Lopes J, Piazza A, Bermejo R, Kriegsman B, Colosio A, Teulade-Fichou MP, Foiani M, Nicolas A
EMBO J. 2011 Oct 5;30(19):4033–46
G-quadruplexes are four-stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G-quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G-quadruplex-prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild-type cells but is very frequently destabilized upon treatment with the potent Phen-DC(3) G-quadruplex ligand, or in the absence of the G-quadruplex-unwinding Pif1 helicase, only when the G-rich strand is the template of leading-strand replication. The orientation-dependent instability is associated with the formation of Rad51-Rad52-dependent X-shaped intermediates during replication detected by two-dimensional (2D) gels, and relies on the presence of intact G-quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G-quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G-rich telomeres.
Sanna ML, Zara S, Zara G, Migheli Q, Budroni M
Fungal Biol. 2012 Jul;116(7):769-77
Pichia fermentans DiSAABA 726 is a biofilm-forming yeast that undergoes dimorphic transition. Under yeast-like morphology it controls brown rot caused by Monilia spp. on apple fruit, while under pseudohyphal form, it shows pathogenic behaviour itself on peach fruit. The present study investigates the nutritional factors that induce and separate yeast-like and pseudohyphal morphologies under laboratory conditions. We show that P.fermentans DiSAABA 726 produces mainly yeast-like cells on media containing millimolar concentrations of urea and diammonium phosphate, and forms pseudohyphae at micromolar concentrations of these two salts. With ammonium sulphate, yeast-like or pseudohyphal morphology depends on the N concentration and the pH of the culture media. Amino acids such as methionine, valine, and phenylalanine invariably induce pseudohyphal morphology irrespective of the N concentration and the pH of the culture media. Methionol, 1-butanol, isobutanol, and isopropanol induce pseudohyphal growth, while phenylethanol and isoamyl alcohol fail to induce the formation of filaments. Thus, the morphogenesis of P. fermentansDiSAABA 726 depends more on the nitrogen source than on the N concentration, and is regulated by the quorum-sensing molecules that are generally produced from amino-acid assimilation under nitrogen starvation.
Patkar RN, Xue YK, Shui G, Wenk MR, Naqvi NI
PLoS Pathog. 2012;8(8):e1002888
Magnaporthe oryzae, which causes the devastating rice-blast disease, invades its host plants via a specialized infection structure called the appressorium. Previously, we showed that the ATP-Binding Cassette 3 transporter is necessary for appressorial function (host penetration) in M.oryzae. However, thus far, the molecular basis underlying impaired appressorial function in the abc3Δ remains elusive. We hypothesized that the abc3Δ appressoria accumulate excessive amounts of specific efflux substrate(s) of the Abc3 transporter in M. oryzae. We devised an innovative yeast-based strategy and identified Abc3 Transporter efflux Substrate (ATS) to be a digoxin-like endogenous steroidal glycoside that accumulates to inhibitory levels in M. oryzae abc3Δ appressoria. Exogenous ATS altered cell wall biogenesis and viability in wild-type Schizosaccharomyces pombe, but not in S. pombe expressing M. oryzae Abc3. We show that ATS associates with the Translation Elongation factor Tef2 in M. oryzae, and propose that ATS regulates ion homeostasis during pathogenesis. Excessive ATS accumulation, either intracellularly due to impaired efflux in the abc3Δ or when added exogenously to the wild type, renders M. oryzae nonpathogenic. Furthermore, we demonstrate that the host penetration defects in the abc3Δ are due to aberrant F-actin dynamics as a result of altered Tef2 function and/or ion homeostasis defects caused by excess accumulation of ATS therein. Rather surprisingly, excessive exogenous ATS or digoxin elicited the hypersensitive response in rice, even in the absence of the blast fungus. Lastly, reduced disease symptoms in the inoculated host plants in the presence of excessive digoxin suggest a potential use for such related steroidal glycosides in controlling rice-blast disease.
Erjavec N, Bayot A, Gareil M, Camougrand N
Free Radic Biol Med. 2013 Mar;56:9-16
The Saccharomyces cerevisiae homolog of the ATP-dependent Lon protease, Pim1p, is essential for mitochondrial protein quality control, DNA maintenance, and respiration. Here, we demonstrate that Pim1p activity declines in aging cells and that Pim1p deficiency shortens the replicative life span of yeast mother cells. This accelerated aging of pim1Δ cells is accompanied by elevated cytosolic levels of oxidized and aggregated proteins, as well as reduced proteasome activity. Overproduction of Hsp104p greatly diminishes aggregation of oxidized cytosolic proteins, rescuesproteasome activity, and restores life span of pim1Δ cells to near wild-type levels. Our results show that defects in mitochondrial protein quality control have global intracellular effects leading to the increased generation of misfolded proteins and cytosolic protein aggregates, which are linked to a decline in replicative potential.
Boveri S, Rainieri S, Pulvirenti A
Can J Microbiol. 2012 Mar;58(3):350–8
The crossing of Saccharomyces strains by spore conjugation is one of the ways to obtain new starter cultures for the fermentation industry. One of the major difficulties of this practice is the identification of the newly formed hybrids. In this work we describe an effective molecular method for the validation of Saccharomyces intraspecific crosses. The method described is based in the hypothesis that hybrids constructed by spore conjugation contain the sum of the genomes of both parental strains. As a consequence, the conjugation of spores of two yeasts showing different genomic fingerprinting profiles will result in a hybrid culture that will show the sum of both profiles. We demonstrated that the detection of polymorphism in two genes containing minisatellite-like sequences, either SED1 or AGA1, is suitable for this purpose. Using this strategy we were able to validate 15crosses out of 162 hybridization attempts.
