Single Gene Deletion Research Articles

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli.

Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc.

Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1:At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.

Analysis of Two Complementary Single-Gene Deletion Mutant Libraries of Salmonella Typhimurium in Intraperitoneal Infection of BALB/c Mice.

Two pools of individual single gene deletion (SGD) mutants of S. Typhimurium 14028s encompassing deletions of 3,923 annotated non-essential ORFs and sRNAs were screened by intraperitoneal (IP) injection in BALB/c mice followed by recovery from spleen and liver 2 days post infection. The relative abundance of each mutant was measured by microarray hybridization. The two mutant libraries differed in the orientation of the antibiotic resistance cassettes (either sense-oriented KanR, SGD-K, or antisense-oriented CamR, SGD-C). Consistent systemic colonization defects were observed in both libraries and both organs for hundreds of mutants of genes previously reported to be important after IP injection in this animal model, and for about 100 new candidate genes required for systemic colonization. Four mutants with a range of apparent fitness defects were confirmed using competitive infections with the wild-type parental strain: ΔSTM0286, ΔSTM0551, ΔSTM2363, and ΔSTM3356. Two mutants, ΔSTM0286 and ΔSTM2363, were then complemented in trans with a plasmid encoding an intact copy of the corresponding wild-type gene, and regained the ability to fully colonize BALB/c mice systemically. These results suggest the presence of many more undiscovered Salmonella genes with phenotypes in IP infection of BALB/c mice, and validate the libraries for application to other systems.

Finding targets for genome reduction in Streptomyces lividans TK24 using flux balance analysis

Abstract: A genome reduction can be a good starting point for optimizing a wild-type strain for use in an industrial process. In a genome reduction, a selection of genes is removed with the intention of obtaining a robust strain with enhanced production properties. The complex soil bacterium Streptomyces lividans TK24 produces many compounds that can hinder the industrial objective. Genome reduction is often based on phylogenetic conservation and absolute gene expression. Here, flux balance analysis is proposed as an additional and complementary method for identifying potential targets for genome reduction. The effect of deletions of single metabolic genes and selected groups of adjacent metabolic genes on bacterial growth is simulated in a high throughput fashion. Targets identified through this method show lower gene expression and are less likely to be conserved between different Streptomyces strains. Gene expression data are used for assessing the effect of gene deletions in silico to improve prediction accuracy.

A Systems Level Approach for Identification of Molecular Targets for Antimicrobial Intervention against Pseudomonas Aeruginosa, while Predicting Biofilm Formation

In this case study, we aimed at evaluating the suitability of genome-scale metabolic models to identify molecular targets that can potentially enhance antimicrobial effects of chemical preservatives against P. aeruginosa, while minimizing biofilm formation. For the case study, isothiazolinones were selected as a group of microbicides where their mechanism of action is well described in scientific literature. Target identification was carried out in several steps. First, we developed a computational model of P. aeruginosa metabolism under action of isothiazolinones. Action of sub-inhibitory concentrations of isothiazolinones was simulated based on extensive information on their mechanisms of action. Then, simulations of single and double gene deletion(s) were performed in silico to identify genes or combinations of genes that could be targeted to induce further reduction of bacterial growth rate. Finally, we assessed whether total or partial inhibition of these genes might activate biofilm formation.

Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol

Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H+ conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l−1 of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.

Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia: The İstanbul Perspective.

Objective:Alpha thalassemia syndromes are caused by mutations on one or more of the four α-globin genes. Mutations could be either more commonly deletional or non-deletional. As some deletions (3.7 and 4.2) cause α+-thalassemia, some cause (-20.5, MED, THAI, FIL) α0 -thalassemia. The aim of this study was to determine alpha thalassemia mutations in patients with unsolved hypochromic microcytic anemia and to evaluate types of mutations.Material and Methods:Two hundred six patients with hypochromic microcytic anemia were evaluated for alpha thalassemia. A venous blood sample of 2 mL was drawn from each patient for DNA isolation. The samples were investigated for α-thalassemia mutations by using the Vienna Lab α-Globlin StripAssay TM commercial kit.Results:Fourteen different mutations were determined in 95 (46.1%) patients. The most common mutation was the 3.7 single gene deletion and was found in 37 patients (n=37/95, 39%). Others common mutations were the 20.5 kb double gene deletion (n=20 patients, 21%), MED double gene deletion (n=17 patients, 17.9%), α2 IVS1 (n=10 patients, 10.5%), α2 cd142 Hb Koya Dora (n=6 patients, 6.3%), α2 polyA1 (Saudi type) (n=6 patients, 6.3%), 4.2 single gene deletion (n=4 patients, 4.2%), α1 cd14 (n=2 patients, 2.1%), and -FIL mutation (n=2 patients 2.1%), respectively. Hb Adana, Hb Icaria, α2 init cd and α2 polyA2 (Turkish type) were found in 1% of the patients (n=1). Seven patients (7.4%) had α-thalassemia triplication. In our study, three mutations (Hb Icaria, α1 cd14, α2 init.cd) were determined firstly in Turkey. Seven mutations (-SEA, -THAI, Hb Constant Spring, α2 cd19, α2 cd59, α2 cd125, Hb Paksé) were not determined in this study.Conclusion:Alpha thalassemia should be considered in the differential diagnosis of hypochromic microcytic anemia especially in cases without iron deficiency and b-thalassemia carrier state. Genetic testing should be performed for the suspicious cases. We also recommend that a national database with all mutations in Turkey should be created to screen the alpha thalassemia cost-effectively.

