scholarly journals A Genomewide Screen for Tolerance to Cationic Drugs Reveals Genes Important for Potassium Homeostasis in Saccharomyces cerevisiae

2011 ◽  
Vol 10 (9) ◽  
pp. 1241-1250 ◽  
Author(s):  
Lina Barreto ◽  
David Canadell ◽  
Silvia Petrezsélyová ◽  
Clara Navarrete ◽  
Lydie Marešová ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Potassium Concentration ◽  
Growth Defect ◽  
Electrochemical Gradient ◽  
Biochemical Tests ◽  
Content Type ◽  
Potassium Homeostasis ◽  
Moderate Growth ◽  
Number Of Genes ◽  
Mutant Strains

ABSTRACTPotassium homeostasis is crucial for living cells. In the yeastSaccharomyces cerevisiae, the uptake of potassium is driven by the electrochemical gradient generated by the Pma1 H+-ATPase, and this process represents a major consumer of the gradient. We considered that any mutation resulting in an alteration of the electrochemical gradient could give rise to anomalous sensitivity to any cationic drug independently of its toxicity mechanism. Here, we describe a genomewide screen for mutants that present altered tolerance to hygromycin B, spermine, and tetramethylammonium. Two hundred twenty-six mutant strains displayed altered tolerance to all three drugs (202 hypersensitive and 24 hypertolerant), and more than 50% presented a strong or moderate growth defect at a limiting potassium concentration (1 mM). Functional groups such as protein kinases and phosphatases, intracellular trafficking, transcription, or cell cycle and DNA processing were enriched. Essentially, our screen has identified a substantial number of genes that were not previously described to play a direct or indirect role in potassium homeostasis. A subset of 27 representative mutants were selected and subjected to diverse biochemical tests that, in some cases, allowed us to postulate the basis for the observed phenotypes.

scholarly journals Genetic Analysis of the Shared Role of CLN3 and BCK2 at the G1-S Transition in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (3) ◽  
pp. 1131-1143
Author(s):  
Herman Wijnen ◽  
Bruce Futcher
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Target Genes ◽  
S Phase ◽  
Negative Regulator ◽  
Growth Defect ◽  
Uncharacterized Protein ◽  
Number Of Genes ◽  
Transcription Complexes

Abstract The transcription complexes SBF and MBF mediate the G1-S transition in the cell cycle of Saccharomyces cerevisiae. In late G1, SBF and MBF induce a burst of transcription in a number of genes, including G1- and S-phase cyclins. Activation of SBF and MBF depends on the G1 cyclin Cln3 and a largely uncharacterized protein called Bck2. We show here that the induction of SBF/MBF target genes by Bck2 depends partly, but not wholly, on SBF and MBF. Unlike Cln3, Bck2 is capable of inducing its transcriptional targets in the absence of functional Cdc28. Our results revealed promoter-specific mechanisms of regulation by Cln3, Bck2, SBF, and MBF. We isolated high-copy suppressors of the cln3 bck2 growth defect; all of these had the ability to increase CLN2 expression. One of these suppressors was the negative regulator of meiosis RME1. Rme1 induces CLN2, and we show that it has a haploid-specific role in regulating cell size and pheromone sensitivity. Genetic analysis of the cln3 bck2 defect showed that CLN1, CLN2, and other SBF/MBF target genes have an essential role in addition to the degradation of Sic1.

scholarly journals Functional Complementation Analyses Reveal that the Single PRAT Family Protein of Trypanosoma brucei Is a Divergent Homolog of Tim17 in Saccharomyces cerevisiae

2015 ◽  
Vol 14 (3) ◽  
pp. 286-296 ◽  
Author(s):  
Ebony Weems ◽  
Ujjal K. Singha ◽  
VaNae Hamilton ◽  
Joseph T. Smith ◽  
Karin Waegemann ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trypanosoma Brucei ◽  
Mitochondrial Proteins ◽  
Growth Defect ◽  
Heterologous Proteins ◽  
Homologous Proteins ◽  
Content Type ◽  
Mitochondrial Inner Membrane ◽  
Family Protein ◽  
Higher Eukaryotes

ABSTRACT Trypanosoma brucei , a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae . We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. brucei . These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. brucei was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the T. brucei mitochondrion and thus failed to complement the function of TbTim17.

scholarly journals CsrA Supports both Environmental Persistence and Host-Associated Growth of Acinetobacter baumannii

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
John M. Farrow ◽  
Greg Wells ◽  
Samantha Palethorpe ◽  
Mark D. Adams ◽  
Everett C. Pesci
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Bacterial Pathogen ◽  
Multidrug Resistant ◽  
Growth Defect ◽  
Gene Encoding ◽  
Content Type ◽  
Indirect Transmission ◽  
Mutant Strains ◽  
Carbon Storage Regulator

ABSTRACT Acinetobacter baumannii is an opportunistic and frequently multidrug-resistant Gram-negative bacterial pathogen that primarily infects critically ill individuals. Indirect transmission from patient to patient in hospitals can drive infections, supported by this organism’s abilities to persist on dry surfaces and rapidly colonize susceptible individuals. To investigate how A. baumannii survives on surfaces, we cultured A. baumannii in liquid media for several days and then analyzed isolates that lost the ability to survive drying. One of these isolates carried a mutation that affected the gene encoding the carbon storage regulator CsrA. As we began to examine the role of CsrA in A. baumannii, we observed that the growth of ΔcsrA mutant strains was inhibited in the presence of amino acids. The ΔcsrA mutant strains had a reduced ability to survive drying and to form biofilms but an improved ability to tolerate increased osmolarity compared with the wild type. We also examined the importance of CsrA for A. baumannii virulence. The ΔcsrA mutant strains had a greatly reduced ability to kill Galleria mellonella larvae, could not replicate in G. mellonella hemolymph, and also had a growth defect in human serum. Together, these results show that CsrA is essential for the growth of A. baumannii on host-derived substrates and is involved in desiccation tolerance, implying that CsrA controls key functions involved in the transmission of A. baumannii in hospitals.

scholarly journals Lsm12 Mediates Deubiquitination of DNA Polymerase η To Help Saccharomyces cerevisiae Resist Oxidative Stress

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
Rui Yao ◽  
Liujia Shi ◽  
Chengjin Wu ◽  
Weihua Qiao ◽  
Liming Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Cell Growth ◽  
Dna Polymerase ◽  
Genome Stability ◽  
Fold Increase ◽  
Growth Defect ◽  
Physical Interaction ◽  
Content Type

ABSTRACT In Saccharomyces cerevisiae, the Y family DNA polymerase η (Polη) regulates genome stability in response to different forms of environmental stress by translesion DNA synthesis. To elucidate the role of Polη in oxidative stress-induced DNA damage, we deleted or overexpressed the corresponding gene RAD30 and used transcriptome analysis to screen the potential genes associated with RAD30 to respond to DNA damage. Under 2 mM H2O2 treatment, the deletion of RAD30 resulted in a 2.2-fold decrease in survival and a 2.8-fold increase in DNA damage, whereas overexpression of RAD30 increased survival and decreased DNA damage by 1.2- and 1.4-fold, respectively, compared with the wild-type strain. Transcriptome and phenotypic analyses identified Lsm12 as a main factor involved in oxidative stress-induced DNA damage. Deleting LSM12 caused growth defects, while its overexpression enhanced cell growth under 2 mM H2O2 treatment. This effect was due to the physical interaction of Lsm12 with the UBZ domain of Polη to enhance Polη deubiquitination through Ubp3 and consequently promote Polη recruitment. Overall, these findings demonstrate that Lsm12 is a novel regulator mediating Polη deubiquitination to promote its recruitment under oxidative stress. Furthermore, this study provides a potential strategy to maintain the genome stability of industrial strains during fermentation. IMPORTANCE Polη was shown to be critical for cell growth in the yeast Saccharomyces cerevisiae, and deletion of its corresponding gene RAD30 caused a severe growth defect under exposure to oxidative stress with 2 mM H2O2. Furthermore, we found that Lsm12 physically interacts with Polη and promotes Polη deubiquitination and recruitment. Overall, these findings indicate Lsm12 is a novel regulator mediating Polη deubiquitination that regulates its recruitment in response to DNA damage induced by oxidative stress.

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals Discovery of a Novel Class of Orally Active Antifungal β-1,3-d-Glucan Synthase Inhibitors

2011 ◽  
Vol 55 (11) ◽  
pp. 5099-5106 ◽  
Author(s):  
Scott S. Walker ◽  
Yiming Xu ◽  
Ilias Triantafyllou ◽  
Michelle F. Waldman ◽  
Cara Mendrick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Pharmacokinetic Data ◽  
Morphological Response ◽  
Glucan Synthase ◽  
Content Type ◽  
Safety Issues ◽  
Spontaneous Mutants ◽  
Orally Active

ABSTRACTThe echinocandins are a class of semisynthetic natural products that target β-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibitedin vitroactivity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GSin vitro, and there was a strong correlation between enzyme inhibition andin vitroantifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants ofSaccharomyces cerevisiaewith reduced susceptibility to the piperazinyl-pyridazinones had substitutions inFKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model ofCandida glabratainfection.

scholarly journals Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4387-4395 ◽  
Author(s):  
D Mack ◽  
K Nishimura ◽  
B K Dennehey ◽  
T Arbogast ◽  
J Parkinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Lethality ◽  
Cell Polarization ◽  
Growth Defect ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Multicopy Suppressor ◽  
Bud Emergence ◽  
Multicopy Suppressors ◽  
Multiple Copies

