| 1. |
- Andresen, Liis, et al.
(author)
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The Small Toxic Salmonella Protein TimP Targets the Cytoplasmic Membrane and Is Repressed by the Small RNA TimR
- 2020
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In: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 11
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Journal article (peer-reviewed)abstract
- Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae. Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane. IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.
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| 2. |
- Ayala, Julio C., et al.
(author)
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Gonococcal Clinical Strains Bearing a Common gdhR Single Nucleotide Polymorphism That Results in Enhanced Expression of the Virulence Gene lctP Frequently Possess a mtrR Promoter Mutation That Decreases Antibiotic Susceptibility
- 2022
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In: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 13:2
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Journal article (peer-reviewed)abstract
- GdhR is a transcriptional repressor of the virulence factor gene lctP, which encodes a unique l-lactate permease that has been linked to pathogenesis of Neisseria gonorrhoeae, and loss of gdhR can confer increased fitness of gonococci in a female mouse model of lower genital tract infection. In this work, we identified a single nucleotide polymorphism (SNP) in gdhR, which is often present in both recent and historical gonococcal clinical strains and results in a proline (P)-to-serine (S) change at amino acid position 6 (P6S) of GdhR. This mutation (gdhR6) was found to reduce GdhR transcriptional repression at lctP in gonococcal strains containing the mutant protein compared to wild-type GdhR. By using purified recombinant proteins and in vitro DNA-binding and cross-linking experiments, we found that gdhR6 impairs the DNA-binding activity of GdhR at lctP without an apparent effect on protein oligomerization. By analyzing a panel of U.S. (from 2017 to 2018) and Danish (1928 to 2013) clinical isolates, we observed a statistical association between gdhR6 and the previously described adenine deletion in the promoter of mtrR (mtrR-P A-del), encoding the repressor (MtrR) of the mtrCDE operon that encodes the MtrCDE multidrug efflux pump that can export antibiotics, host antimicrobials, and biocides. The frequent association of gdhR6 with the mtrR promoter mutation in these clinical isolates suggests that it has persisted in this genetic background to enhance lctP expression, thereby promoting virulence. IMPORTANCE We report the frequent appearance of a novel SNP in the gdhR gene (gdhR6) possessed by Neisseria gonorrhoeae. The resulting amino acid change in the GdhR protein resulted in enhanced expression of a virulence gene (lctP) that has been suggested to promote gonococcal survival during infection. The mutant GdhR protein expressed by gdhR6 had a reduced ability to bind to its target DNA sequence upstream of lctP. Interestingly, gdhR6 was found in clinical gonococcal strains isolated in the United States and Denmark at a high frequency and was frequently associated with a mutation in the promoter of the gene encoding a repressor (MtrR) of both the mtrCDE antimicrobial efflux pump operon and gdhR. Given this frequent association and the known impact of these regulatory mutations, we propose that virulence and antibiotic resistance properties are often phenotypically linked in contemporary gonococcal strains.
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| 3. |
- Babina, Arianne M, et al.
(author)
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In Vivo Behavior of the Tandem Glycine Riboswitch in Bacillus subtilis.
- 2017
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In: mBio. - 2161-2129 .- 2150-7511. ; 8:5
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Journal article (peer-reviewed)abstract
- In many bacterial species, the glycine riboswitch is composed of two homologous ligand-binding domains (aptamers) that each bind glycine and act together to regulate the expression of glycine metabolic and transport genes. While the structure and molecular dynamics of the tandem glycine riboswitch have been the subject of numerous in vitro studies, the in vivo behavior of the riboswitch remains largely uncharacterized. To examine the proposed models of tandem glycine riboswitch function in a biologically relevant context, we characterized the regulatory activity of mutations to the riboswitch structure in Bacillus subtilis using β-galactosidase assays. To assess the impact disruptions to riboswitch function have on cell fitness, we introduced these mutations into the native locus of the tandem glycine riboswitch within the B. subtilis genome. Our results indicate that glycine does not need to bind both aptamers for regulation in vivo and mutations perturbing riboswitch tertiary structure have the most severe effect on riboswitch function and gene expression. We also find that in B. subtilis, the glycine riboswitch-regulated gcvT operon is important for glycine detoxification.IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets.
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| 4. |
- Boeck, Desiree, et al.
