A common bacterial metabolite elicits prion-based bypass of glucose repression
Small molecules commonly drive productive and destructive relationships between species . The breadth of these molecular messages is vividly illustrated by examples ranging from bacterial control of fungal pathogenesis to programming of multi-cellular development ( Alegado et al . , 2012; Ehrhardt et al . , 1992; Hogan and Kolter , 2002; Hogan et al . , 2004 ) . Small molecule interactions between mammals and their associated gut microbiota may have important consequences for human health ( David et al . , 2014; Lozupone et al . , 2012 ) . Cross-kingdom interactions also have commercial relevance – for example those that occur between yeast and bacteria in the fermentation of alcoholic beverages ( Bisson et al . , 2007; Bokulich et al . , 2012; Boulton et al . , 1996 ) . Pasteur and others since have noted that lactic acid bacteria are a common contaminant in incomplete fermentations ( Boulton et al . , 1996; Pasteur , 1873 ) . Indeed , brewers and vintners have long appreciated that some bacteria have the power to both reduce the ethanol content and spoil the taste profile of fermented beverages . The budding yeast S . cerevisiae is a metabolic specialist: it strongly favors glucose as a carbon source even when other carbohydrates are present ( Johnston , 1999 ) . This is achieved through stringent regulation of enzymes involved in glycolysis and respiration at both the transcriptional and post-transcriptional levels ( Johnston , 1999; Zampar et al . , 2013 ) . Such glucose-associated repression of metabolism is a defining feature of S . cerevisiae , and many other microorganisms ( Gorke and Stulke , 2008 ) . A similar preference for glycolytic metabolism , even in the presence of oxygen , is a defining feature of cancer cells , known as the ‘Warburg effect’ ( Hsu and Sabatini , 2008; Liberti and Locasale , 2016; Vander Heiden et al . , 2009; Warburg , 1956 ) . When yeast cultures switch from metabolizing glucose to utilizing a respirative carbon source such as glycerol , they experience a pronounced lag phase known as the diauxic shift . This pause in growth occurs as a new transcriptional program is engaged to favor respiration ( Galdieri et al . , 2010; Zampar et al . , 2013 ) . The robust nature of this glucose-associated repression can be readily observed by using glucose mimetics . Even in trace quantities , these molecules provide a stable glucose signal , but cannot be metabolized . For example , just 0 . 05% of the mimetic glucosamine prevents yeast cells from utilizing non-fermentable carbon sources present at 40-fold higher concentrations ( Ball et al . , 1976; Brown and Lindquist , 2009; Kunz and Ball , 1977 ) . We recently reported that yeast strains have the ability to overcome such glucose-associated repression of metabolism at frequencies that depend upon the ecological niche from which they were isolated ( Jarosz et al . , 2014a , 2014b ) . This allows them to switch from being metabolic specialists to metabolic generalists , gaining the ability to metabolize a wide variety of carbon sources in the presence of glucose . This heritable change in metabolic program is not accompanied by a change in the yeast genotype . Rather , the trait is cytoplasmically transduced from mother cells to their daughters through the inheritance of an altered protein conformation – a prion known as [GAR+] ( named for its ability to reverse glucose associated repression ) ( Brown and Lindquist , 2009 ) . Although the molecular details of [GAR+] are incompletely understood , several operational tests to identify and modify it are known . [GAR+]’s nomenclature derives from defining characteristics of prion biology: dominance in genetic crosses ( denoted by capital letters ) and transmission to all progeny of meiosis ( denoted by brackets ) . The ability to acquire [GAR+] has been widely conserved among wild S . cerevisiae strains and is also present in fungal species separated by at least two hundred million years of evolution ( Jarosz et al . , 2014b ) . Under monoculture conditions in the laboratory , acquisition of [GAR+] occurs much more frequently than spontaneous mutations that reverse glucose repression ( Brown and Lindquist , 2009 ) . In nature , the precise rates of [GAR+] appearance correlate with environmental niche from which yeast are derived , suggestive of a bet-hedging mechanism for adaptation in environments with fluctuating carbon stores ( Griswold and Masel , 2009; Jarosz et al . , 2014b; Lancaster and Masel , 2009 ) . Perhaps most remarkably , [GAR+] can be induced by cross-kingdom communication with many bacterial species , and this interaction has been conserved over long evolutionary timescales ( Jarosz et al . , 2014a , 2014b ) . Notably , this relationship substantiates a model in which [GAR+]’s benefit as a reversible bet-hedging element would alone be sufficient to motivate its evolutionary retention ( Jarosz et al . , 2014b ) . Induction of [GAR+] by bacteria occurs with an extremely high efficiency – orders of magnitude greater than the spontaneous acquisition rate – and the consequences are mutually beneficial . [GAR+] yeast cells gain the ability to metabolize a wider range of carbohydrates , improved uptake of limiting nutrients , and increased chronological lifespan ( Jarosz et al . , 2014a ) . [GAR+] yeast cells also produce less ethanol , which itself has anti-bacterial properties . This generates a more hospitable environment for the inducing bacteria and allows them to flourish in fermentations where they would otherwise have perished ( Jarosz et al . , 2014a ) ; bacterial species that strongly induce [GAR+] are common contaminants in failed wine fermentations ( Bisson et al . , 2007; Boulton et al . , 1996; Jarosz et al . , 2014a ) . When yeast and inducing bacteria are co-cultured on solid medium , induction of [GAR+] occurs along a spatial gradient emanating