic media, associated with a marked down-regulation of all genes involved in the biosynthesis of the two mycotoxins. Moreover, the null mutant PeΔveA strain was unable to produce patulin on apples. The analysis of gene expression revealed a global impact on secondary metabolism, as 15 of 35 backbone genes showed differential regulation on two different media. These findings support the hypothesis that VeA contributes to the pathogenicity of P.expansum and modulates its secondary metabolism.

Proteomic analysis of a Candida albicans pir32 null strain reveals proteins involved in adhesion, filamentation and virulence.

We have previously characterized Pir32, a Candia albicans cell wall protein that we found to be involved in filamentation, virulence, chitin deposition, and resistance to oxidative stress. Other than defining the cell shape, the cell wall is critical for the interaction with the surrounding environment and the point of contact and interaction with the host surface. In this study, we applied tandem mass spectrometry combined with bioinformatics to investigate cell wall proteome changes in a pir32 null strain. A total of 16 and 25 proteins were identified exclusively in the null mutant strains grown under non-filamentous and filamentous conditions. These proteins included members of the PGA family with various functions, lipase and the protease involved in virulence, superoxide dismutases required for resisting oxidative stress, alongside proteins required for cell wall remodeling and synthesis such as Ssr1, Xog1, Dfg5 and Dcw1. In addition proteins needed for filamentation like Cdc42, Ssu81 and Ucf1, and other virulence proteins such as Als3, Rbt5, and Csa2 were also detected. The detection of these proteins in the mutant and their lack of detection in the wild type can explain the differential phenotypes previously observed.

Biofilm Formation by the Acidophile Bacterium Acidithiobacillus thiooxidans Involves c-di-GMP Pathway and Pel exopolysaccharide.

Acidophile bacteria belonging to the Acidithiobacillus genus are pivotal players for the bioleaching of metallic values such as copper. Cell adherence to ores and biofilm formation, mediated by the production of extracellular polymeric substances, strongly favors bioleaching activity. In recent years, the second messenger cyclic diguanylate (c-di-GMP) has emerged as a central regulator for biofilm formation in bacteria. C-di-GMP pathways have been reported in different Acidithiobacillus species; however, c-di-GMP effectors and signal transduction networks are still largely uncharacterized in these extremophile species. Here we investigated Pel exopolysaccharide and its role in biofilm formation by sulfur-oxidizing species Acidithiobacillus thiooxidans. We identified 39 open reading frames (ORFs) encoding proteins involved in c-di-GMP metabolism and signal transduction, including the c-di-GMP effector protein PelD, a structural component of the biosynthesis apparatus for Pel exopolysaccharide production. We found that intracellular c-di-GMP concentrations and transcription levels of pel genes were higher in At. thiooxidans biofilm cells compared to planktonic ones. By developing an At. thiooxidans ΔpelD null-mutant strain we revealed that Pel exopolysaccharide is involved in biofilm structure and development. Further studies are still necessary to understand how Pel biosynthesis is regulated in Acidithiobacillus species, nevertheless these results represent the first characterization of a c-di-GMP effector protein involved in biofilm formation by acidophile species.

Investigation of Candida parapsilosis virulence regulatory factors during host-pathogen interaction

Invasive candidiasis is among the most life-threatening infections in patients in intensive care units. Although Candida albicans is the leading cause of candidaemia, the incidence of Candida parapsilosis infections is also rising, particularly among the neonates. Due to differences in their biology, these species employ different antifungal resistance and virulence mechanisms and also induce dissimilar immune responses. Previously, it has been suggested that core virulence effecting transcription regulators could be attractive ligands for future antifungal drugs. Although the virulence regulatory mechanisms of C. albicans are well studied, less is known about similar mechanisms in C. parapsilosis. In order to search for potential targets for future antifungal drugs against this species, we analyzed the fungal transcriptome during host-pathogen interaction using an in vitro infection model. Selected genes with high expression levels were further examined through their respective null mutant strains, under conditions that mimic the host environment or influence pathogenicity. As a result, we identified several mutants with relevant pathogenicity affecting phenotypes. During the study we highlight three potentially tractable signaling regulators that influence C. parapsilosis pathogenicity in distinct mechanisms. During infection, CPAR2_100540 is responsible for nutrient acquisition, CPAR2_200390 for cell wall assembly and morphology switching and CPAR2_303700 for fungal viability.

