Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra-or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome.

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits ␥-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ϳ30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other ␥-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

LY294002 and wortmannin are chemical compounds that act as potent inhibitors of phosphoinositide 3-kinases (PI3Ks). Both of them are generally used to inhibit cell proliferation as cancer treatment by inhibiting the PI3K/protein kinase B (AKT) signaling pathway. In this study, LY294002 (but not wortmannin) showed an abnormal ability to enhance AKT phosphorylation (at Ser472) specifically in gemcitabine (GEM)-resistant pancreatic cancer (PC) cell lines PK59 and KLM1-R. LY294002 was shown to activate AKT and accumulate phospho-AKT at the intracellular membrane in PK59, which was abolished by treatment with AKTi-1/2 or wortmannin. Inhibiting AKT phosphorylation by treatment with AKTi-1/2 or wortmannin further enhanced LY294002-induced cell death in PK59 and KLM1-R cells. In addition, treatment with wortmannin alone failed to inhibit cell proliferation in both PK59 and KLM1-R cells. Thus, our results reveal that LY294002 displays the opposite effect on PI3K-dependent AKT phosphorylation, which maintains cell survival from the cytotoxicity introduced by LY294002 itself in GEM-resistant pancreatic cancer cells. We suggest that targeting the PI3K/AKT signaling pathway with inhibitors may be counterproductive for patients with PC who have acquired GEM-resistance.

The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens, and the subunit CdtB has the ability to create DNA lesions, primarily DNA single-strand breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension, and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4,708 nonessential genes. A total of 4,706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups, including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, and nuclear transport, as well as one unknown open reading frame. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create a DNA double-strand break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells, but only marginal defects in diploid yeast cells. The presence and absence of genes known to be involved in DNA repair in these genome-wide data reveal that CdtB-induced DNA damage is specifically repaired well in the diploid by homologous recombination but not by other repair mechanisms. Our present results provide insights into how CdtB pathogenesis is linked to eukaryotic cellular functions.The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by a number of bacterial pathogens, including Escherichia coli, Haemophilus ducreyi, Campylobacter spp., Salmonella enterica serovar Typhi, Actinobacillus actinomycetemcomitans, Shigella dysenteriae, and Helicobacter spp. (42,44,56). CDTs consist of the three subunits CdtA, CdtB, and CdtC and form a ternary complex (40). CdtB shares conserved residues with the active sites of DNase I-like nucleases, and purified CdtB primarily shows single-strand nicking activity on coiled plasmid DNA and subsequently produces linear DNA in vitro (15,35,40). Enzymatically active CdtB induces cell cycle arrest at the G 2 /M phase, inhibits cell proliferation, and causes cellular enlargement in mammalian cells (28,40). Biochemical analysis has also demonstrated that DNA damage checkpoint machineries and Rho-type GTPase function are involved in CdtB-induced cell cycle arrest and cellular enlargement (13,15,16,30,60). However, the entire complement of genes required for the repair of CdtB-induced DNA lesions and also those leading to cell cycle arrest, cellular enlargement, and cell death have not been fully identified. Because Hassane et al. showed that CdtB is active in yeast (Saccharomyces cerevisiae) cells similar to mammalian cells (19), we used the yeast genome to fur...

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We demonstrate the value of the thermotolerant yeast Issatchenkia orientalis as a candidate microorganism for bioethanol production from lignocellulosic biomass with the goal of consolidated bioprocessing. The I. orientalis MF-121 strain is acid tolerant, ethanol tolerant, and thermotolerant, and is thus a multistress-tolerant yeast. To express heterologous proteins in I. orientalis, we constructed a transformation system for the MF-121 strain and then isolated the promoters of TDH1 and PGK1, two genes that were found to be strongly expressed during ethanol fermentation. As a result, expression of beta-glucosidase from Aspergillus aculeatus could be achieved with I. orientalis, demonstrating successful heterologous gene expression in I. orientalis for the first time. The transformant could convert cellobiose to ethanol under acidic conditions and at high temperature. Simultaneous saccharification and fermentation (SSF) was performed with the transformant, which produced 29 g l(-1) of ethanol in 72 h at 40 degrees C even without addition of beta-glucosidase when SSF was carried out in medium containing 100 g l(-1) of microcrystalline cellulose and a commercial cellulase preparation. These results suggest that using a genetically engineered thermotolerant yeast such as I. orientalis in SSF could lead to cost reduction because less saccharification enzymes are required.

Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3′-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall–related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide.