Lack ofHXK2Induces Localization of Active Ras in Mitochondria and Triggers Apoptosis in the YeastSaccharomyces cerevisiae

We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in thehxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis inS. cerevisiae. In particular, cells lackingHXK2and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion ofHXK2causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

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Proteome and metabolome profiling of wild-type and YCA1 -knock-out yeast cells during acetic acid-induced programmed cell death

Journal of Proteomics ◽

10.1016/j.jprot.2015.08.003 ◽

2015 ◽

Vol 128 ◽

pp. 173-188 ◽

Cited By ~ 14

Author(s):

Valentina Longo ◽

Maša Ždralević ◽

Nicoletta Guaragnella ◽

Sergio Giannattasio ◽

Lello Zolla ◽

...

Keyword(s):

Cell Death ◽

Acetic Acid ◽

Programmed Cell Death ◽

Yeast Cells ◽

Wild Type ◽

Knock Out ◽

Metabolome Profiling

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Improvement of Phosphate Solubilization and Medicago Plant Yield by an Indole-3-Acetic Acid-Overproducing Strain of Sinorhizobium meliloti

Applied and Environmental Microbiology ◽

10.1128/aem.02756-09 ◽

2010 ◽

Vol 76 (14) ◽

pp. 4626-4632 ◽

Cited By ~ 80

Author(s):

Carmen Bianco ◽

Roberto Defez

Keyword(s):

Acetic Acid ◽

Plant Growth ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Dry Weight ◽

Limiting Factors ◽

Wild Type ◽

System A ◽

Indole 3 Acetic Acid

ABSTRACT Nitrogen (N) and phosphorus (P) are the most limiting factors for plant growth. Some microorganisms improve the uptake and availability of N and P, minimizing chemical fertilizer dependence. It has been published that the RD64 strain, a Sinorhizobium meliloti 1021 strain engineered to overproduce indole-3-acetic acid (IAA), showed improved nitrogen fixation ability compared to the wild-type 1021 strain. Here, we present data showing that RD64 is also highly effective in mobilizing P from insoluble sources, such as phosphate rock (PR). Under P-limiting conditions, the higher level of P-mobilizing activity of RD64 than of the 1021 wild-type strain is connected with the upregulation of genes coding for the high-affinity P transport system, the induction of acid phosphatase activity, and the increased secretion into the growth medium of malic, succinic, and fumaric acids. Medicago truncatula plants nodulated by RD64 (Mt-RD64), when grown under P-deficient conditions, released larger amounts of another P-solubilizing organic acid, 2-hydroxyglutaric acid, than plants nodulated by the wild-type strain (Mt-1021). It has already been shown that Mt-RD64 plants exhibited higher levels of dry-weight production than Mt-1021 plants. Here, we also report that P-starved Mt-RD64 plants show significant increases in both shoot and root fresh weights when compared to P-starved Mt-1021 plants. We discuss how, in a Rhizobium-legume model system, a balanced interplay of different factors linked to bacterial IAA overproduction rather than IAA production per se stimulates plant growth under stressful environmental conditions and, in particular, under P starvation.

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A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype inGluconobacter frateuriiby Increasing the NADPH/NADP+Ratio via Metabolic Change

Applied and Environmental Microbiology ◽

10.1128/aem.00354-18 ◽

2018 ◽

Vol 84 (10) ◽

Cited By ~ 6

Author(s):

Nami Matsumoto ◽

Hiromi Hattori ◽

Minenosuke Matsutani ◽

Chihiro Matayoshi ◽

Hirohide Toyama ◽

...

Keyword(s):

Acetic Acid ◽

High Temperatures ◽

Type Strain ◽

Wild Type Strain ◽

Metabolic Change ◽

Drug Transporter ◽

Transporter Gene ◽

Wild Type ◽

Content Type ◽

In The Wild

ABSTRACTThermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adaptedGluconobacter frateuriistrain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsABstrain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsABmutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP+ratio was higher in strain Wild-G, and much higher in the ΔotsABstrain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance inG. frateurii.IMPORTANCEThe biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show thatGluconobacterfrateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP+ratios and reductions in ROS levels. We concluded that inG. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP+ratio to enhance thermotolerance.

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Mitochondrial active Ras2 protein promotes apoptosis and regulated cell death in a cAMP/PKA pathway-dependent manner in budding yeast.