Functional Characterization of TvCyt2, a Member of the p450 Monooxygenases From Trichoderma virens Relevant During the Association With Plants and Mycoparasitism.

Secondary metabolites are crucial for the establishment of interactions between plants and microbes, as in the case of Trichoderma-plant interactions. In the biosynthetic pathway of secondary metabolites, specific enzymes participate in the formation of hydroxyl and epoxy groups, belonging to the p450 monooxygenases family. Here, we show that the product of the gene TvCyt2 from Trichoderma virens encodes a new protein homologous to the cytochrome p450, which is down-regulated at the beginning of Trichoderma-Arabidopsis interaction. To investigate its role in the interactions established by Trichoderma spp., we analyzed the metabolic profile obtained from the overexpressing (OETvCyt2) and null mutant (Δtvcyt2) strains, observing that the OETvCyt2 strains produce a higher concentration of some metabolites than the wild-type (WT) strain. Δtvcyt2 strains showed a decreased antagonistic activity against Rhizoctonia solani in antibiosis assays. Arabidopsis plants cocultivated with the OETvCyt2 strains showed stronger induction of systemic acquired resistance than plants cocultivated with the WT strain, as well as increases in biomass and fitness. Our data suggest that the product of the TvCyt2 gene is involved in secondary metabolite biosynthesis, which can increase antagonistic activity with phytopathogenic fungi and the capacity to promote plant growth.

Growth Inhibition and DNA Damage Induced by X-Phenols in Yeast: A Quantitative Structure-Activity Relationship Study.

Phenolic compounds and their derivatives are ubiquitous constituents of numerous synthetic and natural chemicals that exist in the environment. Their toxicity is mostly attributed to their hydrophobicity and/or the formation of free radicals. In a continuation of the study of phenolic toxicity in a systematic manner, we have examined the biological responses of Saccharomyces cerevisiae to a series of mostly monosubstituted phenols utilizing a quantitative structure–activity relationship (QSAR) approach. The biological end points included a growth assay that determines the levels of growth inhibition induced by the phenols as well as a yeast deletion (DEL) assay that assesses the ability of X-phenols to induce DNA damage or DNA breaks. The QSAR analysis of cell growth patterns determined by IC50 and IC80 values indicates that toxicity is delineated by a hydrophobic, parabolic model. The DEL assay was then utilized to detect genomic deletions in yeast. The increase in the genotoxicity was enhanced by the electrophilicity of the phenolic substituents that were strong electron donors as well as by minimal hydrophobicity. The electrophilicities are represented by Brown’s sigma plus values that are a variant of the Hammett sigma constants. A few mutant strains of genes involved in DNA repair were separately exposed to 2,6-di-tert-butyl-4-methyl-phenol (BHT) and butylated hydroxy anisole (BHA). They were subsequently screened for growth phenotypes. BHA-induced growth defects in most of the DNA repair null mutant strains, whereas BHT was unresponsive.

Molecular Regulatory Network Involved in Biofilm Structure Development by <i>Acidithiobacillus</i> <i>thiooxidans</i> Includes Pel Exopolysaccharide Machinery

The Acidithiobacillus genus plays a relevant role in bioleaching. The molecular understanding of biofilm formation has been pointed out to design biological strategies to improve the efficiency of this industrial process and to prevent environmental damages caused by acid mine/rock drainages. In Acidithiobacillus spp., the molecular mechanisms involved in biofilm formation are currently emerging. The second messenger cyclic diguanylate (c-di-GMP) appears as a key player for biofilm formation by Acidithiobacillus sp. Here, results obtained from genomic analysis to characterize c-di-GMP pathway in At. thiooxidans are reported. Intracellular levels of c-di-GMP have been previously measured and data indicated that they are higher in adhered cells than planktonic ones. During the course of characterization of c-di-GMP effectors, a complete pel-like gene cluster has been identified in At. thiooxidans. By using total RNA obtained from planktonic and adhered sulfur-grown cells, transcriptomic analysis revealed that pelA belonging to the pel-like gene cluster is overexpressed in adhered cells. Moreover, genetic experiments were performed to compare wild type and null-mutant strains of At. thiooxidans for assessing the role of Pel exopolysaccharide. All together, the results obtained suggest a specific role for Pel machinery in the attachment to solid energy substrates by At. thiooxidans.