from the bacteria . Conditioned medium generated from cultures of these bacteria also induces the prion ( Jarosz et al . , 2014a ) . These results and others have led us to propose that inducing bacteria produce a diffusible signal that promotes [GAR+] in yeast . Here we report that the common bacterial metabolite lactic acid can serve as this inducing signal . Different concentrations of lactic acid elicit distinct and heritable [GAR+]-like phenotypes that have the properties of ‘strong’ and ‘weak’ variants of the prion . Lactic acid also has the capacity to induce [GAR+]-like epigenetic states in evolutionarily distant organisms in which glucose repression has evolved independently . Our results suggest that [GAR+] induction may provide a molecular explanation for Pasteur’s observations , and indicate that links between lactate and diversification of metabolic strategies have been repeatedly employed to modulate the collective behavior of eukaryotic cells . More importantly , we for the first time uncover a bacterially secreted molecule that induces a prion . We do so using a straightforward approach with few technical prerequisites , which we propose could be used for discovering other natural molecules that potentiate epigenetic transformations .
Diverse bacteria have the capacity to induce [GAR+] in neighboring yeast cells through a cross-kingdom chemical communication . To characterize the diffusible element that was responsible for eliciting this change in yeast metabolism , we employed activity guided fractionation . We first grew large-volume cultures of the inducing bacteria S . gallinarum and L . innocua to saturation in rich media containing glycerol as a principle carbon source ( YP-glycerol ) . After removing the bacteria from the media by filtration to create a sterile conditioned medium , we assessed its capacity to induce growth on media containing glycerol ( 2% ) and glucosamine ( 0 . 05% ) , hereafter abbreviated as GLY + GlcN . GlcN provides a stable , non-metabolizable glucose signal ( Ball et al . , 1976; Kunz and Ball , 1977 ) . Serial dilutions of naïve [gar−] yeast did not grow on GLY + GlcN alone over a five day period , but pre-treatment with the conditioned medium caused the yeast cells to grow robustly . We then purified the extracts from each inducing bacterium using reversed-phase high performance liquid chromatography ( HPLC ) on a C18 column . We collected fractions and monitored their ability to induce yeast cells to grow in GLY + GlcN . Pre-treatment with this purified extract afforded growth to a similar degree as S . gallinarum itself ( Figure 1A ) . After these two rounds of purification , the inducing activity eluted in a single fraction as a homogenous peak . We combined active fractions from multiple injections and analyzed this peak by NMR and mass spectrometry . Both analyses revealed that the active fraction contained high concentrations of lactic acid ( Figure 1B; Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 17978 . 003Figure 1 . Identification of the small molecule that induces [GAR+] in a concentration dependent manner . ( A ) S . gallinarum spotted next to yeast on GLY + GlcN ( YP with 2% glycerol and 0 . 05% glucosamine ) induced growth of naïve S . cerevisiae in a spatially dependent manner . An extract prepared from a S . gallinarum conditioned medium induced [GAR+] with similar strength . Each panel shows two biological replicates of yeast colonies spotted in five-fold serial dilutions from saturated cultures . ( Bacterial spots are undiluted . ) ( B ) Multiple-reaction monitoring-mass spectrometry ( m/z 89 . 0200 , 43 . 1000 ) traces of pure lactic acid ( top ) , the purified active fraction from S . gallinarum conditioned medium ( middle ) and a co-injection of active fraction spiked with pure lactic acid ( bottom ) . Results indicate that lactic acid is present in the active fraction ( see Materials and methods ) . Note that y-axis scale differs between the top two traces due to the difference of lactic acid concentration in the pure standard , but they are normalized here for ease of viewing . ( C ) Plating assay ( see Materials and methods ) showing that both L- and D- isomers of lactic acid strongly induce [GAR+] in a concentration dependent manner . Plotted are the fraction of total colony forming units ( CFUs ) that grew on GLY + GlcN ( mean with standard deviation from three biological replicates ) . Ten-fold dilutions were used for plates lacking lactic acid; 1000-fold and 10 , 000-fold dilutions were used for plates containing 0 . 025% LA and 0 . 625% LA , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 00310 . 7554/eLife . 17978 . 004Figure 1—figure supplement 1 . Flow chart depicting the activity guided fractionation strategy used to identify the small molecule that induces [GAR+] . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 00410 . 7554/eLife . 17978 . 005Figure 1—figure supplement 2 . Growth curves—of cells that start as [gar−] at the outset of the experiment—in synthetic complete media ( Sigma-Aldrich ) containing glucosamine , with or without 0 . 1% L-lactic acid added . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 00510 . 7554/eLife . 17978 . 006Figure 1—figure supplement 3 . Growth rates of [GAR+] and [gar−] cells do not differ in the presence of lactic acid . Four biological replicates of [GAR+] or [gar−] cells were grown in GLY or GLY + 0 . 