Genome-Wide Screen for Oxalate-Sensitive Mutants of Saccharomyces cerevisiae

Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a Saccharomyces cerevisiae deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the rgp1Δ, ric1Δ, snf7Δ, vps16Δ, vps20Δ, and vps51Δ mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2Δ, vps16Δ, vps51Δ, ric1Δ, and rib4Δ mutants, was lethal. With the exception of the rib4Δ mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4Δ mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5.     

Comparative Genome-Wide Screening Identifies a Conserved Doxorubicin Repair Network That Is Diploid Specific in Saccharomyces cerevisiae

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Δ and hfi1Δ) or deficient (tho2Δ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Δ, thp1Δ, thp2Δ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases.     

Role of DNA mismatch repair and double-strand break repair in genome stability and antifungal drug resistance in Candida albicans

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Δ/mre11Δ, rad50Δ/rad50Δ, and rad52Δ/rad52Δ mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Δ/msh2Δ, pms1Δ/pms1Δ, and yku80Δ/yku80Δ mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Δ/mre11Δ and rad50Δ/rad50Δ mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Δ/mre11Δ, rad50Δ/rad50Δ, or rad52Δ/rad52Δ leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Δ/msh2Δ and pms1Δ/pms1Δ (MMR mutants) and rad50Δ/rad50Δ (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.     

Development of a Biofilm Production-Deficient Escherichia coli Strain as a Host for Biotechnological Applications

Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient Escherichia coli strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Δ(csgG-csgC); colanic acid biosynthesis and assembly, Δ(wcaL-wza); and type I pilus biosynthesis, Δ(fimB-fimH). E. coli BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type E. coli MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were ~16% and ~25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production-deficient strain of E. coli displays several key properties that substantially enhance its utility in the biotechnology arena.     

DNA Repair Pathway Selection Caused by Defects in TEL1, SAE2, and De Novo Telomere Addition Generates Specific Chromosomal Rearrangement Signatures

Whole genome sequencing of cancer genomes has revealed a diversity of recurrent gross chromosomal rearrangements (GCRs) that are likely signatures of specific defects in DNA damage response pathways. However, inferring the underlying defects has been difficult due to insufficient information relating defects in DNA metabolism to GCR signatures. By analyzing over 95 mutant strains of Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants. The hph-retaining GCRs isolated from tel1Δ mutants contained either an interstitial deletion dependent on non-homologous end-joining or an inverted duplication that appeared to be initiated from a double strand break (DSB) on chrV L followed by hairpin formation, copying of chrV L from the DSB toward the centromere, and homologous recombination to capture the hph-containing end of chrV L. In contrast, hph-containing GCRs from other mutants were primarily interstitial deletions (mrc1Δ tof1Δ) or inverted duplications (sae2Δ and rad53Δ sml1Δ). Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs. Both types of hph-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of hph retention were due to the relative efficiencies of competing DNA repair pathways. Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.     

Integration of Metabolic Modeling and Phenotypic Data in Evaluation and Improvement of Ethanol Production Using Respiration-Deficient Mutants of Saccharomyces cerevisiae

Flux balance analysis and phenotypic data were used to provide clues to the relationships between the activities of gene products and the phenotypes resulting from the deletion of genes involved in respiratory function in Saccharomyces cerevisiae. The effect of partial or complete respiratory deficiency on the ethanol production and growth characteristics of hap4Δ/hap4Δ, mig1Δ/mig1Δ, qdr3Δ/qdr3Δ, pdr3Δ/pdr3Δ, qcr7Δ/qcr7Δ, cyt1Δ/cyt1Δ, and rip1Δ/rip1Δ mutants grown in microaerated chemostats was investigated. The study provided additional evidence for the importance of the selection of a physiologically relevant objective function, and it may improve quantitative predictions of exchange fluxes, as well as qualitative estimations of changes in intracellular fluxes. Ethanol production was successfully predicted by flux balance analysis in the case of the qdr3Δ/qdr3Δ mutant, with maximization of ethanol production as the objective function, suggesting an additional role for Qdr3p in respiration. The absence of similar changes in estimated intracellular fluxes in the qcr7Δ/qcr7Δ mutant compared to the rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants indicated that the effect of the deletion of this subunit of complex III was somehow compensated for. Analysis of predicted flux distributions indicated self-organization of intracellular fluxes to avoid NAD⁺/NADH imbalance in rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants, but not the qcr7Δ/qcr7Δ mutant. The flux through the glycerol efflux channel, Fps1p, was estimated to be zero in all strains under the investigated conditions. This indicates that previous strategies for improving ethanol production, such as the overexpression of the glutamate synthase gene GLT1 in a GDH1 deletion background or deletion of the glycerol efflux channel gene FPS1 and overexpression of GLT1, are unnecessary in a respiration-deficient background.     

