Adaptive Resistance Mutations at Suprainhibitory Concentrations Independent of SOS Mutagenesis.

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.

Genomic structural plasticity of rodent-associated Bartonella in nature.

Rodent-associated Bartonella species have shown a remarkable genetic diversity and pathogenic potential. To further explore the extent of the natural intraspecific genomic variation and its potential role as an evolutionary driver, we focused on a single genetically diverse Bartonella species, Bartonella krasnovii, which circulates among gerbils and their associated fleas. Twenty genomes from 16 different B. krasnovii genotypes were fully characterized through a genome sequencing assay (using short and long read sequencing), pulse field gel electrophoresis (PFGE), and PCR validation. Genomic analyses were performed in comparison to the B. krasnovii strain OE 1-1. While, single nucleotide polymorphism represented only a 0.3% of the genome variation, structural diversity was identified in these genomes, with an average of 51 ± 24 structural variation (SV) events per genome. Interestingly, a large proportion of the SVs (>40%) was associated with prophages. Further analyses revealed that most of the SVs, and prophage insertions were found at the chromosome replication termination site (ter), suggesting this site as a plastic zone of the B. krasnovii chromosome. Accordingly, six genomes were found to be unbalanced, and essential genes near the ter showed a shift between the leading and lagging strands, revealing the SV effect on these genomes. In summary, our findings demonstrate the extensive genomic diversity harbored by wild B. krasnovii strains and suggests that its diversification is initially promoted by structural changes, probably driven by phages. These events may constantly feed the system with novel genotypes that ultimately lead to inter- and intraspecies competition and adaptation.

The Goldilocks effect of respiration on canavanine tolerance in Saccharomyces cerevisiae.

When glucose is available, Saccharomyces cerevisiae prefers fermentation to respiration. In fact, it can live without respiration at all. Here, we study the role of respiration in stress tolerance in yeast. We found that colony growth of respiratory-deficient yeast (petite) is greatly inhibited by canavanine, the toxic analog of arginine that causes proteotoxic stress. We found lower amounts of the amino acids involved in arginine biosynthesis in petites compared with WT. This finding may be explained by the fact that petite cells exposed to canavanine show reduction in the efficiency of targeting of proteins required for arginine biosynthesis. The retrograde (RTG) pathway signals mitochondrial stress. It positively controls production of arginine precursors. We show that canavanine abrogates RTG signaling especially in petite cells, and mutants in the RTG pathway are extremely sensitive to canavanine. We suggest that petite cells are naturally ineffective in production of some amino acids; combination of this fact with the effect of canavanine on the RTG pathway is the simplest explanation why petite cells are inhibited by canavanine. Surprisingly, we found that canavanine greatly inhibits colony formation when WT cells are forced to respire. Our research proposes a novel connection between respiration and proteotoxic stress.

Untangling the knots: Co-infection and diversity of Bartonella from wild gerbils and their associated fleas.

Based on molecular data, previous studies have suggested a high overall diversity and co-infection rates of Bartonella bacteria in wild rodents and their fleas. However, partial genetic characterization of uncultured co-infecting bacteria limited sound conclusions concerning intra- and inter-specific diversity of the circulating Bartonella. To overcome this limitation, Bartonella infections of wild populations of two sympatric gerbil species and their fleas were explored by multiple isolations of Bartonella organisms. Accordingly, 448 pure Bartonella isolates, obtained from 20 rodent blood and 39 flea samples, were genetically characterized to the genotype and species levels. Results revealed a remarkable diversity and co-infection rates of Bartonella among these sympatric rodents and their associated fleas. Specifically, 38 genotypes, classified into four main Bartonella species, were identified. Co-infection was confirmed in 56% of the samples, which contained two to four Bartonella genotypes per sample, belonging to up to three different species. Recombination within and between these species was demonstrated, serving as a direct evidence of the frequent bacteria-bacteria interactions. Moreover, despite the noticeable interchange of common Bartonella genotypes between rodents and fleas, the co-occurrence of genotypes was not random and differences in the overall diversity, and the ecological and phylogenetic similarities of the infection compositions were significantly associated with the carrier type (rodent vs. flea) and the rodent species. Thus, comprehensive identification of the co-infecting organisms enabled the elucidation of ecological factors affecting the Bartonella distribution among reservoirs and vectors. This study may serve as a model for the investigation of other vector-borne organisms and their relationships with Bartonella.

