ABSTRACT Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to the yeast Saccharomyces cerevisiae for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle quickly and exhibit higher resistance to harmful chemicals than single cells. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation or about the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential for both floc formation and survival in a minimal aerobic medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in a minimal aerobic medium, providing a physiological role for this enzyme, which has previously been found to be disadvantageous in a rich aerobic medium. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation. IMPORTANCE The bacterium Zymomonas mobilis is best known for its anaerobic fermentative lifestyle, in which it converts glucose into ethanol at a yield surpassing that of yeast. However, Z. mobilis also has an aerobic lifestyle, which has confounded researchers with its attributes of poor growth, accumulation of toxic acetic acid and acetaldehyde, and respiratory enzymes that are detrimental for aerobic growth. Here we show that a major Z. mobilis respiratory enzyme and the ability to form multicellular aggregates, called flocs, are important for survival, but only during aerobic growth in a medium containing a minimum set of nutrients required for growth. Supplements, such as vitamins or yeast extract, promote aerobic growth and, in some cases, inhibit floc formation. We propose that Z. mobilis likely requires aerobic respiration and floc formation in order to survive in natural environments that lack protective factors found in supplements such as yeast extract.
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Independent Mechanisms for Acquired Salt Tolerance versus Growth Resumption Induced by Mild Ethanol Pretreatment in Saccharomyces cerevisiae
ABSTRACT All living organisms must recognize and respond to various environmental stresses throughout their lifetime. In natural environments, cells frequently encounter fluctuating concentrations of different stressors that can occur in combination or sequentially. Thus, the ability to anticipate an impending stress is likely ecologically relevant. One possible mechanism for anticipating future stress is acquired stress resistance, where cells preexposed to a mild sublethal dose of stress gain the ability to survive an otherwise lethal dose of stress. We have been leveraging wild strains of Saccharomyces cerevisiae to investigate natural variation in the yeast ethanol stress response and its role in acquired stress resistance. Here, we report that a wild vineyard isolate possesses ethanol-induced cross protection against severe concentrations of salt. Because this phenotype correlates with ethanol-dependent induction of the ENA genes, which encode sodium efflux pumps already associated with salt resistance, we hypothesized that variation in ENA expression was responsible for differences in acquired salt tolerance across strains. Surprisingly, we found that the ENA genes were completely dispensable for ethanol-induced survival of high salt concentrations in the wild vineyard strain. Instead, the ENA genes were necessary for the ability to resume growth on high concentrations of salt following a mild ethanol pretreatment. Surprisingly, this growth acclimation phenotype was also shared by the lab yeast strain despite lack of ENA induction under this condition. This study underscores that cross protection can affect both viability and growth through distinct mechanisms, both of which likely confer fitness effects that are ecologically relevant. IMPORTANCE Microbes in nature frequently experience “boom or bust” cycles of environmental stress. Thus, microbes that can anticipate the onset of stress would have an advantage. One way that microbes anticipate future stress is through acquired stress resistance, where cells exposed to a mild dose of one stress gain the ability to survive an otherwise lethal dose of a subsequent stress. In the budding yeast Saccharomyces cerevisiae , certain stressors can cross protect against high salt concentrations, though the mechanisms governing this acquired stress resistance are not well understood. In this study, we took advantage of wild yeast strains to understand the mechanism underlying ethanol-induced cross protection against high salt concentrations. We found that mild ethanol stress allows cells to resume growth on high salt, which involves a novel role for a well-studied salt transporter. Overall, this discovery highlights how leveraging natural variation can provide new insights into well-studied stress defense mechanisms.
