Meeting
Report
Microbial
stress:
From
molecules
to
systems
(Sitges,
November
2015)
Francesca
Martani
1,
Nadia
Maria
Berterame
1,
Paola
Branduardi
*
UniversityofMilano-Bicocca,DepartmentofBiotechnologyandBiosciences,Milano,Italy
ARTICLE INFO
Articlehistory: Received21July2016
Receivedinrevisedform30October2016 Accepted16November2016
Availableonline25November2016
ABSTRACT
Themeeting“MicrobialStress:fromMoleculestoSystems”–thethirdinthisseries–washeldinSitges (Spain)inNovember2015.Themeetingofferedtheopportunityforinternationalscientiststosharetheir viewpointsandrecentoutcomesconcerningmicrobialstressresponses.Particularattentionwasgivento thecharacterisationofmechanismstriggeredbystress,fromdetailedmolecularbiologythroughwhole organismsystemsbiologyuptothelevelofpopulations.Adeeperunderstandingofmicrobialresponses tostressisindeedattainableonlyconsideringthephenomenonasawhole.Exhaustiveknowledgeofthe variousstressresponsesystems,andoftheirinterconnections,isimportantfordifferentapplications, fromthepreventionandcounteractionofbacterialinfectiousdiseasestotheengineeringofrobustcell factories. The presentations covered all of these aspects, enabling an active interaction among participants.Italsostimulateddiscussionsandcross-fertilisationamongdisciplines,whichwasoneof theaimsofthemeeting.Moreover,sincemanystressresponsemechanismsarebroadlyconserved,data obtainedatthemicrobialscalemayfacilitatethecomprehensionofcomplexphenomena,suchasaging, evolutionofneurologicaldiseasesandcancer.
ã2016PublishedbyElsevierB.V.
TheconferencewaschairedbyMichaelSauer(BOKUUniversity, NaturalResourcesandLifeSciencesVienna,Austria),withthe co-chairingofPaolaBranduardi(UniversityofMilano-Bicocca,Italy) andPeterLund(UniversityofBirmingham,UK),onbehalfofthe Microbial Physiology Section of the European Federation of Biotechnology.
Almost90delegates,bothfromAcademiaandfromIndustry, attendedtheconferencewithrepresentativesfromEurope,USA, Japan,Korea,China,BrazilandPakistan.
Cross-fertilisationofideasandtechniquestounearthmicrobial stressresponse
The programmewas composed of threesessionsconcerning differentaspectsofthemicrobialstressresponse,consideredasa key element for (i) the adaptation to intra-and extra-cellular
environmentalchanges,(ii)cellperformanceinindustrial produc-tionprocessesand (iii)cross talkwithhigher organisms.A key feature of the meeting was toemphasise the relevance of the microbialphysiologyinrelationtothebioprocessingindustry.
Among the most relevant factors that impair microbial metabolism,andconsequentlytheabilitytoproducecompounds or proteins of interest efficiently, are the stressing conditions imposedbyindustrialbioprocesses,oftenrelatedtothesubstrates used. Presentations during the second session illustrated the effectsofsuchstressorsonmicroorganismphysiology,aswellas different strategies to improve cellular properties in order to increase the production and the yield of fermentations. These enhancements can only be achieved through an in-depth knowledge of the mechanisms that steer the microbial stress response.Theinvestigationofreactionsandnetworksevolvedby microorganismstodealwithstressesencounteredinnatural or artificial habitats, explored in the first and third sessions, are thereforeessential.Beyonddoubt,cross-fertilisationofideasand techniques is indispensable togain a greater understandingof microbialresponsetostress(Fig. 1).Accordingly,thedivisionofthe programmeintothreeresearchthemescoveringbroadaspectsof stress responses, rather than focusing on single stresses or
*Correspondingauthorat:PiazzadellaScienza2,20126,Milano,Italy. E-mailaddresses:francesca.martani@unimib.it(F.Martani),
n.berterame1@campus.unimib.it(N.M.Berterame),paola.branduardi@unimib.it (P.Branduardi).
1
Theseauthorscontributedequallytothiswork. http://dx.doi.org/10.1016/j.nbt.2016.11.001 1871-6784/ã2016PublishedbyElsevierB.V.