Dynamic responses of Fusarium mangiferae to ultra-violet radiation.

The repair capacity of ultra-violet (UV) light DNA damage is important for adaptation of fungi to different ecological niches. We previously showed that in the soil-borne pathogen Fusarium oxysporum photo-reactivation dependent UV repair is induced at the germling stage and reduced at the filament stage. Here, we tested the developmental control of the transcription of photolyase, UV survival, UV repair capacity, and UV induced mutagenesis in the foliar pathogen Fusarium mangiferae. Unlike F. oxysporum, neither did we observe developmental control over photo-reactivation dependent repair nor the changes in gene expression of photolyase throughout the experiment. Similarly, photo-reactivation assisted reduction in UV induced mutagenesis was similar throughout the development of F. mangiferae but fluctuated during the development of F. oxysporum. To generate hypotheses regarding the recovery of F. mangiferae after UV exposure, an RNAseq analysis was performed after irradiation at different timepoints. The most striking effect of UV on F. mangiferae was developmental-dependent induction of translation related genes. We further report a complex response that changes during recovery time and involves translation, cell cycle and lipid biology related genes.

A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds.

BACKGROUND: Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. RESULTS: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. CONCLUSIONS: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterization of constitutive CTCF/cohesin loci: a possible role in establishing topological domains in mammalian genomes.

BACKGROUND: Recent studies suggested that human/mammalian genomes are divided into large, discrete domains that are units of chromosome organization. CTCF, a CCCTC binding factor, has a diverse role in genome regulation including transcriptional regulation, chromosome-boundary insulation, DNA replication, and chromatin packaging. It remains unclear whether a subset of CTCF binding sites plays a functional role in establishing/maintaining chromatin topological domains. RESULTS: We systematically analysed the genomic, transcriptomic and epigenetic profiles of the CTCF binding sites in 56 human cell lines from ENCODE. We identified ~24,000 CTCF sites (referred to as constitutive sites) that were bound in more than 90% of the cell lines. Our analysis revealed: 1) constitutive CTCF loci were located in constitutive open chromatin and often co-localized with constitutive cohesin loci; 2) most constitutive CTCF loci were distant from transcription start sites and lacked CpG islands but were enriched with the full-spectrum CTCF motifs: a recently reported 33/34-mer and two other potentially novel (22/26-mer); 3) more importantly, most constitutive CTCF loci were present in CTCF-mediated chromatin interactions detected by ChIA-PET and these pair-wise interactions occurred predominantly within, but not between, topological domains identified by Hi-C. CONCLUSIONS: Our results suggest that the constitutive CTCF sites may play a role in organizing/maintaining the recently identified topological domains that are common across most human cells.

Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes.

Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage-induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G(2)/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G(2)/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage-induced recombinants in G(2)/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.

Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells.

Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5' overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ.

Numt-mediated double-strand break repair mitigates deletions during primate genome evolution.

Non-homologous end joining (NHEJ) is the major mechanism of double-strand break repair (DSBR) in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs). Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler DNA, prevents nuclear processing of DSBs that could result in deleterious repair.

Genomic Instability in Fungal Plant Pathogens.

Fungi and fungal-like organisms (oomycetes) that cause diseases in plants have impacted human communities for centuries and probably from the dawn of agriculture. In modern agriculture, there is a constant race between new strategies to manage fungal plant pathogens and their ability to adapt. An important component in this race is fungal genetic diversity. Mechanisms such as sexual and parasexual recombination that contribute to the creation of novel allele combinations in fungal plant pathogens are briefly discussed in the first part of this review. Advances in genomics have enabled the investigation of chromosomal aberrations of agriculturally important fungal isolates at the nucleotide level. Some of these cases are summarized in the second part of this review; it is claimed that the effect of chromosomal aberrations on pathogenicity should be studied mechanistically. More data on the effect of gene copy number variations on phenotypes that are relevant to agriculture are especially needed. Genome rearrangements through translocations have shaped the genome of fungal plant pathogens by creating lineage-specific chromosome territories encoding for genes participating in plant diseases. Pathogenicity chromosomes are unique cases of such lineage-specific genetic elements, interestingly these chromosomes can be transferred horizontally and thus transforming a non-pathogenic strain to a pathogenic one. The third part of this review describes our attempts to reveal mutators in fungal plant pathogens by identifying fungi that lack important DNA repair genes or respond to DNA damage in an unconventional way. We found that a group of fungal plant pathogens lack conserved genes that are needed for an important Holliday junction resolution pathway. In addition, in Fusarium oxysporum, the rate-limiting step in dNTP production is not induced under DNA replication stress. This is very different from organisms from bacteria to humans. It remains to be seen if these mechanisms promote genetic instability in fungal plant pathogens.