10.1101/2021.10.13.464237 ◽

2021 ◽

Author(s):

Barbara Bonomelli ◽

Enzo Martegani ◽

Sonia Colombo

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Acetic Acid ◽

Dependent Manner ◽

Ras Proteins ◽

Wild Type ◽

High Affinity ◽

Regulated Cell Death ◽

Molecular Machinery ◽

Pka Pathway

In previous papers, using the eGFP-RBD3 probe, which binds Ras-GTP with high affinity, we showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type Saccharomyces cerevisiae cells growing exponentially on glucose, while an aberrant accumulation of activated Ras in mitochondria correlates to mitochondrial dysfunction, accumulation of ROS and an increase of apoptosis. In this paper, we show that lack of TPS1, which is known to trigger apoptosis in S. cerevisiae, induces localization of active Ras proteins in mitochondria, confirming the above-mentioned correlation. Next, by characterizing the ras1Δ and ras2Δ mutants concerning localization of active Ras proteins and propensity to undergo cell death, we show that active Ras2 proteins, which accumulate in the mitochondria following addition of acetic acid, a well-known pro-apoptotic stimulus, might be the GTPases involved in regulated cell death, while active Ras1 proteins, constitutively localized in mitochondria, might be involved in a pro-survival molecular machinery. Finally, by characterizing the gpa2Δ and cyr1Δ mutants concerning the propensity to undergo cell death, we show that active mitochondrial Ras proteins promote apoptosis through the cAMP/PKA pathway.

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Novel Secretory Protein Ss-Caf1 of the Plant-Pathogenic Fungus Sclerotinia sclerotiorum Is Required for Host Penetration and Normal Sclerotial Development

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-13-0145-r ◽

2014 ◽

Vol 27 (1) ◽

pp. 40-55 ◽

Cited By ~ 45

Author(s):

Xueqiong Xiao ◽

Jiatao Xie ◽

Jiasen Cheng ◽

Guoqing Li ◽

Xianhong Yi ◽

...

Keyword(s):

Cell Death ◽

Sclerotinia Sclerotiorum ◽

Type Strain ◽

Wild Type Strain ◽

Tobacco Rattle Virus ◽

Secretory Protein ◽

Host Cells ◽

Wild Type ◽

Appressorium Formation ◽

Sclerotial Development

To decipher the mechanism of pathogenicity in Sclerotinia sclerotiorum, a pathogenicity-defective mutant, Sunf-MT6, was isolated from a T-DNA insertional library. Sunf-MT6 could not form compound appressorium and failed to induce lesions on leaves of rapeseed though it could produce more oxalic acid than the wild-type strain. However, it could enter into host tissues via wounds and cause typical necrotic lesions. Furthermore, Sunf-MT6 produced fewer but larger sclerotia than the wild-type strain Sunf-M. A gene, named Ss-caf1, was disrupted by T-DNA insertion in Sunf-MT6. Gene complementation and knockdown experiments confirmed that the disruption of Ss-caf1 was responsible for the phenotypic changes of Sunf-MT6. Ss-caf1 encodes a secretory protein with a putative Ca2+-binding EF-hand motif. High expression levels of Ss-caf1 were observed at an early stage of compound appressorium formation and in immature sclerotia. Expression of Ss-caf1 without signal peptides in Nicotiana benthamiana via Tobacco rattle virus-based vectors elicited cell death. These results suggest that Ss-caf1 plays an important role in compound appressorium formation and sclerotial development of S. sclerotiorum. In addition, Ss-Caf1 has the potential to interact with certain host proteins or unknown substances in host cells, resulting in subsequent host cell death.

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Transcriptome Analysis of Trichothecene-Induced Gene Expression in Barley

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-11-1364 ◽

2007 ◽

Vol 20 (11) ◽

pp. 1364-1375 ◽

Cited By ~ 77

Author(s):

Jayanand Boddu ◽

Seungho Cho ◽

Gary J. Muehlbauer

Keyword(s):

Cell Death ◽

Defense Response ◽

Type Strain ◽

Wild Type Strain ◽

Loss Of Function ◽

Wild Type ◽

Genes Encoding ◽

Basal Defense ◽

Related Proteins ◽

The Impact

Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

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Prodiginine Production inStreptomyces coelicolorCorrelates Temporally and Spatially to Programmed Cell Death

10.1101/240689 ◽

2018 ◽

Cited By ~ 3

Author(s):