PyMut: A Web Tool for Overlapping Gene Loss-of-Function Mutation Design.

Loss-of-function study is an effective approach to research gene functions. However, currently most of such studies have ignored an important problem (in this paper, we call it "off-target" problem), that is, if the target gene is an overlapping gene (A gene whose expressible nucleotides overlaps with that of another one), loss-of-function mutation by deleting the complete open reading frame (ORF) may also cause the gene it overlaps lose function, resulting a phenotype which may be rather different from that of single gene deletion. Therefore, when doing such studies, the loss-of-function mutations should be carefully designed to guarantee only the function of the target gene will be abolished. In this paper, we present PyMut, an easy-to-use web tool for biologists to design such mutations. To the best of our knowledge, PyMut is the first tool that aims to solve the "off-target" problem regarding the overlapping genes. Our web server is freely available at http://www.bioinfo.tsinghua.edu.cn/∼liuke/PyMut/index.html.

Identification of the phd gene cluster responsible for phenylpropanoid utilization in Corynebacterium glutamicum.

Phenylpropanoids as abundant, lignin-derived compounds represent sustainable feedstocks for biotechnological production processes. We found that the biotechnologically important soil bacterium Corynebacterium glutamicum is able to grow on phenylpropanoids such as p-coumaric acid, ferulic acid, caffeic acid, and 3-(4-hydroxyphenyl)propionic acid as sole carbon and energy sources. Global gene expression analyses identified a gene cluster (cg0340-cg0341 and cg0344-cg0347), which showed increased transcription levels in response to phenylpropanoids. The gene cg0340 (designated phdT) encodes for a putative transporter protein, whereas cg0341 and cg0344-cg0347 (phdA-E) encode enzymes involved in the β-oxidation of phenylpropanoids. The phd gene cluster is transcriptionally controlled by a MarR-type repressor encoded by cg0343 (phdR). Cultivation experiments conducted with C. glutamicum strains carrying single-gene deletions showed that loss of phdA, phdB, phdC, or phdE abolished growth of C. glutamicum with all phenylpropanoid substrates tested. The deletion of phdD (encoding for putative acyl-CoA dehydrogenase) additionally abolished growth with the α,β-saturated phenylpropanoid 3-(4-hydroxyphenyl)propionic acid. However, the observed growth defect of all constructed single-gene deletion strains could be abolished through plasmid-borne expression of the respective genes. These results and the intracellular accumulation of pathway intermediates determined via LC-ESI-MS/MS in single-gene deletion mutants showed that the phd gene cluster encodes for a CoA-dependent, β-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicum.

Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events—disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.

Molecular nature of alpha-globin genes in the Saudi population.

Alpha-thalassemia (α-thal) is a disorder caused by the deletion of single or double α-globin genes, and/or point mutations in the α-globin genes. There are 2 common types of α-globin genes; HBA2 and HBA1. Recently, it has been discovered that the HBA2 gene is replaced by a unique HBA12 gene convert in 5.7% of the Saudi population. The α-globin genes have been emerging as a molecular target for the treatment of β-thalassemia (β-thal). Hence, it is essential to understand the molecular nature of α-globin genes to treat the most prevalent hemoglobin disorders, such as sickle cell disease, α-thal, and β-thal prevalent in the Kingdom of Saudi Arabia. Thirty-two different α-globin genotypes have been observed in the Saudi population. This review outlines the classification of the α-globin genes on the basis of their molecular nature and complex combinations of α-globin genes, and their variants predominant in Saudis.

The trans-kingdom identification of negative regulators of pathogen hypervirulence.

Modern society and global ecosystems are increasingly under threat from pathogens, which cause a plethora of human, animal, invertebrate and plant diseases. Of increasing concern is the trans-kingdom tendency for increased pathogen virulence that is beginning to emerge in natural, clinical and agricultural settings. The study of pathogenicity has revealed multiple examples of convergently evolved virulence mechanisms. Originally described as rare, but increasingly common, are interactions where a single gene deletion in a pathogenic species causes hypervirulence. This review utilised the pathogen–host interaction database (www.PHI-base.org) to identify 112 hypervirulent mutations from 37 pathogen species, and subsequently interrogates the trans-kingdom, conserved, molecular, biochemical and cellular themes that cause hypervirulence. This study investigates 22 animal and 15 plant pathogens including 17 bacterial and 17 fungal species. Finally, the evolutionary significance and trans-kingdom requirement for negative regulators of hypervirulence and the implication of pathogen hypervirulence and emerging infectious diseases on society are discussed.