ContentslistsavailableatScienceDirect
New
Biotechnology
organisms, promoted stimulating discussion and an unbiased point of view and comments from scientists not necessarily belongingtothesamefieldofresearch.
Intracellularvs.environmentalstresses:differencesand commonlines
The survival of microorganisms under stressful conditions relies on regulatory networks that enable the sensing of environmentaland/orintracellularchangesandtheactivationof anappropriate response. This complexregulation relieson the interactionofdifferentmoleculesofalltypes,includingDNA,RNA, proteins,lipidsandmetabolites.Thepresentationsofthissession showeddifferent(andcomplementary)mechanismsexploitedby microbialcellstocopewithextracellularaswellasintracellular stress, providing many examples of the complexity of stress responses.
HiroshiTakagi(NaraInstituteofScienceandTechnology,Japan) intriguinglydescribedhowthenitricoxide(NO)-dependent anti-oxidativesystemismediatedinbaker’syeastbytheactivationof thecopper-sensingtranscriptionfactorMac1[1].Microarrayand real-timequantitativePCRanalysesrevealedthatcopper metabo-lism-relatedgenesregulatedbyMac1areinducedbyexogenous NOtreatment;accordingly,NOsynthesisedunder high-tempera-ture stress increases the level of the CTR1 (high-affinity Cu2+ transporter 1) transcript, intracellular copper content, copper-dependent Sod1 (superoxide dismutase 1) activity, and the consequent cell viability. Interestingly, yeasts engineered to over-produceNOshowed improvedfermentationabilitieswhen exposedtoair-drying,highsugarconcentrationsandfreeze-thaw stresses. When present at low concentrations, NO acts as a signalingmoleculepreservingcellsfromdeath,buthigh concen-trationsofNOaretoxic.ThesensingofNOlevels,aswellasthe activationofmechanismstocounteractitstoxicity,aretherefore crucialindeterminingthetriggeringofthestressresponse.
JeffCole(Universityof Birmingham,UK)presenteda coordi-nated system based on proteins YtfE and Hcp-Hcr for the prevention and repair of nitrosative damage during anaerobic growth, a physiologically relevant condition for pathogenic
gastrointestinal bacteria [2]. He demonstrated that the hybrid cluster protein, Hcp, is a high affinity NO reductase that is inactivatedbyitstoxicsubstrateunlessprotectedbyitspartner protein,Hcr.
Decreased cytosolic pH, generally recognised as stress, was shownbyGertienSmits(UniversityofAmsterdam,Netherlands)to beasignalthroughwhichcellsexperiencetheenvironment[3].It determines whether energy is used for growth or for stress resistance.Indeed,sheshowedthatyeastmutantswithdefective cellwallintegrityordeficientinCa2+sensingweresensitivetopH 2.5,butwereabletosurviveatpH1.5,thankstotheintracellular pH decrease occurringonly atextremely low external pH.Cell survival at low pH was also illustrated by Daniela De Biase (Sapienza University of Rome, Italy) who found a connection between the more acid-resistant phenotype of a new Brucella species and the presence of a functional GAD (glutamate decarboxylase)system,which enablesthesepathogenstoadapt incertainnaturalhabitatsand/orinthegastrointestinaltractof theirhosts[4].