Elodie Tenconi ◽

Matthew F. Traxler ◽

Charline Hoebreck ◽

Gilles P. Van Wezel ◽

Sébastien Rigali

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Genetic Basis ◽

Wild Type Strain ◽

Streptomyces Coelicolor ◽

Morphological Differentiation ◽

Wild Type ◽

Synthesis Inhibitor ◽

Dna Damaging Agents ◽

Spatio Temporal

AbstractProgrammed cell death (PCD) is a common feature of multicellularity and morphogenesis in bacteria. While cell death has been well documented whenStreptomycesspecies switch from vegetative (nutrition) to aerial (reproduction) growth, lethal determinants are yet to be discovered to unveil the genetic basis of PCD in mycelial bacteria. In this work we used prodiginines ofStreptomyces coelicoloras model to test the hypothesis that a bacterium uses ‘self-made’ antiproliferative DNA-damaging agents as toxins of their PCD process. Spatio-temporal visualisation of the autofluorescence of prodiginines reveals that their biosynthesis is triggered in the dying zone of the colony prior to morphological differentiation of the mycelium. A prodiginine nonproducer showed hyper-accumulation of viable filaments, with increased RNA and proteins synthesis when most of the mycelium of the wild-type strain was dead when prodiginine accumulated. Addition of a prodiginine synthesis inhibitor also strongly favoured viable over dead filaments. As self-toxicity has also been reported for other producers of DNA-damaging agents we propose that cytotoxic metabolites synthetized during the morphological transition of filamentous bacteria may be used to execute PCD.Significance StatementActinobacteria are prolific producers of compounds with antiproliferative activity, but why these bacteria synthetize metabolites with this bioactivity has so far remained a mystery. Using prodiginines (PdGs) as model system, we revealed that the spatio-temporal synthesis of these molecules correlates to cell death of the producerStreptomyces coelicolorand that inhibition of their synthesis results in hyper-accumulation of viable filaments. Since PdGs potentiate death ofS. coelicolorrecurrently prior to morphological differentiation, this is a form of programmed cell death (PCD). Hence, next to weapons in competition between organisms or signals in inter- and intra-species communications, we propose a third role for secondary metabolites i.e., elements required for self-toxicity in PCD processes.

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Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.2.383 ◽

1996 ◽

Vol 142 (2) ◽

pp. 383-391 ◽

Cited By ~ 2

Author(s):

Yasumasa Tsukamoto ◽

Jun-ichi Kato ◽

Hideo Ikeda

Keyword(s):

Saccharomyces Cerevisiae ◽

Quantitative Analysis ◽

Structural Analysis ◽

Recombination Rate ◽

Type Strain ◽

Negative Selection ◽

Wild Type Strain ◽

Illegitimate Recombination ◽

Wild Type ◽

In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

BMC Microbiology ◽

10.1186/s12866-020-02083-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Nayeong Kim ◽

Hyo Jeong Kim ◽

Man Hwan Oh ◽

Se Yeon Kim ◽

Mi Hyun Kim ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Mutant Strain ◽

Antimicrobial Susceptibility ◽

Type Strain ◽

Wild Type Strain ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Wild Type ◽

Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

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Translation machinery reprogramming in programmed cell death in Saccharomyces cerevisiae

Cell Death Discovery ◽

10.1038/s41420-020-00392-x ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Francesco Monticolo ◽

Emanuela Palomba ◽

Maria Luisa Chiusano

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Acetic Acid ◽

Programmed Cell Death ◽

Differential Expression ◽

Ribosomal Proteins ◽

Relative Proportion ◽

Duplication Event ◽

Genome Duplication Event ◽

Cell Death Pathway

AbstractProgrammed cell death involves complex molecular pathways in both eukaryotes and prokaryotes. In Escherichia coli, the toxin–antitoxin system (TA-system) has been described as a programmed cell death pathway in which mRNA and ribosome organizations are modified, favoring the production of specific death-related proteins, but also of a minor portion of survival proteins, determining the destiny of the cell population. In the eukaryote Saccharomyces cerevisiae, the ribosome was shown to change its stoichiometry in terms of ribosomal protein content during stress response, affecting the relative proportion between ohnologs, i.e., the couple of paralogs derived by a whole genome duplication event. Here, we confirm the differential expression of ribosomal proteins in yeast also during programmed cell death induced by acetic acid, and we highlight that also in this case pairs of ohnologs are involved. We also show that there are different trends in cytosolic and mitochondrial ribosomal proteins gene expression during the process. Moreover, we show that the exposure to acetic acid induces the differential expression of further genes coding for products related to translation processes and to rRNA post-transcriptional maturation, involving mRNA decapping, affecting translation accuracy, and snoRNA synthesis. Our results suggest that the reprogramming of the overall translation apparatus, including the cytosolic ribosome reorganization, are relevant events in yeast programmed cell death induced by acetic acid.

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