(author)
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The Polar Legionella Icm/Dot T4SS Establishes Distinct Contact Sites with the Pathogen Vacuole Membrane
- 2021
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In: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 12:5
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Journal article (peer-reviewed)abstract
- Legionella pneumophila, the causative agent of Legionnaires disease, is a facultative intracellular pathogen that survives inside phagocytic host cells by establishing a protected replication niche, termed the "Legionella-containing vacuole" (LCV). To form an LCV and subvert pivotal host pathways, L pneumophila employs a type IV secretion system (T4SS), which translocates more than 300 different effector proteins into the host cell. The L. pneumophila T4SS complex has been shown to span the bacterial cell envelope at the bacterial poles. However, the interactions between the T4SS and the LCV membrane are not understood. Using cryo-focused ion beam milling, cryo-electron tomography, and confocal laser scanning fluorescence microscopy, we show that up to half of the intravacuolar L. pneumophila bacteria tether their cell pole to the LCV membrane. Tethering coincides with the presence and function of T4SSs and likely promotes the establishment of distinct contact sites between T4SSs and the LCV membrane. Contact sites are characterized by indentations in the limiting LCV membrane and localize juxtaposed to T4SS machineries. The data are in agreement with the notion that effector translocation occurs by close membrane contact rather than by an extended pilus. Our findings provide novel insights into the interactions of the L. pneumophila T4SS with the LCV membrane in situ. IMPORTANCE Legionnaires disease is a life-threatening pneumonia, which is characterized by high fever, coughing, shortness of breath, muscle pain, and headache. The disease is caused by the amoeba-resistant bacterium L. pneumophila found in various soil and aquatic environments and is transmitted to humans via the inhalation of small bacteria-containing droplets. An essential virulence factor of L pneumophila is a so-called "type IV secretion system" (T4SS), which, by injecting a plethora of "effector proteins" into the host cell, determines pathogen-host interactions and the formation of a distinct intracellular compartment, the "Legionella-containing vacuole" (LCV). It is unknown how the T4SS makes contact to the LCV membrane to deliver the effectors. In this study, we identify indentations in the host cell membrane in close proximity to functional T4SSs localizing at the bacterial poles. Our work reveals first insights into the architecture of Legionella-LCV contact sites.
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| 5. |
- Bolisetty, M., et al.
(author)
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Unexpected diversity and expression of avian endogenous retroviruses
- 2012
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In: mBio. - 2161-2129. ; 3:5, s. e00344-12
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Journal article (peer-reviewed)abstract
- Endogenous retroviruses (ERVs) were identified and characterized in three avian genomes to gain insight into early retroviral evolution. Using the computer program RetroTector to detect relatively intact ERVs, we identified 500 ERVs in the chicken genome, 150 in the turkey genome, and 1,200 in the zebra finch genome. Previous studies suggested that endogenous alpharetroviruses were present in chicken genomes. In this analysis, a small number of alpharetroviruses were seen in the chicken and turkey genomes; however, these were greatly outnumbered by beta-like, gamma-like, and alphabeta proviruses. While the avian ERVs belonged to the same major groups as mammalian ERVs, they were more heterogeneous. In particular, the beta-like viruses revealed an evolutionary continuum with the gradual acquisition and loss of betaretroviral markers and a transition from beta to alphabeta and then to alpharetroviruses. Thus, it appears that birds may resemble a melting pot for early ERV evolution. Many of the ERVs were integrated in clusters on chromosomes, often near centromeres. About 25% of the chicken ERVs were in or near cellular transcription units; this is nearly random. The majority of these integrations were in the sense orientation in introns. A higher-than-random number of integrations were >100 kb from the nearest gene. Deep-sequencing studies of chicken embryo fibroblasts revealed that about 20% of the 500 ERVs were transcribed and translated. A subset of these were also transcribed in vivo in chickens, showing tissue-specific patterns of expression.
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| 6. |
- Bueno, Emilio, et al.
(author)
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Transient glycolytic complexation of arsenate enhances resistance in the enteropathogen Vibrio cholerae
- 2022
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In: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 13:5
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Journal article (peer-reviewed)abstract
- The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we found that the cholera pathogen V. cholerae exhibits increased resistance to arsenate compared to closely related enteric pathogens. Such resistance is promoted not by ArsC-dependent reduction of arsenate to arsenite but by an operon encoding an arsenate transporter (ArsJ), an alternative glyceraldehyde 3-phosphate dehydrogenase (VarG), and a putative, uncharacterized phosphatase (VarH). Mechanistically, we demonstrate that V. cholerae detoxifies arsenate by complexing it with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). 1As3PG is not transported outside the cell; instead, it is subsequently dissociated by VarH to enable extrusion of free arsenate through ArsJ. Collectively, this study proposes a novel mechanism for arsenate detoxification, entirely independent of arsenate reduction and arsenite extrusion, that enhances V. cholerae resistance to this metalloid compared to other enteric pathogens.
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| 7. |
- Bäckström, Disa, et al.