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- Award ID(s):
- 1656602
- NSF-PAR ID:
- 10084171
- Date Published:
- Journal Name:
- mSphere
- Volume:
- 3
- Issue:
- 6
- ISSN:
- 2379-5042
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Most organisms must cope with temperature changes. This involves genes and gene networks both as subjects and agents of cellular protection, creating difficulties in understanding. Here, we study how heating and cooling affect expression of single genes and synthetic gene circuits in
Saccharomyces cerevisiae . We discovered that nonoptimal temperatures induce a cell fate choice between stress resistance and growth arrest. This creates dramatic gene expression bimodality in isogenic cell populations, as arrest abolishes gene expression. Multiscale models incorporating population dynamics, temperature-dependent growth rates, and Arrhenius scaling of reaction rates captured the effects of cooling, but not those of heating in resistant cells. Molecular-dynamics simulations revealed how heating alters the conformational dynamics of the TetR repressor, fully explaining the experimental observations. Overall, nonoptimal temperatures induce a cell fate decision and corrupt gene and gene network function in computationally predictable ways, which may aid future applications of engineered microbes in nonstandard temperatures. -
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Abstract The probiotic yeast
Saccharomyces boulardii (Sb ) is a promising chassis to deliver therapeutic proteins to the gut due toSb ’s innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineerSb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in bothcis- (i.e. to the expression cassette of the secreted protein) andtrans- (i.e. to theSb genome) that enhanceSb ’s ability to secrete proteins, taking aClostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76–458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge ofS. cerevisiae ’s secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficientSb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-typeSb . Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion inSb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability ofSb to deliver therapeutics to the gut and other settings to which it is adapted. -
Abstract Protein stability is a major constraint on protein evolution. Molecular chaperones, also known as heat-shock proteins, can relax this constraint and promote protein evolution by diminishing the deleterious effect of mutations on protein stability and folding. This effect, however, has only been stablished for a few chaperones. Here, we use a comprehensive chaperone-protein interaction network to study the effect of all yeast chaperones on the evolution of their protein substrates, that is, their clients. In particular, we analyze how yeast chaperones affect the evolutionary rates of their clients at two very different evolutionary time scales. We first study the effect of chaperone-mediated folding on protein evolution over the evolutionary divergence of Saccharomyces cerevisiae and S. paradoxus. We then test whether yeast chaperones have left a similar signature on the patterns of standing genetic variation found in modern wild and domesticated strains of S. cerevisiae. We find that genes encoding chaperone clients have diverged faster than genes encoding nonclient proteins when controlling for their number of protein-protein interactions. We also find that genes encoding client proteins have accumulated more intra-specific genetic diversity than those encoding nonclient proteins. In a number of multivariate analyses, controlling by other well-known factors that affect protein evolution, we find that chaperone dependence explains the largest fraction of the observed variance in the rate of evolution at both evolutionary time scales. Chaperones affecting rates of protein evolution mostly belong to two major chaperone families: Hsp70s and Hsp90s. Our analyses show that protein chaperones, by virtue of their ability to buffer destabilizing mutations and their role in modulating protein genotype-phenotype maps, have a considerable accelerating effect on protein evolution.more » « less
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Cellular aging in
Saccharomyces cerevisiae can lead to genomic instability and impaired mitotic asymmetry. To investigate the role of oxidative stress in cellular aging, we examined the effect of exogenous hydrogen peroxide on genomic instability and mitotic asymmetry in a collection of yeast strains with diverse backgrounds. We treated yeast cells with hydrogen peroxide and monitored the changes of viability and the frequencies of loss of heterozygosity (LOH) in response to hydrogen peroxide doses. The mid-transition points of viability and LOH were quantified using sigmoid mathematical functions. We found that the increase of hydrogen peroxide dependent genomic instability often occurs before a drop in viability. We previously observed that elevation of genomic instability generally lags behind the drop in viability during chronological aging. Hence, onset of genomic instability induced by exogenous hydrogen peroxide treatment is opposite to that induced by endogenous oxidative stress during chronological aging, with regards to the midpoint of viability. This contrast argues that the effect of endogenous oxidative stress on genome integrity is well suppressed up to the dying-off phase during chronological aging. We found that the leadoff of exogenous hydrogen peroxide induced genomic instability to viability significantly correlated with replicative lifespan (RLS), indicating that yeast cells’ ability to counter oxidative stress contributes to their replicative longevity. Surprisingly, this leadoff is positively correlated with an inverse measure of endogenous mitotic asymmetry, indicating a trade-off between mitotic asymmetry and cell’s ability to fend off hydrogen peroxide induced oxidative stress. Overall, our results demonstrate strong associations of oxidative stress to genomic instability and mitotic asymmetry at the population level of budding yeast.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/mSphere.00574-18