35% D-lactic acid liquid media , with OD600 measurements taken every six minutes over four days using an automated Eon microplate reader ( BioTek Instruments , Winooski , VT ) . [GAR+] cells neither grew faster than [gar−] cells in glycerol alone nor when lactic acid was added . ( Note that [GAR+] cells did have a slightly longer lag time , i . e . slightly more time passed before they entered exponential phase , even though their growth rate during exponential phase was equivalent to that of [gar−] cells . ) Solid lines mark mean values , dashed lines standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 006 Lactic acid , a small organic molecule , exists in two distinct forms: L- ( + ) -lactic acid ( L-LA ) and its mirror image D- ( – ) -lactic acid ( D-LA ) . Both occur naturally . Some bacteria , including the class known as lactic acid bacteria ( LAB ) , have the ability to produce one or both forms from carbohydrates ( Eiteman and Ramalingam , 2015; Salminen et al . , 2004 ) . Eukaryotes typically can metabolize both isomers , sometimes to varying degrees ( Adeva-Andany et al . , 2014; Pajot and Claisse , 1974 ) . Malolactic fermentation by LAB in winemaking can typically yield up to 2 . 5 g/L of lactic acid , or 0 . 25% ( Boulton et al . , 1996 ) ; in other fermented foods , concentrations can exceed 10 g/L , or >1% ( Fleming et al . , 1988; Plengvidhya et al . , 2007 ) . Moreover , many bacterial species that we previously found to strongly induce [GAR+] are known LAB ( Jarosz et al . , 2014a ) . To establish whether lactic acid was sufficient to induce heritable reversal of glucose repression in S . cerevisiae , we tested the ability of yeast to grow on GLY + GlcN agar plates supplemented with concentrations of L- or D-lactic acid at 0 . 025 or 0 . 625% . We saw robust reversal of glucose repression in this range from both stereoisomers , in a concentration dependent manner ( Figure 1C ) . D-lactic acid induced cells to acquire the phenotype slightly more rapidly ( data not shown ) . Addition of 0 . 05% lactic acid did not increase growth on YP alone , eliminating the possibility that the yeast were simply metabolizing the lactic acid ( data not shown ) . Induction was also robust in synthetic complete medium , further establishing that it was due to the added lactic acid , rather than unknown components of the medium ( e . g . yeast extract , peptone , or an unknown metabolite therein; Figure 1—figure supplement 2 ) . Finally , we addressed the possibility that the presence of lactic acid could select for pre-existing [GAR+] cells , as opposed to true induction of [gar−] cells into the [GAR+] state . We examined the growth rates of isogenic [GAR+] cells and [gar−] cells in liquid YP-glycerol ( GLY ) alone or in YP-glycerol supplemented with 0 . 35% lactic acid . We did not observe any significant prion-dependent differences in growth ( Figure 1—figure supplement 3 ) , supporting an induction mechanism rather than selection of a pre-existing population . Glucose repression can be commonly circumvented in S . cerevisiae by induction of the [GAR+] prion , but it can less frequently arise from genetic mutations ( Ball et al . , 1976; Kunz and Ball , 1977 ) and , in principle , from other heritable epigenetic modifications ( Jarosz et al . , 2014a , 2014b ) . We thus tested whether the reversal of glucose repression that we observed in response to lactic acid was due to [GAR+] . To do so we examined hallmarks of prion-based inheritance: dominance in genetic crosses and the ability to be heritably reduced or eliminated by perturbations in chaperone activity ( Shorter and Lindquist , 2005; Wickner et al . , 2004 ) . We first mated yeast cells that had been induced with 0 . 1% lactic acid to naïve isogenic mating partners and selected for diploids . All diploids we tested grew on GLY + GlcN medium , and moreover exhibited the same unusual pattern of semi-dominance ( Figure 2A ) – in which the diploid cells exhibit a strength of reversal of glucose repression that is intermediate between the two haploid parents – that is highly characteristic of both spontaneous and bacterially-induced [GAR+] ( Brown and Lindquist , 2009; Jarosz et al . , 2014a ) . We next sporulated diploid cells induced with lactic acid , dissected multiple tetrads , and examined the ability of the resulting spores to grow on GLY + GlcN . Nearly all spores we tested ( 29 of 31 ) exhibited the [GAR+] phenotype , in agreement with the non-Mendelian , dominant inheritance previously noted for both spontaneous and bacterially induced [GAR+] cells ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 17978 . 007Figure 2 . Lactic acid induced [GAR+] has the same prion-like features as spontaneous [GAR+] . ( A ) Dominance in crosses between [GAR+] strains and [gar−] strains is seen in colonies isolated from a GLY + GlcN plate ( spontaneous [GAR+] ) , as well as those induced with 0 . 1% D-lactic acid ( LA-induced [GAR+] ) . Five-fold serial dilutions are shown for three biological replicates; orange coloring indicates strains exhibiting the [GAR+] phenotype . ( B ) Lactic acid-induced cells can be cured of the [GAR+] phenotype with transient expression of a dominant negative Hsp70 variant ( Ssa1-K69M or Hsp70DN ) . Single colonies were propagated alone or with a plasmid expressing Hsp70DN for ~125 generations , bottlenecked each ~25 generations . After verifying plasmid loss , colonies were again tested for their capacity to grow on GLY + GlcN medium . Each image shows five-fold serial dilutions for three biological replicates . ( C ) A strain with diminished expression of PMA1 , pma1-DAmP , has reduced spontaneous [GAR+] acquisition and is not induced by lactic acid . Lactic acid-induction frequencies differ from those in Figure 1C due to strain background BY4741 , which has a lower frequency of [GAR+] acquisition ( Brown and Lindquist , 2009 ) . Plotted as in Figure 1C . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 00710 . 7554/eLife . 17978 . 008Figure 2—figure supplement 1 . Both spontaneous and lactic acid-induced [GAR+] diploids transmit the phenotype through meiosis to nearly all spores , demonstrating non-Mendelian inheritance . Representative spores from control ( 4 ) , spontaneous [GAR+] ( 8 ) , and lactic acid-induced [GAR+] diploids ( 16 ) are shown . Five-fold serial dilutions are shown for each spore . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 00810 . 7554/eLife . 17978 . 