Metal availabilityrepresents another criticalfactor in deter-mining the outcome of host-pathogen interactions. John D. Helmann (Cornell University, USA) described a Bacillus subtilis modelforthemaintenanceofzinc(Zn2+)homeostasis,which is regulatedbythemetalloregulatoryproteinsZurandCzrAthatare sensitivetoZndeficiencyandexcess,respectively.Heshowedthat Zurcontrolsribosomalproteinsthatdonotcontainazincbinding motif,butwhichmightfunctiontomaintaintheproperassembly of the ribosome during zinc limitation [5]. Understanding the mechanismsinvolvedinthiskindofregulationcouldbecriticalfor thedevelopmentofnewantimicrobialcompoundsthataltermetal ionhomeostasis.IncontrasttoZn,silver(Ag)isametalwithno biological functionsand istoxictomicroorganismsevenat low concentrations.PetekCakar(IstanbulTechnicalUniversity,Turkey) showedthesuccessfulapplicationofevolutionaryengineeringfor theachievementoftheupto250
m
MAgNO3-resistant Saccharo-mycescerevisiae strains,resulting fromtheupregulation of 739 genesandthedownregulationof853others(unpublisheddata). Metal response mechanisms provided examples of cross-talk amongdifferentstressresponsepathways.Anotherexampleofthe highcomplexityofcellularfunctionswasprovidedbyKyung-Tae Lee(YonseiUniversity,Korea),whodescribedthemultiplerolesof thecytoskeleton-associatedproteinglycine-richCgp1in regulat-inggrowth,stressresponse,differentiationandpathogenicityof Cryptococcusneoformans(unpublisheddata).The production of specific metabolites represents another efficientstrategyadoptedbysomemicroorganismstocounteract cellularstress.Asanexample,trehaloseisanosmolyteproducedas astress-responsivemoleculebyseveralorganismsinresponseto environmentalstressessuchasheat,cold,desiccationandosmotic stress.EwaLaskowska(UniversityofGdansk,Poland)reportedthat aggregatedproteinsareremovedbytrehaloseinEscherichia coli cellsduringrecoveryfromstationaryphaseandafterheatshock.In addition,otsAmutants,defectiveintrehalosesynthesis,displayed various markers of oxidative stress and were unable to form biofilm[6].StanislavObru9ca(BrnoUniversityoftechnology,Czech Republic)showedthecryoprotectantfunctionofanotherchemical chaperone,3-hydroxybutyrate(3HB),in CupriavidusnecatorH16 [7].3HBmonomersformpoly-hydroxybutyrate(PHB),astorage materialproducedbydifferentbacteria.Thisdefencemechanism might therefore be exploited for industrial applications for biomaterialproduction.
Bacterialcellenveloperearrangementrepresentsthefirstline ofcellulardefenceagainststress.Changesinthefattyacidprofileof thecellularmembraneofRhodococcuserythropoliscellsexposedto variousstresseswerereportedbyCarladeCarvalho(Universityof Lisbon,Portugal).Inparticular,undernutrientlimitingconditions
anddesiccation,R.erythropolisproducesspecialisedlipids,suchas triacylglycerolsandwaxesters[8].AsinthecaseofPHBdescribed byObru9ca,thesecompoundsaccumulatedunderstresscondition may have potential industrial applications, for example in biodieselproduction.
Microbialstressinindustrialproductionprocesses
Oneofthemajorobstaclesforthedevelopmentofcompetitive industrial bioprocesses is the reduced performance of micro-organismsduetostressencounteredduringfermentation.Stress mayoccurbecauseofthepresence ofinhibitors,oftenderiving fromthesubstrate usedfor mediaformulation, or from unfav-ourablegrowthconditions.Frequently,theproductitselfbecomes toxic when accumulated at high concentration. Among the undesirableeffects caused bystressat both the molecularand physiologicallevels,thedecreaseingeneticstabilityofindustrial strainsmaydecreaseproduction,productivityandprocessyields. WandaDischert(METabolicEXplorer,France, http://www.meta-bolic-explorer.com) presented different strategies adopted by METEXtodelaythepressureonbacteria,includingthe develop-ment of a stable genetically engineered E. coli strain for the productionofglycolicacid,amonomerforbiodegradableplastics. Amongdifferentapproaches,glycolicacidyieldscanbeincreased byreducingtheexpressionoftheicd(isocitratedehydrogenase) gene;however,strainswithanattenuatedexpressionofthisgene were unstable when grown for many generations, a strong disadvantage for industrial use. Strain stability was therefore achievedbyMETEXthroughthemodulationoficdexpressionby replacing the natural promoter of this gene with a thermo-induciblepromoter.Consequently,theoptimisedprocessconsists ofafirstphaseat37C,whereicdisexpressedtoallowbiomass production, followed by a second step at 30C, where icd is repressedandglyconicacidisproduced.