Two Histone Deacetylases,FfHda1 and FfHda2, Are Important for Fusarium fujikuroi Secondary Metabolism and Virulence

Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn²⁺-dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as Δffhda1 Δffhda2 mutants resemble the uninfected control plant. Microarray analysis with a Δffhda1 mutant that has lost the major HDAC revealed differential expression of secondary-metabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the Δffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.     

The MET13 Methylenetetrahydrofolate Reductase Gene Is Essential for Infection-Related Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae

Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.     

The Expression of Superoxide Dismutase (SOD) and a Putative ABC Transporter Permease Is Inversely Correlated during Biofilm Formation in Listeria monocytogenes 4b G

Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.     

Genetic and Biochemical Characterizations of Enzymes Involved in Streptococcus pneumoniae Serotype 2 Capsule Synthesis Demonstrate that Cps2T (WchF) Catalyzes the Committed Step by Addition of β1-4 Rhamnose,the Second Sugar Residue in the Repeat Unit

Five genes (cps2E, cps2T, cps2F, cps2G, and cps2I) are predicted to encode the glycosyltransferases responsible for synthesis of the Streptococcus pneumoniae serotype 2 capsule repeat unit, which is polymerized to yield a branched surface structure containing glucose-glucuronic acid linked to a glucose-rhamnose-rhamnose-rhamnose backbone. Cps2E is the initiating glycosyltransferase, but experimental evidence supporting the functions of the remaining glycosyltransferases is lacking. To biochemically characterize the glycosyltransferases, the donor substrate dTDP-rhamnose was first synthesized using recombinant S. pneumoniae enzymes Cps2L, Cps2M, Cps2N, and Cps2O. In in vitro assays with each of the glycosyltransferases, only reaction mixtures containing recombinant Cps2T, dTDP-rhamnose, and the Cps2E product (undecaprenyl pyrophosphate glucose) generated a new product, which was consistent with lipid-linked glucose-rhamnose. cps2T, cps2F, and cps2I deletion mutants produced no detectable capsule, but trace amounts of capsule were detectable in Δcps2G mutants, suggesting that Cps2G adds a nonbackbone sugar. All Δcps2F, Δcps2G, and Δcps2I mutants contained different secondary suppressor mutations in cps2E, indicating that the initial mutations were lethal in the absence of reduced repeat unit synthesis. Δcps2T mutants did not contain secondary mutations affecting capsule synthesis. The requirement for secondary mutations in mutants lacking Cps2F, Cps2G, and Cps2I indicates that these activities occur downstream of the committed step in capsule synthesis and reveal that Cps2T catalyzes this step. Therefore, Cps2T is the β1-4 rhamnosyltransferase that adds the second sugar to the repeat unit and, as the committed step in type 2 repeat unit synthesis, is predicted to be an important point of capsule regulation.     

Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70⁺ strain due to the low frequency of homologous recombination were efficiently obtained from Δku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.     

Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways

Poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B) is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV) genes (C6L and K7R) coding for inhibitors of interferon (IFN) signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R) in human macrophages and monocyte-derived dendritic cells (moDCs) showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4⁺ and CD8⁺ T cell adaptive and memory immune responses, which were mostly mediated by CD8⁺ T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4⁺ T cell responses were mainly directed against Env, while GPN-specific CD8⁺ T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B. These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the immunogenicity of poxvirus-based vaccine candidates.     

Induction of Oxidative Stress by High Hydrostatic Pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation

Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.     