009Figure 2—figure supplement 2 . Both spontaneous [GAR+] cells and lactic acid-induced [GAR+] S . cerevisiae cells exhibited sensitivity to the Hsp70 inhibitor myricetin ( 50 μM ) on GLY + GlcN medium . Each image shows five-fold serial dilutions for two representative biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 009 [GAR+] is unusual among known prions in that it is not influenced by perturbation of the Hsp104 disaggregase ( Brown and Lindquist , 2009; Jarosz et al . , 2014a ) . Instead it is sensitive to perturbation of Hsp70 function ( Brown and Lindquist , 2009; Jarosz et al . , 2014a , 2014b ) . We assembled multiple colonies that had been induced with lactic acid to grow on GLY + GlcN plates . We then transformed them with a plasmid that constitutively expressed a dominant negative Hsp70 variant ( Ssa1-K69M , or Hsp70DN ) . We passaged these cells five times on media that selected for the plasmid . We then passaged them three times on non-selective plates ( and confirmed plasmid loss ) before retesting them on GLY + GlcN plates . This regimen cured multiple independent clones of their induced [GAR+] phenotype , indicating that the lactic acid induced colonies have the same Hsp70-dependency as spontaneous and bacterially induced [GAR+] cells ( Figure 2B ) . We also grew induced cells on plates containing an Hsp70 inhibitor , myricetin ( Chang et al . , 2011; Koren et al . , 2010 ) , at concentrations that did not affect growth on GLY medium . This regimen , which has been shown to inhibit the growth of both spontaneous and bacterially induced [GAR+] on GLY + GlcN ( Jarosz et al . , 2014b ) , also reduced the ability of these colonies to circumvent glucose repression that had been induced by lactic acid ( Figure 2—figure supplement 2 ) . We next tested whether our lactic acid induced isolates exhibited genetic characteristics related to proteins originally linked to spontaneous [GAR+] . PMA1 encodes the essential major plasma membrane proton pump in yeast; in [GAR+] cells Pma1 adopts an altered conformation and associates more strongly with another protein , Std1 ( Brown and Lindquist , 2009 ) . Because PMA1 is essential , we employed pma1-DAmP , a degron allele that reduces the amount of protein expressed ( Breslow et al . , 2008 ) . Cells harboring this allele had a low frequency of spontaneous prion acquisition , and could not be induced to acquire [GAR+] by exposure to lactic acid ( Figure 2C ) . Thus , the heritable bypass of glucose repression induced by lactic acid has the same prion-like patterns of inheritance and depends upon the same proteins as spontaneous and bacterially induced [GAR+] . Although this lactic acid-induced epigenetic state could in principle differ from the spontaneous prion in other unknown ways , for simplicity we hereafter refer to it as [GAR+] . Lactic acid can be imported into yeast via saturable ( “active”' ) and unsaturable ( 'passive' ) mechanisms ( Casal et al . , 1999; Pacheco et al . , 2012; Paiva et al . , 2013 ) . Both depend upon the transcription factor SOK2 , and the active mechanism is ultimately mediated by the monocarboxylate transporter JEN1 . Jen1 is required for yeast to grow on lactic acid as a sole carbon source , but is down-regulated during glucose repression ( Casal et al . , 1999; Pacheco et al . , 2012 ) , and its transcript was not abundant in [GAR+] cells compared to control cells ( Brown and Lindquist , 2009; Jarosz et al . , 2014a ) . Deletion of JEN1 greatly reduces growth on glycerol compared to wild-type cells , precluding its emergence in the previous screens for genes affecting [GAR+] ( Brown and Lindquist , 2009; Jarosz et al . , 2014a ) . We therefore tested if lactic acid could induce jen1∆ cells to acquire [GAR+] . Deletion of JEN1 did not influence [GAR+] induction by lactic acid at either low or high concentrations ( Figure 3A , B ) , indicating that active transport is not required . We also tested whether enzymes reported to be involved in lactic acid metabolism in the Saccharomyces Genome Database ( Cherry et al . , 2012 ) could influence the ability of this molecule to induce [GAR+] . Individual deletion of these various lactic acid dehydrogenases and transhydrogenases ( DLD1 , DLD2 , DLD3 , and CYB2 ) also had no significant effect on [GAR+] induction by lactic acid ( Figure 3A , B ) . Thus , the cellular mechanisms engaged by lactic acid to induce [GAR+] do not require its active import or its metabolism . 10 . 7554/eLife . 17978 . 010Figure 3 . Analysis of induction frequencies in deletion mutants of genes with roles in lactic acid metabolism . ( A ) Plating assay ( see Materials and methods ) measuring induction of [GAR+] at a low ( left ) or high concentration ( right ) of L-lactic acid . Strains are in the W303 background , and cell dilutions match those in Figure 1C . We note that annotations of DLD2 and DLD3 as D-lactate dehydrogenases have recently been challenged by data suggesting they are instead transhydrogenases ( Becker-Kettern et al . , 2016 ) . Only DLD1 is required for yeast to grow on D-lactic acid as a sole carbon source . Plotted the same as in Figure 1C . B . ) Plating assay measuring induction of [GAR+] by D-lactic acid . Otherwise as in ( A ) . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 010 SOK2 previously emerged as a hit in the screen for yeast mutants that were impaired in their ability to acquire [GAR+] upon induction by the bacterium Staphylococcus hominis ( Jarosz et al . , 2014a ) . We replicated this result using S . gallinarum in a co-plating assay on GLY + GlcN ( Figure 4A ) . We then tested whether loss of SOK2 impaired the capacity of lactic acid alone to induce [GAR+] in yeast cells . It did: yeast cells in which SOK2 was deleted were not induced by lactic acid ( Figure 4B ) . Thus , the previously described passive import of lactic acid ( Paiva et al . , 2013 ) is likely the dominant means through which this inducing signal is transmitted from bacteria to neighboring fungi . 