In thenaturalenvironment,butalsoinlargescaleindustrial cultivation,organismsareexposed torapiddynamicconditions, which are repetitive. Relevant cultivation parameters, such as dissolvedoxygen,substrateconcentration,pHandtemperaturedo varyinlargescalebioreactorsbecauseoflongmixingtimes.Thus, microorganismsinthebioreactorexperiencerapidenvironmental changes that may affect performance. How to study these fluctuations is not trivial. Aljoscha Wahl (Delft University of Technology, Netherlands) presented an experimental approach basedonadynamicshiftbetweenfeastandfamineregimes(400s and1800s)thatledtorepetitivecycleswithmoderatechangesin substrate availability in an aerobic glucose cultivation of S. cerevisiae [9]. After a few cycles, the feast/famine switches produced a stable and repetitive pattern with a reproducible metabolic response time, thus providing a robust platform for studying the microorganism’s physiology under dynamic con-ditions.Heshowedthatthebiomassyieldwasslightlydecreased (by 5% to 25%) under the feast/famine regime, while the averagedsubstrateandoxygenconsumptionaswellasthecarbon dioxideproductionrateswerecomparable.Comparingtheshort and long cycles, he showed that despite a 10-fold change in extracellularglucose concentration, the intracellular concentra-tionandfluxesofglycolyticintermediatesonlyrespondedwitha changeof1.2–1.5-fold.Incontrast,intermediatesofcarbohydrate storagemetabolism,suchastrehalose6-phosphate(T6P),reacted moredramatically.Therefore,theseresultsconfirmedthe adapt-abilityofcellularmetabolismandshowthatstoragecarbohydrates arekeymetabolitesforyeastrobustness.
Anotherlimitationduetolarge-scalemixingthatcanberelated tofluctuationsis thatthecellsarerepeatedlyexposedtozones withvarying substrate concentrations. Thus, heterogeneities in largereactorsmightimposestressonthemicrobialhostsresulting
inperformancelosses.Inparticular,JohanaSimen(Universityof Tuebingen,Germany)describedtheimpactoflarge-scalecarbon and nitrogen gradients onE. coli K-12 W3110 [10]. Short-term dynamicswerefoundnotonlyfor intracellularpoolsof related nucleotidesandalarmones,butalsoformRNAlevels.Inall,about 300 genes were found to be differentially expressed after a residencetimeof110sinthestarvationzone.Analysisoflong-term adaptationrevealedcellularstrategiestocopewiththeimposed oscillatingstressconditions.
However,copingwithstressissomethingthatcellsexperience innaturalenvironmentsaswell,offeringthepossibilitytostudy how to turn this ability into an industrial advantage. Joseph Shiloach (National Institute of Diabetes Digestive and Kidney Disease/NIH,USA)reportedanexampleof howoxidativestress facedbycellsduringgrowthcanbeexploitedfortheproductionof heterologousproteins.Heshowedthatinresponsetohighoxygen concentrations,soxSexpressionincreased16-foldinE.coli,making itspromoterapossiblecandidateforinducingrecombinantprotein expressionundertheseconditions[11].Inductionofrecombinant proteinexpressionbyincreasingthedissolvedoxygen concentra-tionwasfoundtobeasimpleandefficientalternativeexpression strategy thatexcludestheuse ofchemical, nutrientor thermal inducersthathaveapotentialnegativeeffectoncellgrowthoron productrecovery.
Withasimilarperspective,BettinaLoràntfy(Chalmers Univer-sityofTechnology,Sweden)illustratedhowthepresenceofoxygen canbeadvantageousandinparticularcanimproveacidification capacityofsomelacticacidbacteria(LAB).LABhavebeenusedin cheesemakingforthousandsofyearsbyexploitingtheircapability fortheanaerobicproductionofacidicby-productsderivedfrom thefermentationofmilksugar.Arecentdiscoveryhasshownthat someLAB,suchasLactococcuslactis,areabletosustainrespiration under aerobic conditions when hemin is added tothe growth medium,sinceitcompletestheelectrontransportchain,whichis otherwisedefective.Sheshowedthat,althoughoxygenrepresents anadditionalstressforLAB,inthepresenceofheminunderaerobic conditionstheyperformedsurprisinglybetterintermsofhigher lactateyield(unpublisheddata).