A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging

Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C.elegans and M.musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C.elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.

Photocatalytic inactivation of Escherichia coli—The roles of genes in β-oxidation of fatty acid degradation

A genetic study on the relationship between cell membrane fatty acid profile and photocatalytic inactivation efficiency of Escherichia coli was conducted. Photocatalytic inactivation of a parental strain (E. coli BW25113) and its seven isogenic mutants with deletion of single gene involving in β-oxidation of fatty acid degradation (fad) were compared. Most of the mutants involved in fatty acid degradation did not show significant difference in susceptibility towards photocatalytic inactivation compared with the parental strain, except that E. coli JW1176 (fadR mutant) and E. coli JW3935 (fabR mutant) showed a lower and higher sensitivity than the parental strain, respectively. Fluorescence microscopic analysis showed that the loss of cell permeability preceded the inactivation of bacterial cells. The results of temperature pre-treatment and fatty acid profiles of the parental strain and fadR mutant indicated that the alteration in cell membrane fatty acid composition played an important role, but not the most crucial one to affect the susceptibility of bacterial cell towards photocatalytic inactivation. The results in this study demonstrated the importance of cell membrane in the bacterial defense system against photocatalytic inactivation.

New cassettes for single-step drug resistance and prototrophic marker switching in fission yeast.

Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein-protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4(+) gene or the G418-resistance kanMX as marker. Because it is rather cumbersome to produce multiply deleted or tagged strains using the same marker, or to completely reconstruct a particular strain with a different marker, single-step exchange protocols of markers are a time-saving alternative. In recent years, dominant drug resistance markers (DDRMs) against clonNAT, hygromycin B and bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes, natMX, hphMX and bleMX, carry the TEF promotor and terminator sequences from Ashbya gossypii as kanMX; this provides flanking homologies to enable single-step marker swapping by homologous gene targeting. To expand this very useful toolset for single-step marker exchange, I constructed MX cassettes containing the nutritional markers arg3(+), his3(+), leu1(+) and ura4(+). Furthermore, a set of constructs was created to enable single-step exchange of ura4(+) to kanMX6, natMX4 and hphMX4. The functionality of the cassettes is demonstrated by successful single-step marker swapping at several loci. These constructs allow straightforward and rapid remarking of existing ura4(+) - and MX-deleted and -tagged strains.

Deletion of homologs of the SREBP pathway results in hyper-production of cellulases in Neurospora crassa and Trichoderma reesei.

BackgroundThe filamentous fungus Neurospora crassa efficiently utilizes plant biomass and is a model organism for genetic, molecular and cellular biology studies. Here, a set of 567 single-gene deletion strains was assessed for cellulolytic activity as compared to the wild-type parental strain. Mutant strains included were those carrying a deletion in: (1) genes encoding proteins homologous to those implicated in the Saccharomyces cerevisiae secretion apparatus; (2) genes that are homologous to those known to differ between the Trichoderma reesei hyper-secreting strain RUT-C30 and its ancestral wild-type strain; (3) genes encoding proteins identified in the secretome of N. crassa when cultured on plant biomass and (4) genes encoding proteins predicted to traverse the secretory pathway.ResultsThe 567 single-gene deletion collection was cultured on crystalline cellulose and a comparison of levels of secreted protein and cellulase activity relative to the wild-type strain resulted in the identification of seven hyper-production and 18 hypo-production strains. Some of these deleted genes encoded proteins that are likely to act in transcription, protein synthesis and intracellular trafficking, but many encoded fungal-specific proteins of undetermined function. Characterization of several mutants peripherally linked to protein processing or secretion showed that the hyper- or hypo-production phenotypes were primarily a response to cellulose. The altered secretome of these strains was not limited to the production of cellulolytic enzymes, yet was part of the cellulosic response driven by the cellulase transcription factor CLR-2. Mutants implicated the loss of the SREBP pathway, which has been found to regulate ergosterol biosynthesis genes in response to hypoxic conditions, resulted in a hyper-production phenotype. Deletion of two SREBP pathway components in T. reesei also conferred a hyper-production phenotype under cellulolytic conditions.ConclusionsThese studies demonstrate the utility of screening the publicly available N. crassa single-gene deletion strain collection for a particular phenotype. Mutants in a predicted E3 ligase and its target SREBP transcription factor played an unanticipated role in protein production under cellulolytic conditions. Furthermore, phenotypes similar to those observed in N. crassa were seen following the targeted deletion of orthologous SREBP pathway loci in T. reesei, a fungal species commonly used in industrial enzyme production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0297-9) contains supplementary material, which is available to authorized users.