10 . 7554/eLife . 17978 . 011Figure 4 . Full induction by S . gallinarum or lactic acid requires the yeast transcription factor SOK2 . ( A ) S . gallinarum spotted next to either wild type or sok2∆ yeast cells on GLY + GlcN , or control medium GLY . ( B ) Plating assay ( see Materials and methods ) comparing spontaneous and lactic acid-induced [GAR+] frequency in wild type and sok2∆ cells . Lactic acid-induction frequencies differ from those in Figure 1C due to strain background BY4741 , which has a lower frequency of [GAR+] acquisition ( Brown and Lindquist , 2009 ) . Plotted the same as in Figure 1C . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 011 Since environmental conditions can exert a strong influence on the amount of lactic acid produced by bacteria , we next tested differing amounts of lactic acid , over a larger concentration regime . Even the lowest concentration we tested in this experiment , 0 . 015% , increased the number of colonies that arose on a GLY + GlcN plate by nearly three orders of magnitude , such that approximately 5% of cells grew into medium-sized colonies ( Figure 5A ) . Increasing concentrations of lactic acid up to 0 . 25% did not strongly increase the degree of induction . However , 1% lactic acid caused an even stronger induction , such that nearly all cells in the population grew into large colonies on GLY + GlcN media ( Figure 5A ) . 10 . 7554/eLife . 17978 . 012Figure 5 . The strength of the [GAR+] phenotype depends on the concentration of lactic acid used for induction . ( A ) Plating assay ( see Materials and methods ) showing that L-lactic acid strongly induced [GAR+] over a wide concentration range . Plotted the same as in Figure 1C . Ten-fold dilutions were used for plates lacking lactic acid and 10 , 000-fold dilutions were used for plates containing lactic acid ( B ) Single [GAR+] colonies were picked from two representative concentrations in ( A ) , propagated ( restreaked ) on non-selective medium ( GLY ) for approximately 75 generations , and then re-spotted on GLY + GlcN in five-fold serial dilutions . Four biological replicates are shown for each induced group , and two for uninduced cells . Note the growth in the third spots ( highest dilution ) of 0 . 0625% L-LA induced cells on GLY + GlcN . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 012 Like virtually all prions , [GAR+] can exist in multiple ‘strains’ – different heritable and stable activity states . When they arise spontaneously on selective GLY + GlcN plates , ‘strong’ [GAR+] colonies are larger than ‘weak’ [GAR+] colonies ( Brown and Lindquist , 2009; Jarosz et al . , 2014a ) . Both activity states are heritable over long biological timescales ( Brown and Lindquist , 2009; Jarosz et al . , 2014a ) . We picked small-sized colonies that had been induced with low concentrations of lactic acid , and large colonies that had been induced with higher concentrations of lactic acid and propagated them for ~75 generations on non-selective rich medium ( YPD ) . We then returned them to GLY + GlcN plates . The small colonies that had originally been induced with low concentrations of lactic acid grew modestly on GLY + GlcN , in a manner that mimicked ‘weak’ [GAR+] strains ( Figure 5B ) . The large colonies that had originally been induced with high concentrations of lactic acid grew robustly on GLY + GlcN , in a manner that mimicked ‘strong’ [GAR+] strains ( Figure 5B ) . Thus , alterations in the concentration of lactic acid produced by inducing bacteria may engage different heritable activity states , or so-called ‘strains’ , of the [GAR+] prion . Glucose repression has evolved multiple times in the fungal lineage ( Hagman and Piskur , 2015 ) . Dekkera bruxellensis , an organism used in the production of lambic beers that diverged from S . cerevisiae 200 million years ago , uses a distinct mechanism for engaging stringent glucose repression ( Rozpedowska et al . , 2011; Woolfit et al . , 2007 ) . We previously established that despite this evolutionary distance , D . bruxellensis can spontaneously acquire a [GAR+]-like state to become a metabolic generalist ( Jarosz et al . , 2014b ) . This [GAR+]-like state can also be induced by cross-kingdom chemical communication with bacteria . Although the molecular lexicon of cross-kingdom communication can evolve extraordinarily rapidly , the simplicity and ubiquity of lactic acid led us to investigate whether it might also influence heritable reversal of stringent glucose repression in D . bruxellensis . We exposed naïve D . bruxellensis strains to lactic acid , guided by our results from S . cerevisiae . Concentrations well within the physiological range of lactic acid produced by bacteria ( 0 . 02% ) induced robust growth on GLY + GlcN ( Figure 6A ) . We picked colonies from these selections and propagated them for ~75 generations on non-selective rich medium ( YPD ) . In each case , the trait was heritable: induced colonies maintained the ability to circumvent glucose repression ( Figure 6B ) . 10 . 7554/eLife . 17978 . 013Figure 6 . Induction , maintenance , and chaperone dependence of a lactic acid-induced [GAR+]-like state is conserved in the evolutionary distant yeast Dekkera bruxellensis . ( A ) Induction of a [GAR+]-like state in D . bruxellensis . [gar−] cells were plated on GLY + GlcN ( containing 0 . 1–0 . 15% glucosamine ) medium with or without 0 . 02% D-lactic acid . Control cells harbor spontaneous [GAR+] . For each of the treatment images , four biological replicates are shown , in five-fold serial dilutions . ( B ) Maintenance and chaperone dependence of the lactic acid-induced [GAR+]-like state in D . bruxellensis . Cells induced in A . were propagated non-selectively for ~75 generations by restreaking on YPD plates , then subsequently retested for maintenance of the prion phenotype on GLY + GlcN ( 0 . 1–0 . 