NADPH Oxidases Are Required for Appressorium-Mediated Penetration in Colletotrichum scovillei-Pepper Fruit Pathosystem

NADPH oxidase (Nox) complexes are known to play essential roles in differentiation and proliferation of many filamentous fungi. However, the functions of Noxs have not been elucidated in Colletotrichum species. Therefore, we set out to characterize the roles of Nox enzymes and their regulators in Colletotrichum scovillei, which causes serious anthracnose disease on pepper fruits in temperate and subtropical and temperate region. In this study, we generated targeted deletion mutants for CsNox1, CsNox2, CsNoxR, and CsNoxD via homologous recombination. All deletion mutants were normal in mycelial growth, conidiation, conidial germination, and appressorium formation, suggesting that CsNox1, CsNox2, CsNoxR, and CsNoxD are not involved in those developmental processes. Notably, conidia of ΔCsnox2 and ΔCsnoxr, other than ΔCsnox1 and ΔCsnoxd, failed to cause anthracnose on intact pepper fruits. However, they still caused normal disease on wounded pepper fruits, suggesting that Csnox2 and CsnoxR are essential for penetration-related morphogenesis in C. scovillei. Further observation proved that ΔCsnox2 and ΔCsnoxr were unable to form penetration peg, while they fully developed appressoria, revealing that defect of anthracnose development by ΔCsnox2 and ΔCsnoxr resulted from failure in penetration peg formation. Our results suggest that CsNox2 and CsNoxR are critical for appressorium-mediated penetration in C. scovillei-pepper fruit pathosystem, which provides insight into understanding roles of Nox genes in anthracnose disease development.     

DdrA,DdrD, and PprA: Components of UV and Mitomycin C Resistance in Deinococcus radiodurans R1

Mutants created by deleting the ddrA, ddrB, ddrC, ddrD, and pprA loci of Deinococcus radiodurans R1alone and in all possible combinations of pairs revealed that the encoded gene products contribute to this species’ resistance to UV light and/or mitomycin C. Deleting pprA from an otherwise wild type cell sensitizes the resulting strain to UV irradiation, reducing viability by as much as eight fold relative to R1. If this deletion is introduced into a ΔddrA or ΔddrD background, the resulting strains become profoundly sensitive to the lethal effects of UV light. At a fluence of 1000 Jm^-2, the ΔddrA ΔpprA and ΔddrD ΔpprA strains are 100- and 1000-fold more sensitive to UV relative to the strain that has only lost pprA. Deletion of ddrA results in a 100 fold increase in strain sensitivity to mitomycin C, but in backgrounds that combine a deletion of ddrA with deletions of either ddrC or ddrD, mitomycin resistance is restored to wild type levels. Inactivation of ddrB also increases D. radiodurans sensitivity to mitomycin, but unlike the ddrA mutant deleting ddrC or ddrD from a ΔddrB background further increases that sensitivity. Despite the effect that loss of these gene products has on DNA damage resistance, none appear to directly affect either excision repair or homologous recombination suggesting that they participate in novel processes that facilitate tolerance to UV light and interstrand crosslinks in this species.     

Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrF ΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.     

Hir Proteins Are Required for Position-Dependent Gene Silencing in Saccharomyces cerevisiae in the Absence of Chromatin Assembly Factor I

Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacΔ mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacΔ mutants, although hirΔ mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacΔ mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.     

Identification of Highly Attenuated Mutants of Simian Immunodeficiency Virus

Deletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missing nef, vpr, and upstream sequences (US) in the U3 region of the LTR (SIVmac239Δ3), nef, vpx, and US (SIVmac239Δ3x), and nef, vpr, vpx, and US (SIVmac239Δ4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking vif sequences (SIVmac239Δvif) could be consistently grown only in a vif-complementing cell line. This Δvif virus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239Δvif were apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: Δvpr > Δvpx > ΔvprΔvpx Δnef > Δ3 > Δ3x ≥ Δ4 > Δvif > Δ5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (Δvpr and Δvpx) to not detectably infectious (Δ5), simply by varying the number and location of deletions in these five loci.