Frequencies of HKαα and anti-HKαα Alleles in Chinese Carriers of Silent Deletional α-Thalassemia

The HKαα (HongKongαα) allele is an unusual rearrangement of the α-globin gene cluster containing both the −α3.7 (rightward) and αααanti 4.2 crossover deletion/duplication. The anti-HKαα (anti-HongKongαα) allele is the reciprocal product containing both the −α4.2 (leftward) and αααanti 3.7 unequal crossover deletion/duplication. In clinical practice of thalassemia screening, gap-polymerase chain reaction (gap-PCR) approaches are used to detect the common −α3.7 and −α4.2 deletions of α-thalassemia (α-thal). Because the HKαα and anti-HKαα alleles also contain the single α-globin gene deletion, individuals with these alleles would be misdiagnosed as −α3.7 or −α4.2 carriers. This would likely produce misleading or incorrect information in genetic counseling. In this study, we investigated the HKαα and anti-HKαα alleles in Chinese carriers of silent deletional α-thal, and reported their frequencies to be 2.27 and 0.35% in −α3.7 and −α4.2 carriers, respectively. Given the rarity of the HKαα and anti-HKαα alleles, a routine screening for these two rearrangements are unlikely to be necessary on most occasions.

Characterization of Novel Factors Involved in Swimming and Swarming Motility in Salmonella enterica Serovar Typhimurium.

Salmonella enterica utilizes flagellar motility to swim through liquid environments and on surfaces. The biosynthesis of the flagellum is regulated on various levels, including transcriptional and posttranscriptional mechanisms. Here, we investigated the motility phenotype of 24 selected single gene deletions that were previously described to display swimming and swarming motility effects. Mutations in flgE, fliH, ydiV, rfaG, yjcC, STM1267 and STM3363 showed an altered motility phenotype. Deletions of flgE and fliH displayed a non-motile phenotype in both swimming and swarming motility assays as expected. The deletions of STM1267, STM3363, ydiV, rfaG and yjcC were further analyzed in detail for flagellar and fimbrial gene expression and filament formation. A ΔydiV mutant showed increased swimming motility, but a decrease in swarming motility, which coincided with derepression of curli fimbriae. A deletion of yjcC, encoding for an EAL domain-containing protein, increased swimming motility independent on flagellar gene expression. A ΔSTM1267 mutant displayed a hypermotile phenotype on swarm agar plates and was found to have increased numbers of flagella. In contrast, a knockout of STM3363 did also display an increase in swarming motility, but did not alter flagella numbers. Finally, a deletion of the LPS biosynthesis-related protein RfaG reduced swimming and swarming motility, associated with a decrease in transcription from flagellar class II and class III promoters and a lack of flagellar filaments.

Abstract 3038: G quadruplex stabilization induces mitophagy in S. cerevisiae

Abstract G-quadruplex (G4) DNA structures are stable planar structures held together by multiple G-G base pairs. G4 structures are thought to affect transcription and other chromosomal processes but their presence in vivo is also expected to slow replication fork progression. Previous work from our lab demonstrates that the S. cerevisiae Pif1 5′ to 3′ DNA helicase is a particularly potent unwinder of G4 structures in vitro1. In addition, cells lacking the Pif1 helicase exhibit replication fork slowing and DNA breakage at G4 motifs, chromosomal sequences that form intra-molecular G4 structures in vitro1,2. In addition, treating yeast in vivo with the G4 stabilizing drug PhenDC3 increases G4-mediated genome instability3. We treated a collection of 4,783 S. cerevisiae strains that each contained a single gene deletion with PhenDC3 to search for genes that are sensitive or resistant to PhenDC3. We will discuss results showing that several yeast genes that affect maintenance of mitochondrial DNA and autophagy induction are hypersensitive to PhenDC3. Using fluorescent microscopy and immunoblotting, we further demonstrate that treatment of wild type S. cerevisiae cells with PhenDC3 induces autophagy, and more specifically, mitophagy. Moreover, cells lacking Pif1 are themselves activated for autophagy. Reference: 1 Paeschke, K. et al. Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature 497, 458-+, doi:10.1038/nature12149 (2013). 2 Paeschke, K., Capra, J. A. & Zakian, V. A. DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145, 678-691, doi:10.1016/j.cell.2011.04.015 (2011). 3 Piazza, A. et al. Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae. Nucleic Acids Res 38, 4337-4348, doi:10.1093/nar/gkq136 (2010). Citation Format: Jennifer Stundon. G quadruplex stabilization induces mitophagy in S. cerevisiae. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3038. doi:10.1158/1538-7445.AM2015-3038