(author)
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Virus Genomes from Deep Sea Sediments Expand the Ocean Megavirome and Support Independent Origins of Viral Gigantism
- 2019
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In: mBio. - 2161-2129 .- 2150-7511. ; 10:2
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Journal article (peer-reviewed)abstract
- The nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes (proposed order, “Megavirales”) include the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, and Mimiviridae, as well as still unclassified pithoviruses, pandoraviruses, molliviruses, and faustoviruses. Several of these virus groups include giant viruses, with genome and particle sizes exceeding those of many bacterial and archaeal cells. We explored the diversity of the NCLDV in deep sea sediments from the Loki’s Castle hydrothermal vent area. Using metagenomics, we reconstructed 23 high-quality genomic bins of novel NCLDV, 15 of which are related to pithoviruses, 5 to marseilleviruses, 1 to iridoviruses, and 2 to klosneuviruses. Some of the identified pithovirus-like and marseillevirus-like genomes belong to deep branches in the phylogenetic tree of core NCLDV genes, substantially expanding the diversity and phylogenetic depth of the respective groups. The discovered viruses, including putative giant members of the family Marseilleviridae, have a broad range of apparent genome sizes, in agreement with the multiple, independent origins of gigantism in different branches of the NCLDV. Phylogenomic analysis reaffirms the monophyly of the pithovirus-iridovirus-marseillevirus branch of the NCLDV. Similarly to other giant viruses, the pithovirus-like viruses from Loki’s Castle encode translation systems components. Phylogenetic analysis of these genes indicates a greater bacterial contribution than had been detected previously. Genome comparison suggests extensive gene exchange between members of the pithovirus-like viruses and Mimiviridae. Further exploration of the genomic diversity of Megavirales in additional sediment samples is expected to yield new insights into the evolution of giant viruses and the composition of the ocean megavirome.Importance: Genomics and evolution of giant viruses are two of the most vigorously developing areas of virus research. Lately, metagenomics has become the main source of new virus genomes. Here we describe a metagenomic analysis of the genomes of large and giant viruses from deep sea sediments. The assembled new virus genomes substantially expand the known diversity of the nucleocytoplasmic large DNA viruses of eukaryotes. The results support the concept of independent evolution of giant viruses from smaller ancestors in different virus branches.
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| 8. |
- Campbell, Christopher, et al.
(author)
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Accumulation of succinyl coenzyme a perturbs the methicillin-resistant staphylococcus aureus (Mrsa) succinylome and is associated with increased susceptibility to beta-lactam antibiotics
- 2021
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In: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 12:3
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Journal article (peer-reviewed)abstract
- Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics.
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| 9. |
- Cao, Sha, et al.
(author)
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Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus
- 2017
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In: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 8:3
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Journal article (peer-reviewed)abstract
- Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD(+), thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections. IMPORTANCE Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.
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| 10. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(author)
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Thermotolerant Yeast Strains Adapted by Laboratory Evolution Show Trade-Off at Ancestral Temperatures and Preadaptation to Other Stresses
- 2015
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In: mBio. - 2150-7511 .- 2161-2129. ; 6:4
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Journal article (peer-reviewed)abstract
- A major challenge for the production of ethanol from biomass-derived feedstocks is to develop yeasts that can sustain growth under the variety of inhibitory conditions present in the production process, e.g., high osmolality, high ethanol titers, and/or elevated temperatures (>40 degrees C). Using adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40 degrees C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses. Thermotolerant yeast strains showed horizontal displacement of their thermal reaction norms to higher temperatures. Hence, their optimal and maximum growth temperatures increased by about 3 degrees C, whereas they showed a growth trade-off at temperatures below 34 degrees C. Computational analysis of the physical properties of proteins showed that the lethal temperature for yeast is around 49 degrees C, as a large fraction of the yeast proteins denature above this temperature. Our analysis also indicated that the number of functions involved in controlling the growth rate decreased in the thermotolerant strains compared with the number in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures. In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance to inhibitory conditions found in industrial ethanol production processes. IMPORTANCE Yeast thermotolerance can significantly reduce the production costs of biomass conversion to ethanol. However, little information is available about the underlying genetic changes and physiological functions required for yeast thermotolerance. We recently revealed the genetic changes of thermotolerance in thermotolerant yeast strains (TTSs) generated through adaptive laboratory evolution. Here, we examined these TTSs' physiology and computed their proteome stability over the entire thermal niche, as well as their preadaptation to other stresses. Using this approach, we showed that TTSs exhibited evolutionary trade-offs in the ancestral thermal niche, as well as reduced numbers of growth functions and preadaptation to other stresses found in ethanol production processes. This information will be useful for rational engineering of yeast thermotolerance for the production of biofuels and chemicals.
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