Developmentally Regulated Oscillations in the Expression of UV Repair Genes in a Soilborne Plant Pathogen Dictate UV Repair Efficiency and Survival.

The ability to withstand UV damage shapes the ecology of microbes. While mechanisms of UV tolerance were extensively investigated in microorganisms regularly exposed to the sun, far less is known about UV repair of soilborne microorganisms. Fusarium oxysporum is a soilborne fungal plant pathogen that is resistant to UV light. We hypothesized that its UV repair capacity is induced to deal with irregular sun exposure. Unlike the SOS paradigm, our analysis revealed only sporadic increases and even decreases in UV repair gene expression following UVC irradiation or exposure to visible light. Strikingly, a major factor determining the expression of UV repair genes was the developmental status of the fungus. At the early stages of germination, the expression of photolyase increased while the expression of UV endonuclease decreased, and then the trend was reversed. These gene expression oscillations were dependent on cell cycle progression. Consequently, the contribution of photoreactivation to UV repair and survival was stronger at the beginning of germination than later when a filament was established. F. oxysporum germinates following cues from the host. Early on in germination, it is most vulnerable to UV; when the filament is established, the pathogen is protected from the sun because it is already within the host tissue.IMPORTANCEFusarium oxysporum infects plants through the roots and therefore is not exposed to the sun regularly. However, the ability to survive sun exposure expands the distribution of the population. UV from the sun is toxic and mutagenic, and to survive sun exposure, fungi encode several DNA repair mechanisms. We found that Fusarium oxysporum has a gene expression program that activates photolyase at the first hours of germination when the pathogen is not established in the plant tissue. Later on, the expression of photolyase decreases, and the expression of a light-independent UV repair mechanism increases. We suggest a novel point of view to a very fundamental question of how soilborne microorganisms defend themselves against sudden UV exposure.

Limited DNA Repair Gene Repertoire in Ascomycete Yeast Revealed by Comparative Genomics.

Ascomycota is the largest phylogenetic group of fungi that includes species important to human health and wellbeing. DNA repair is important for fungal survival and genome evolution. Here, we describe a detailed comparative genomic analysis of DNA repair genes in Ascomycota. We determined the DNA repair gene repertoire in Taphrinomycotina, Saccharomycotina, Leotiomycetes, Sordariomycetes, Dothideomycetes, and Eurotiomycetes. The subphyla of yeasts, Saccharomycotina and Taphrinomycotina, have a smaller DNA repair gene repertoire comparing to Pezizomycotina. Some genes were absent from most, if not all, yeast species. To study the conservation of these genes in Pezizomycotina, we used the Gain Loss Mapping Engine algorithm that provides the expectations of gain or loss of genes given the tree topology. Genes that were absent from most of the species of Taphrinomycotina or Saccharomycotina showed lower conservation in Pezizomycotina. This suggests that the absence of some DNA repair in yeasts is not random; genes with a tendency to be lost in other classes are missing. We ranked the conservation of DNA repair genes in Ascomycota. We found that Rad51 and its paralogs were less conserved than other recombinational proteins, suggesting that there is a redundancy between Rad51 and its paralogs, at least in some species. Finally, based on the repertoire of UV repair genes, we found conditions that differentially kill the wine pathogen Brettanomyces bruxellensis and not Saccharomyces cerevisiae. In summary, our analysis provides testable hypotheses to the role of DNA repair proteins in the genome evolution of Ascomycota.

Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress.

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed.

Alternative Functional rad21 Paralogs in Fusarium oxysporum.