15% glucosamine ) , with or without the Hsp70 inhibitor myricetin ( 50 μM ) . Four biological replicates are shown for each induced group , and two shown for the uninduced and propagated [gar−] cells . DOI: http://dx . doi . org/10 . 7554/eLife . 17978 . 013 The [GAR+]-like states that arise spontaneously or are induced by bacteria in D . bruxellensis depend strongly on the activity of Hsp70 chaperones ( Jarosz et al . , 2014b ) . We therefore tested whether the D . bruxellensis colonies induced by lactic acid to circumvent glucose repression were sensitive to Hsp70 inhibition . We plated serial dilutions of D . bruxellensis cells that had been induced with lactic acid on GLY + GlcN plates and on GLY + GlcN plates containing the Hsp70 inhibitor myricetin ( Chang et al . , 2011; Koren et al . , 2010 ) . We used a concentration of this inhibitor that did not significantly affect growth on GLY alone . This treatment significantly reduced the ability of these induced colonies to circumvent glucose repression ( Figure 6B ) , just as it does for the [GAR+]-like states that arise spontaneously in this organism ( Jarosz et al . , 2014b ) . Thus , across a vast evolutionary distance , bacterial production of lactic acid can engage [GAR+]-like mechanisms to heritably convert neighboring fungi from metabolic specialists to metabolic generalists .
More than a century ago , Louis Pasteur documented what vintners have long since appreciated: the presence of lactic acid producing bacteria in fermentations can often lead to their failure ( Pasteur , 1873 ) . Our observations suggest that the ability of lactic acid to elicit the [GAR+] prion could provide a molecular mechanism for those observations . The [GAR+] prion is found in evolutionarily distant fungal species , and can confer strong selective advantage – converting metabolic specialists to metabolic generalists in a manner that improves fitness in complex microbial communities ( Jarosz et al . , 2014a , 2014b ) . Our data link the common bacterial metabolite lactic acid , both structurally simple and widespread in nature , to this ancient switch in metabolic strategies . Recent evidence points to important signaling roles for lactic acid in human cancer cells , in the context of the Warburg effect ( Adeva-Andany et al . , 2014; Dhup et al . , 2012; Fiaschi et al . , 2012; Liberti and Locasale , 2016 ) . Other studies have linked LAB to benefits for human health and disease prevention , however it remains unclear how much these effects are due to the effects of lactic acid on host cells ( Kim et al . , 2007; Masood et al . , 2011; Tiptiri-Kourpeti et al . , 2016 ) . In at least some cases , LAB appear to exert their health effects by influencing other microbes , for example , modulating the virulence of Clostridium difficile ( Lee et al . , 2013; Yun et al . , 2014 ) . Many mysteries remain regarding the genetic and biochemical features that lead to the [GAR+] prion; findings have so far been limited to its initial characterization ( Brown and Lindquist , 2009 ) and discovery of its regulation by environmental cues ( Jarosz et al . , 2014a ) . Our discovery of a connection between passive lactic acid uptake and the ability to induce [GAR+] ( via our results with SOK2 ) provides new insight into the initiation of this complex cross-kingdom chemical communication . Further molecular understanding of induction by lactic acid , and its links to the unusual biochemistry that fuels [GAR+] , stands as a goal for future studies . Several lines of evidence suggest that other molecules may modulate [GAR+] induction by lactic acid . Although we were able to fully reconstitute induction with lactic acid alone , the kinetics were slightly faster in conditioned media . Moreover , some of the bacteria that induce [GAR+] in S . cerevisiae do not induce the [GAR+]-like state in D . bruxellensis , even though high levels of lactic acid can induce the prion in each of these organisms . An intriguing but plausible explanation is that other bacterially secreted factors influence perception of the lactic acid signal , and that the effects of these molecules may also be species specific . Indeed , there is precedent for this behavior in choanoflagellates , where two bacterially secreted lipids were required to recapitulate full induction of rosette , multicellular colony formation ( Alegado et al . , 2012; Woznica et al . , 2016 ) . Finally , although we have shown that S . gallinarium induces [GAR+] via lactic acid , it is entirely possible that other molecules could also fuel this cross-kingdom interaction in diverse ecological niches . There is also precedent in mechanisms of quorum sensing for competition among inducers secreted by different bacteria ( Drescher et al . , 2014; Even-Tov et al . , 2016 ) . Perhaps most remarkably , our study raises the possibility that other prion-like epigenetic states could be regulated by common metabolites , at times in service of cross-species communication . Prior studies have highlighted the fact that general stressors that perturb protein homeostasis can modestly elevate the frequency of cells harboring the [PSI+] or [MOT3+] prions ( Holmes et al . , 2013; Tyedmers et al . , 2008 ) . These intrinsic links between stress responses and prion switching ensure that subpopulations of cells can diversify their phenotypes when they are ill-suited to the environment . Our findings extend this biology to a more concerted , switch-like behavior in which prions can serve as a target for rapid and effective cross-species communication . Biological niches like the human body harbor microbial communities that are far more complex than those we have studied here ( Lozupone et al . , 2012 ) . It is striking that the robust connection we have discovered between a simple metabolite and a heritable change in metabolic strategy has been missed despite a multitude studies in this model organism . Our observations thus underscore the potential of cross-kingdom molecular communication to illuminate biological processes , in particular its capacity to elicit heritable , epigenetic transformations of biological traits . Recent progress in metabolomics and metagenomics motivate deeper exploration of such interactions between species , at both the molecular and systems levels .