Belyi Y, Tartakovskaya D, Tais A, Fitzke E, Tzivelekidis T, Jank T, Rospert S, Aktories K
J Biol Chem. 2012 Jul 27;287(31):26029–37
Legionella is a pathogenic Gram-negative bacterium that can multiply inside of eukaryotic cells. It translocates numerous bacterial effector proteins into target cells to transform host phagocytes into a niche for replication. One effector of Legionella pneumophila is the glucosyltransferase Lgt1, which modifies serine 53 in mammalian elongation factor 1A (eEF1A), resulting in inhibition of protein synthesis and cell death. Here, we demonstrate that similar to mammalian cells, Lgt1 was severely toxic when produced in yeast and effectively inhibited in vitro protein synthesis. Saccharomyces cerevisiae strains, which were deleted of endogenous eEF1A but harbored a mutant eEF1A not glucosylated by Lgt1, were resistant toward the bacterial effector. In contrast, deletion of Hbs1, which is also an in vitro substrate of the glucosyltransferase, did not influence the toxic effects of Lgt1. Serial mutagenesis in yeast showed that Phe(54), Tyr(56) and Trp(58), located immediately downstream of serine 53 of eEF1A, are essential for the function of the elongation factor. Replacement of serine 53 by glutamic acid, mimicking phosphorylation, produced a non-functional eEF1A, which failed to support growth of S. cerevisiae. Our data indicate that Lgt1-induced lethal effect in yeast depends solely on eEF1A. The region of eEF1A encompassing serine 53 plays a critical role in functioning of the elongation factor.
Gutiérrez A, Beltran G, Warringer J, Guillamón JM
PLoS ONE. 2013;8(6):e67166
The capacity of wine yeast to utilize the nitrogen available in grape must directly correlates with the fermentation and growth rates of all wine yeastfermentation stages and is, thus, of critical importance for wine production. Here we precisely quantified the ability of low complexity nitrogencompounds to support fast, efficient and rapidly initiated growth of four commercially important wine strains. Nitrogen substrate abundance in grape must failed to correlate with the rate or the efficiency of nitrogen source utilization, but well predicted lag phase length. Thus, human domestication of yeast for grape must growth has had, at the most, a marginal impact on wine yeast growth rates and efficiencies, but may have left a surprising imprint on the time required to adjust metabolism from non growth to growth. Wine yeast nitrogen source utilization deviated from that of the lab strain experimentation, but also varied between wine strains. Each wine yeast lineage harbored nitrogen source utilization defects that were private to that strain. By a massive hemizygote analysis, we traced the genetic basis of the most glaring of these defects, near inability of the PDM wine strain to utilize methionine, as consequence of mutations in its ARO8, ADE5,7 and VBA3 alleles. We also identified candidate causative mutations in these genes. The methionine defect of PDM is potentially very interesting as the strain can, in some circumstances, overproduce foul tasting H2S, a trait which likely stems from insufficient methionine catabolization. The poor adaptation of wine yeast to the grape must nitrogen environment, and the presence of defects in each lineage, open up wine strain optimization through biotechnological endeavors.
Fernández-Vázquez J, Vargas-Pérez I, Sansó M
PLoS Genet. 2013;9(7):e1003647
The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNA(Lys) UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery.
Sengupta S, Kaufmann A, T S C
J Basic Microbiol. 2013 Apr 2;54(1):81–7
Ashbya gossypii is a riboflavin overproducing filamentous fungus. RIB1 and RIB3 genes encode GTP-cyclohydrolase II (GCH II) and DHBP synthase, respectively, the two rate limiting enzymes of the riboflavin biosynthetic pathway. The genes encoding yeast enhanced green fluorescent protein (yEGFP) and mCherry were fused with RIB1 and RIB3 genes, under their native promoters by PCR-based gene tagging method for their earlyin vivo expression and cellular localization in A. gossypii. In the integrative transformants, the fusion proteins were expressed as cytoplasmic proteins from the germ bubble stage, in all the cells throughout the hypha. This was evident from the observation that mCherry fusion proteins were seen outside the vacuoles in the cytoplasm. The older matured cells of 14 h hyphae developed large vacuoles which showed green autofluorescence due to riboflavin. It is concluded that RIB1 and RIB3 genes are constitutively expressed in all the cells of this multicellular multinucleate fungus.
Shimizu K, Imanishi Y, Toh-e A, Uno J, Chibana H, Hull CM, Kawamoto S
Fungal Genet Biol. 2014 Aug;69:13–22
Diazobenzoic acid B (DBB), also known as diazonium blue B or fast blue B, can be used to distinguish basidiomycetous yeasts from ascomycetes. This chemical has long been used for the taxonomic study of yeast species at the phylum level, but the mechanism underlying the DBB staining remains unknown. To identify molecular targets of DBB staining, we isolated Agrobacterium tumefaciens-mediated insertional mutants of Cryptococcus neoformans, a basidiomycetous pathogenic yeast, which were negative to DBB staining. In one of these mutants, we found that the PMT2 gene, encoding a protein-O-mannosyltransferase, was interrupted by a T-DNA insertion. A complete gene knockout of the PMT2 gene revealed that the gene was responsible for DBB staining in C. neoformans, suggesting that one of the targets of Pmt2-mediated glycosylation is responsible for interacting with DBB. We also determined that Cryptococcus gattii, a close relative of C. neoformans, was not stained by DBB when the PMT2 gene was deleted. Our finding suggests that the protein-O-mannosylation by the PMT2 gene product is required for DBB staining in Cryptococcus species in general. We also showed that glycosylation in Cryptococcus by Pmt2 plays important roles in controlling cell size, resistance to high temperature and osmolarity, capsule formation, sexual reproduction, and virulence.