Cohesin, the sister chromatid cohesion complex, is an essential complex that ensures faithful sister chromatid segregation in eukaryotes. It also participates in DNA repair, transcription and maintenance of chromosome structure. Mitotic cohesin is composed of Smc1, Smc3, Scc3, and Rad21/Mcd1. The meiotic cohesin complex contains Rec8, a Rad21 paralog and not Rad21 itself. Very little is known about sister chromatid cohesion in fungal plant pathogens. Fusarium oxysporum is an important fungal plant pathogen without known sexual life cycle. Here, we describe that F. oxysporum encodes for three Rad21 paralogs; Rad21, Rec8, and the first alternative Rad21 paralog in the phylum of ascomycete. This last paralog is found only in several fungal plant pathogens from the Fusarium family and thus termed rad21nc (non-conserved). Conserved rad21 (rad21c), rad21nc, and rec8 genes are expressed in F. oxysporum although the expression of rad21c is much higher than the other paralogs. F. oxysporum strains deleted for the rad21nc or rec8 genes were analyzed for their role in fungal life cycle. Δrad21nc and Δrec8 single mutants were proficient in sporulation, conidia germination, hyphal growth and pathogenicity under optimal growth conditions. Interestingly, Δrad21nc and Δrec8 single mutants germinate less effectively than wild type (WT) strains under DNA replication and mitosis stresses. We provide here the first genetic analysis of alternative rad21nc and rec8 paralogs in filamentous fungi. Our results suggest that rad21nc and rec8 may have a unique role in cell cycle related functions of F. oxysporum.

How Does Fusarium oxysporum Sense and Respond to Nicotinaldehyde, an Inhibitor of the NAD+ Salvage Biosynthesis Pathway?

Plant pathogenic fungi are a major threat to food security and impose a severe economic burden, thus there is a continuous need to develop new strategies to manage them. NAD+ is a co-factor in numerous enzymatic activities and determines the metabolic fate of the cell. Therefore, maintenance of NAD+ concentration is important for cellular viability. Consequently, the NAD+ biosynthetic pathway and redox homeostasis was suggested as a target for antifungal development. We aimed to study how Fusarium oxysporum senses and responds to nicotinaldehyde (NA), an inhibitor of Pnc1, a key enzyme in the salvage pathway of NAD+ biosynthesis. We were able to show that NA was inhibitory in high concentrations to several fungal plant pathogens, with much milder effects on tomato growth. Under low nutrient conditions NA reduced the total amounts of NAD+ in the fungal cell, a trend that was also observed in rich media, although without statistical significance. In low and high nutrient availability NA dramatically reduced the NAD+/NADH ratio. After exposure to NA, NADH levels were increased and NAD+ levels and the biomass were greatly reduced. Cells responded to NA by up-regulation of oxidoreductases, with hardly any up-regulation of the classic response to oxidative stress. Direct measurement of oxidative stress response showed that unlike formaldehyde and hydrogen peroxide, NA caused reductive rather than oxidative stress. Surprisingly, alcohol dehydrogenases were significantly up-regulated more than any other dehydrogenases, including aldehyde dehydrogenases. We propose that conidia of F. oxysporum efficiently detoxified the aldehyde group of NA by reducing NAD+ to NADH; the high concentrations of the latter provoked the expression of alcohol dehydrogenases that in yeast can act to reduce NADH and increase NAD+ amounts, respectively. Overall, the results suggest that targeting NAD+ biosynthesis pathway and redox homeostasis can be a potential approach to manage fungal plant pathogens. Many of the natural antifungal compounds produced by bio-control agents or even the natural biome are aldehydes, and thus the results presented here predict the possible response of Fusarium to wide sources of toxicity in the environment.

The response to the DNA damaging agent methyl methanesulfonate in a fungal plant pathogen.

DNA damage can cause mutations that in fungal plant pathogens lead to hypervirulence and resistance to pesticides. Almost nothing is known about the response of these fungi to DNA damage. We performed transcriptomic and phosphoproteomic analyses of Fusarium oxysporum exposed to methyl methanesulfonate (MMS). At the RNA level we observe massive induction of DNA repair pathways including the global genome nucleotide excision. Cul3, Cul4, several Ubiquitin-like ligases and components of the proteasome are significantly induced. In agreement, we observed drug synergism between a proteasome inhibitor and MMS. While our data suggest that Yap1 and Xbp1 networks are similarly activated in response to damage in yeast and F. oxysporum we were able to observe modules that were MMS-responsive in F. oxysporum and not in yeast. These include transcription/splicing modules that are upregulated and respiration that is down-regulated. In agreement, MMS treated cells are much more sensitive to a respiration inhibitor. At the phosphoproteomic level, Adenylate cyclase, which generates cAMP, is phosphorylated in response to MMS and forms a network of phosphorylated proteins that include cell cycle regulators and several MAPKs. Our analysis provides a starting point in understanding how genomic changes in response to DNA damage occur in Fusarium species.

Prophage-Driven Genomic Structural Changes Promote Bartonella Vertical Evolution.