Construction of a fur null mutant and RNA-sequencing provide deeper global understanding of the Aliivibrio salmonicida Fur regulon.

BackgroundThe ferric uptake regulator (Fur) is a transcription factor and the main regulator of iron acquisition in prokaryotes. When bound to ferric iron, Fur recognizes its DNA binding site and generally executes its function by repressing transcription of its target genes. Due to its importance in virulence, the Fur regulon is well studied for several model bacteria. In our previous work, we used computational predictions and microarray to gain insights into Fur-regulation in Aliivibrio salmonicida, and have identified a number of genes and operons that appear to be under direct control of Fur. To provide a more accurate and deeper global understanding of the biological role of Fur we have now generated an A. salmonicida fur knock-out strain and used RNA-sequencing to compare gene expression between the wild-type and fur null mutant strains.ResultsAn A. salmonicida fur null mutant strain was constructed. Biological assays demonstrate that deletion of fur results in loss of fitness, with reduced growth rates, and reduced abilities to withstand low-iron conditions, and oxidative stress. When comparing expression levels in the wild-type and the fur null mutant we retrieved 296 differentially expressed genes distributed among 18 of 21 functional classes of genes. A gene cluster encoding biosynthesis of the siderophore bisucaberin represented the highest up-regulated genes in the fur null mutant. Other highly up-regulated genes all encode proteins important for iron acquisition. Potential targets for the RyhB sRNA was predicted from the list of down-regulated genes, and significant complementarities were found between RyhB and mRNAs of the fur, sodB, cysN and VSAL_I0422 genes. Other sRNAs with potential functions in iron homeostasis were identified.ConclusionThe present work provides by far the most comprehensive and deepest understanding of the Fur regulon in A. salmonicida to date. Our data also contribute to a better understanding of how Fur plays a key role in iron homeostasis in bacteria in general, and help to show how Fur orchestrates iron uptake when iron levels are extremely low.

Balanced CoQ6 biosynthesis is required for lifespan and mitophagy in yeast.

Coenzyme Q is an essential lipid with redox capacity that is present in all organisms. In yeast its biosynthesis depends on a multiprotein complex in which Coq7 protein has both catalytic and regulatory functions. Coq7 modulates CoQ6 levels through a phosphorylation cycle, where dephosphorylation of three amino acids (Ser/Thr) by the mitochondrial phosphatase Ptc7 increases the levels of CoQ6. Here we analyzed the role of Ptc7 and the phosphorylation state of Coq7 in yeast mitochondrial function. The conversion of the three Ser/Thr to alanine led to a permanently active form of Coq7 that caused a 2.5-fold increase of CoQ6 levels, albeit decreased mitochondrial respiratory chain activity and oxidative stress resistance capacity. This resulted in an increase in endogenous ROS production and shortened the chronological life span (CLS) compared to wild type. The null PTC7 mutant (ptc7∆) strain showed a lower biosynthesis rate of CoQ6 and a significant shortening of the CLS. The reduced CLS observed in ptc7Δ was restored by the overexpression of PTC7 but not by the addition of exogenous CoQ6. Overexpression of PTC7 increased mitophagy in a wild type strain. This finding suggests an additional Ptc7 function beyond the regulation of CoQ biosynthesis. Genetic disruption of PTC7 prevented mitophagy activation in conditions of nitrogen deprivation. In brief, we show that, in yeast, Ptc7 modulates the adaptation to respiratory metabolism by dephosphorylating Coq7 to supply newly synthesized CoQ6, and by activating mitophagy to remove defective mitochondria at stationary phase, guaranteeing a proper CLS in yeast.