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

scholarly journals Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

2013 ◽  
Vol 13 (1) ◽  
pp. 2-9 ◽  
Author(s):  
Frans M. Klis ◽  
Chris G. de Koster ◽  
Stanley Brul
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Candida Albicans ◽  
Cell Size ◽  
Pathogenic Fungus ◽  
Turgor Pressure ◽  
Hyphal Growth ◽  
Biological Research ◽  
Content Type ◽  
Starting Point

ABSTRACTBionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeastSaccharomyces cerevisiaeand the polymorphic, pathogenic fungusCandida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation ofin vivovalues. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allowsC. albicansto cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.

scholarly journals Subtelomeric Repeat Amplification Is Associated With Growth at Elevated Temperature in yku70 Mutants of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1039-1051
Author(s):  
Barbara Fellerhoff ◽  
Friederike Eckardt-Schupp ◽  
Anna A Friedl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperature ◽  
Alternative Pathway ◽  
Dna Amplification ◽  
Strand Break ◽  
Significant Loss ◽  
Growth Defect ◽  
Slight Reduction ◽  
Telomeric Sequences ◽  
Repair Defect

Abstract Inactivation of the Saccharomyces cerevisiae gene YKU70 (HDF1), which encodes one subunit of the Ku heterodimer, confers a DNA double-strand break repair defect, shortening of and structural alterations in the telomeres, and a severe growth defect at 37°. To elucidate the basis of the temperature sensitivity, we analyzed subclones derived from rare yku70 mutant cells that formed a colony when plated at elevated temperature. In all these temperature-resistant subclones, but not in cell populations shifted to 37°, we observed substantial amplification and redistribution of subtelomeric Y′ element DNA. Amplification of Y′ elements and adjacent telomeric sequences has been described as an alternative pathway for chromosome end stabilization that is used by postsenescence survivors of mutants deficient for the telomerase pathway. Our data suggest that the combination of Ku deficiency and elevated temperature induces a potentially lethal alteration of telomere structure or function. Both in yku70 mutants and in wild type, incubation at 37° results in a slight reduction of the mean length of terminal restriction fragments, but not in a significant loss of telomeric (C1-3A/TG1-3)n sequences. We propose that the absence of Ku, which is known to bind to telomeres, affects the telomeric chromatin so that its chromosome end-defining function is lost at 37°.

Leucobacter soli sp. nov., from soil amended with humic acid

2021 ◽  
Vol 71 (12) ◽  
Author(s):  
Peter Kämpfer ◽  
John A. McInroy ◽  
Dominique Clermont ◽  
Meina Neumann-Schaal ◽  
Alexis Criscuolo ◽  
...  
Keyword(s):  
Humic Acid ◽  
Type Strain ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Polar Lipid Profile ◽  
Phenotypic Differentiation ◽  
Content Type ◽  
Link Type ◽  
Diamino Acid

A Gram-positive, non-spore-forming actinobacterium (IMT-300T) was isolated from soil amended with humic acid in Malvern, AL, USA. This soil has been used for 50+years for the cultivation of earthworms for use as fish bait. Based on 16S rRNA gene sequence similarity studies, strain IMT-300T was shown to belong to the genus Leucobacter and was closely related to the type strain of ‘Leucobacter margaritiformis’ L1T (97.8%). Similarity to all other type strains of Leucobacter species was lower than 97.2 %. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the IMT-300T genome assembly and those of the closest relative Leucobacter type strain were 81.4 and 23.3 % ( Leucobacter chironomi ), respectively. The peptidoglycan of strain IMT-300T contained l-2,4-diaminobutyric acid as the diagnostic diamino acid. In addition, glycine, d- and l-alanine and d-glutamic acid were found. The peptidoglycan type represents a variant of B2δ (B11). The major quinones were menaquinones MK-10 and MK-11. The polar lipid profile consisted of the major lipids diphosphatidylglycerol, phosphatidylglycerol and moderate to minor amounts of two unidentified phospholipids, two unidentified glycolipids and an unidentified aminophospholipid. The polyamine pattern contained major amounts of spermidine and spermine. Strain IMT-300T contained the major fatty acids C15 : 0 anteiso, C16 : 0 iso and C17 : 0 anteiso, like other members of the genus Leucobacter . The results of ANI and dDDH analyses and physiological and biochemical tests allowed a genotypic and phenotypic differentiation of strain IMT-300T from the most closely related Leucobacter species. Strain IMT-300T represents a novel Leucobacter species, for which we propose the name Leucobacter soli sp. nov., with the type strain IMT-300T (CIP 111803T=DSM 110505T=CCM 9020T=LMG 31600T).

Sign in / Sign up

Export Citation Format

Share Document