Bacteria strains were cultured on LB agar ( Research Products International , Mt . Prospect , IL and BD-Difco , Franklin Lakes , NJ ) plates . Yeast strains were cultured on either: ( 1 ) YPD agar or liquid ( RPI and BD-Difco ) , ( 2 ) YP-Glycerol agar ( 1% Yeast Extract ( BD Bacto ) , 2% Peptone ( BD BBL/Difco ) , 2% glycerol ( Sigma-Aldrich , St . Louis , MO and Amresco , Solon , OH ) , 2% agar ( IBI Scientific , Peosta , IA] ) , or ( 3 ) “'GLY + GlcN”' agar ( same mixture as YP-Glycerol ) which is supplemented with 0 . 05% glucosamine hydrochloride ( Sigma-Aldrich ) ( 0 . 1–0 . 15% for D . bruxellensis ) that was added from a filter-sterilized 5% stock once media cooled to ~60°C after autoclaving . We noticed that the stringency of selection for [GAR+] cells on GLY + GlcN media was dependent on the source of yeast extract and peptone , and therefore recommend the aforementioned suppliers , as other sources for unknown reasons sometimes imposed much weaker selection . Myricetin ( Sigma-Aldrich ) was made into a stock solution of 15 . 7 mM in 100% ethanol , and then diluted to a final concentration of 50 μM in agar medium once cooled to ~60°C after autoclaving . Strains were maintained using standard microbiological techniques . Bacteria and S . cerevisiae strains were grown up in 2–5 mL cultures at 30°C shaking or rotating overnight for most experiments; for experiments requiring smaller volumes they were grown in 200 μL volumes in microtiter plates . All D . bruxellensis strains were grown at room temperature until saturation for liquid or sufficiently large colonies for agar plates , typically requiring at least 5 days of growth . Serial dilutions , starting from saturated cultures , were performed in 96-well plates using multichannel pipettes , and cells were spotted gently onto agar plates using a blot replicating tool ( V&P Scientific VP404A , San Diego , CA ) . For S . cerevisiae strains , GLY plates were grown for 2 days before imaging . GLY + GlcN plates were grown 5–7 days before imaging . D . bruxellensis strains required several extra days of growth for single colonies to become visible . Bacterial strains that induced yeast growth on GLY + GlcN media were grown in large scale to purify the active component . Strains were grown from single colonies in 5 mL LB cultures overnight at 30°C and with 250 rpm shaking . The inoculum was then transferred to Erlenmeyer flasks containing 200 mL YP-Glycerol and incubated for 72 hr at 250 rpm , 30°C . The media were centrifuged ( 30 min at 6000 rpm , Beckman ( Brea , CA ) JLA 8 . 1 rotor ) , and passed through a 0 . 22 μm filter . The filter-sterilized media was acidified to pH ~1 using 2M HCl , and extracted with a threefold volumetric excess of ethyl acetate . The organic extracts were dried over sodium sulfate , concentrated on a rotary evaporator , suspended in 2–4 mL water , and neutralized with sodium hydroxide . The extract was purified by preparative HPLC using a gradient of acetonitrile ( solvent B , Fisher Scientific ( Waltham , MA ) , containing 0 . 1% formic acid ) in milli-Q water ( solvent A , containing 0 . 1% formic acid ) on a reversed-phase C18 column ( Phenomenex ( Torrance , CA ) Luna 5U , 100A , 250 × 21 . 29 mm ) . The following program was used: 10 mL/min; ramp from 0 to 20 min of 2% to 35% B , followed by a 10 min wash cycle; collect 0 . 5 min fractions from 7 to 13 min . Collected fractions were dried in a speed-vac , suspended in water ( ~5 mL ) and passed through a 0 . 22 μm filter . Active fractions were identified by assaying their ability to induce growth of YDJ1034 in typically non-permissive GLY + GlcN media containing crude bacterial extracts or HPLC fractions ( 10 to 25% of total media volume ) . Fractions that supported yeast growth in GLY + GlcN were combined and subjected to subsequent rounds of semi-preparative HPLC using a gradient of acetonitrile ( containing 0 . 1% formic acid – solvent B ) in water ( containing 0 . 1% formic acid – solvent A ) on a reversed-phase C18 column ( Phenomenex Luna 5U , 100A , 250 x 10 . 00 mm ) . The following program was used: 2 . 4 mL/min; ramp from 0 to 20 min of 2% to 10% B , followed by a 15 min wash cycle; collect 0 . 2 min fractions from 8 . 4 to 9 . 4 min . Fractions were analyzed by high-resolution HPLC-MRM-mass spectrometry and NMR spectroscopy . The active fractions contained high levels of lactic acid , which was confirmed by NMR and by co-injection with a purified standard ( Figure 1B ) . Multiple sources of lactic acid yielded similar results . We tested sodium D-lactate ( Sigma-Aldrich 71716 ) , sodium L-lactate ( Sigma-Aldrich L7022 ) , D-lactic acid zinc salt ( MP Biomedicals , Santa Ana , CA ) , and L- ( + ) -lactic acid ( Sigma-Aldrich L6402 ) . Excess NaCl was tested in control reactions to mimic the sodium chloride produced during naturalization of sodium lactate and found to produce no phenotypic differences . Lactic acid was added to GLY + GlcN medium at indicated concentrations before autoclaving , and pH was balanced if necessary to ensure it was within ~0 . 1 units or less of control media lacking lactic acid ( pH ~6 . 