Bartonella is a genetically diverse group of vector-borne bacteria. Over 40 species have been characterized to date, mainly from mammalian reservoirs and arthropod vectors. Rodent reservoirs harbor one of the largest Bartonella diversity described to date, and novel species and genetic variants are continuously identified from these hosts. Yet, it is still unknown if this significant genetic diversity stems from adaptation to different niches or from intrinsic high mutation rates. Here, we explored the vertical occurrence of spontaneous genomic alterations in 18 lines derived from two rodent-associated Bartonella elizabethae-like strains, evolved in nonselective agar plates under conditions mimicking their vector- and mammalian-associated temperatures, and the transmission cycles between them (i.e., 26 °C, 37 °C, and alterations between the two), using mutation accumulation experiments. After ∼1,000 generations, evolved genomes revealed few point mutations (average of one-point mutation per line), evidencing conserved single-nucleotide mutation rates. Interestingly, three large structural genomic changes (two large deletions and an inversion) were identified over all lines, associated with prophages and surface adhesin genes. Particularly, a prophage, deleted during constant propagation at 37 °C, was associated with an increased autonomous replication at 26 °C (the flea-associated temperature). Complementary molecular analyses of wild strains, isolated from desert rodents and their fleas, further supported the occurrence of structural genomic variations and prophage-associated deletions in nature. Our findings suggest that structural genomic changes represent an effective intrinsic mechanism to generate diversity in slow-growing bacteria and emphasize the role of prophages as promoters of diversity in nature.

Whole Genome Sequence Analysis of Mutations Accumulated in rad27Δ Yeast Strains with Defects in the Processing of Okazaki Fragments Indicates Template-Switching Events.

Okazaki fragments that are formed during lagging strand DNA synthesis include an initiating primer consisting of both RNA and DNA. The RNA fragment must be removed before the fragments are joined. In Saccharomyces cerevisiae, a key player in this process is the structure-specific flap endonuclease, Rad27p (human homolog FEN1). To obtain a genomic view of the mutational consequence of loss of RAD27, a S. cerevisiae rad27Δ strain was subcultured for 25 generations and sequenced using Illumina paired-end sequencing. Out of the 455 changes observed in 10 colonies isolated the two most common types of events were insertions or deletions (INDELs) in simple sequence repeats (SSRs) and INDELs mediated by short direct repeats. Surprisingly, we also detected a previously neglected class of 21 template-switching events. These events were presumably generated by quasi-palindrome to palindrome correction, as well as palindrome elongation. The formation of these events is best explained by folding back of the stalled nascent strand and resumption of DNA synthesis using the same nascent strand as a template. Evidence of quasi-palindrome to palindrome correction that could be generated by template switching appears also in yeast genome evolution. Out of the 455 events, 55 events appeared in multiple isolates; further analysis indicates that these loci are mutational hotspots. Since Rad27 acts on the lagging strand when the leading strand should not contain any gaps, we propose a mechanism favoring intramolecular strand switching over an intermolecular mechanism. We note that our results open new ways of understanding template switching that occurs during genome instability and evolution.

Increased LOH due to Defective Sister Chromatid Cohesion Is due Primarily to Chromosomal Aneuploidy and not Recombination.

Loss of heterozygosity (LOH) is an important factor in cancer, pathogenic fungi, and adaptation to changing environments. The sister chromatid cohesion process (SCC) suppresses aneuploidy and therefore whole chromosome LOH. SCC is also important to channel recombinational repair to sister chromatids, thereby preventing LOH mediated by allelic recombination. There is, however, insufficient information about the relative roles that the SCC pathway plays in the different modes of LOH. Here, we found that the cohesin mutation mcd1-1, and other mutations in SCC, differentially affect the various types of LOH. The greatest effect, by three orders of magnitude, was on whole chromosome loss (CL). In contrast, there was little increase in recombination-mediated LOH, even for telomeric markers. Some of the LOH events that were increased by SCC mutations were complex, i.e., they were the result of several chromosome transactions. Although these events were independent of POL32, the most parsimonious way to explain the formation of at least some of them was break-induced replication through the centromere. Interestingly, the mcd1-1 pol32Δ double mutant showed a significant reduction in the rate of CL in comparison with the mcd1-1 single mutant. Our results show that defects in SCC allow the formation of complex LOH events that, in turn, can promote drug or pesticide resistance in diploid microbes that are pathogenic to humans or plants.