8 ) . For the plating assay , S . cerevisiae W303 strains were plated with 100 μL volumes for all plates . For BY4741 strains , 250 μL volumes were plated for GLY + GlcN plates +/– lactic acid ( no cell dilution ) , while 100 μL was used for GLY plates . Cell dilution was empirically determined to obtain plates containing between approximately 25 and 300 colonies per plate , which were counted using an aCOLyte colony counter ( Synbiosis , Cambridge , England ) . For all GLY plates , a 100 , 000-fold dilution of saturated culture was plated . For GLY + GlcN plates for W303 strains , a 10-fold dilution of saturated culture was plated; for GLY + GlcN plates containing lactic acid , dilutions of 1000-fold or 10 , 000-fold were plated to achieve a suitable colony number for W303 strains . Induction frequency was calculated by normalizing the number of colony forming units ( CFUs ) on the GLY + GlcN plates to the total possible number on GLY plates after accounting for cell dilutions , reported as [GAR+] cfu/total cfu . We noted some variability in values of [GAR+] cfu/total cfu from experiment to experiment ( e . g . for spontaneous rate between Figure 1C and Figure 5A ) , however the qualitative relationship between lactic acid concentration and increased [GAR+] cfu/total cfu was maintained across all dilutions tested . For induction of D . bruxellensis strains , cells were spotted in serial dilutions on plates containing lactic acid . Single colonies were picked from the highest dilution and propagated as described in figure legends . Diploids were constructed by crossing indicated BY4741 haploids to the BY4742 parental strain ( ATCC , Manassas , VA ) by mixing a bead of cells of each strain together on a YPD plate and growing overnight . A small globule of this cell mixture was then restreaked to single colonies on SD-Lys-Met agar plates to select for diploids . Deletion mutants in the W303 background were created by amplifying a KanMX construct from corresponding gene deletion mutants from the BY4741 library , using primers listed in Supplementary file 1C . Approximately 500 ng of purified construct was transformed into YDJ1034 , and integrants were selected on agar plates containing G418 ( Fisher Scientific ) . All gene deletions were confirmed by PCR using primers listed in Supplementary file 1C . Curing of the [GAR+] phenotype ( Figure 2B ) was achieved by transformation of lactic acid induced cells with PDJ281 ( Hygromycin resistant , Supplementary file 1D ) , restreaking multiple transformants to single colonies on YPD plates containing 400 ug/mL hygromycin ( Gold Biotechnology , Olivette , MO ) a total of five times , then restreaking in the same manner on YPD plates twice before confirming each clone had lost the plasmid by testing on YPD+hygromycin . These clones were grown in liquid YPD cultures before testing on GLY + GlcN media . Spontaneous and lactic acid-induced [GAR+] diploids ( W303 background , Figure 2—figure supplement 1 ) were isolated from single colonies grown on GLY + GlcN or GLY + GlcN + lactic acid plates and grown in Pre-SPO liquid media ( YPD with 6% glucose ) for 3 days at room temperature . Cells were then pelleted and washed twice in SPO media ( 1% Potassium Acetate ( Sigma-Aldrich ) , 320 mg CSM-Met powder ( Sunrise Science , San Diego , CA ) , 20 mg Methionine ( Sigma-Aldrich ) per liter ) , and then diluted ten-fold to obtain 3 mL cultures in SPO media . These cultures were incubated on a rotary wheel for one week at room temperature , before dissecting tetrads on a Singer Instruments MSM400 ( Somerset , UK ) . Recovered spores were grown to saturation in liquid YPD before testing on GLY + GlcN media . Yeast plates were imaged using an Epson Perfection V700 Photo Scanner ( Long Beach , CA ) . Plots were made using PRISM 6 software ( GraphPad , La Jolla , CA ) .
Robust preference for fermentative glucose metabolism has motivated domestication of the budding yeast Saccharomyces cerevisiae . This program can be circumvented by a protein-based genetic element , the [GAR+] prion , permitting simultaneous metabolism of glucose and other carbon sources . Diverse bacteria can elicit yeast cells to acquire [GAR+] , although the molecular details of this interaction remain unknown . Here we identify the common bacterial metabolite lactic acid as a strong [GAR+] inducer . Transient exposure to lactic acid caused yeast cells to heritably circumvent glucose repression . This trait had the defining genetic properties of [GAR+] , and did not require utilization of lactic acid as a carbon source . Lactic acid also induced [GAR+]-like epigenetic states in fungi that diverged from S . cerevisiae ~200 million years ago , and in which glucose repression evolved independently . To our knowledge , this is the first study to uncover a bacterial metabolite with the capacity to potently induce a prion .
We communicate with each other using speech , writing and physical gestures . But how do bacteria , yeast and other single-celled microbes communicate ? In 2014 , researchers reported a new example of communication between bacteria and yeast in which the bacteria send a chemical message that has a very long-lasting effect on how the yeast grow in certain environments . This in turn also affected the ability of the bacteria to survive in these environments . The identity of the chemical message produced by the bacteria , however , was not known . Garcia , Dietrich et al . – including one of the researchers from the previous study – used biochemical and genetic approaches to identify the chemical message . The experiments show that the message is a molecule called lactic acid , which is very common in nature and is produced by many bacteria . Garcia , Dietrich et al . found out how much lactic acid is needed to alter the growth of brewer’s yeast , and which genes in yeast are involved in responding to the message from the bacteria . Further experiments suggest that the ability of yeast and bacteria to communicate using lactic acid is likely to have existed for hundreds of millions of years . The next step following this work will be to identify other chemical messages used by microbes . The human body is packed with billions of bacterial cells , and in some cases yeast can also take up residence . A future challenge will be to find out if bacteria and yeast inside the human body are able to communicate with each other in ways that could affect our health .