Asmentionedabove,stressorsthatnegativelyaffect produc-tion parametersare often derived fromthe substrateused for mediaformulation.Thisisoneofthechallengestotackleforthe viability of a bio-based economy, where fuels, chemicals and materials are supposed to be produced from lignocellulosic biomass.Thesesubstratesareoftenproblematic,bothbecauseof theirheterogeneousnatureandtheircomplexity.Inaddition,the necessary pre-treatments of lignocellulosic biomasses release inhibitory compounds, in the form of furans, weak acids and phenolic derivatives, which impair cell growth and, in conse-quence, theyield andproduction of the process.Strategies to improve cellular robustnessmaygenerallycomprisedecreased uptake or increased efflux of inhibitory compounds,increased detoxificationreactionsandabilitytorepairthedamagecaused bystressfulconditions.
Pseudomonasincreasesendogenousconversionofconiferyl alde-hyde into the less toxic ferulate, and that the endogenous acetaldehyde dehydrogenase (Ald5) is actively involved in the catabolismofthephenoliccompoundinS.cerevisiae[13].Inthe latterexample,attentionwasfocusedonaceticacid,oneof the mostabundanttoxic compoundsreleased duringlignocellulose pre-treatments, and its effect on the plasma membrane. This cellularstructure playsa crucial role as a flexible but effective elementinthecellularresponsetostress.Studieson Zygosacchar-omyces bailii and S. cerevisiaecell membranes highlightedhow sphingolipid content affects acetic acid permeability, making membraneengineeringapossibleapproachtoincreasingcellular robustnesstowardinhibitorycompounds[14].
WeakacidtolerancewasalsoinvestigatedbyNurzhan Kuany-shev(UniversityofMilano-Bicocca,Italy)whofocusedattentionon theindustriallyrelevantyeastKluyveromycesmarxianus.Genome analysisrevealedthepresenceof additionalcopiesofthegenes PDR12 and PDR5 compared to S. cerevisiae (unpublisheddata): thesegenesencodeweakacidtransportersinbaker’syeastandthe geneamplificationsuggestsadifferentevolutionthatoccurredin K.marxianus.Aspecifictranscriptomicanalysisshowedapeculiar inductionprofileof these transporters that was extended from exposuretoacetictootherorganicacids.Theseresultshighlight oncemoretheimportantroleofthecellmembraneinorganicacid tolerance/intracellularconcentration,aspreviouslypresentedby LisbethOlsson.
Marco Brambilla (University of Milano-Bicocca, Italy) gave another example of cellular rewiring connected to strain robustness.He showed thatthe alteration of a post-transcrip-tional master regulator element might represent a novel approach to unlock industrially promising phenotypes. The modulationofthelevelsofthepoly(A)bindingproteinPab1(a stress granules component) increased S. cerevisiae robustness againstaceticacid,H2O2andheatstress.Inaddition,mutagenesis of PAB1and theapplication of a specificscreening protocolon aceticacidenrichedmediumallowedtheisolationofthefurther amelioratedmutantpab1A60-9,characterisedbydefectsinmRNA deadenylation[15].
Inadditiontothetoxicityofthecompoundsreleasedfromthe treatmentofbiomass, oneof themajor limitations fora viable productionisthetoxiceffectoftheveryhighconcentrationsof productreachedduring thefermentativeprocess. NunoPereira Mira(UniversityofLisbon,Portugal)presentedanexperimental system to identify, at a genome-wide scale, the genes and pathwaysrequiredfor maximal tolerance ofS. cerevisiaetothe stressinducedbyanimportantbuildingblockmolecule,itaconic acid. He showed an enrichment of genes involved in different pathways, including the identification of a plasma membrane transporter that serves as an itaconic acid/itaconate exporter (unpublisheddata).
Whateverthenatureofthestress,theuseofbiosensorsoffers possibilitiesforimprovingcellsforthesynthesisofmoleculesof interest.JanDinesKnudsen(LundUniversity,Sweden)described an efficient tool to measure the impact of environmental perturbationsorheterologousgeneexpressiononcellularNADH levels [16]. The biosensoris based onthe expression of green fluorescent protein (GFP) under the control of the glycerol-3-phospate dehydrogenase (GPD) 2 promoter, responding to changesintheNADH/NAD+ratio.Thereportersystemdisplayed high resolution in distinguishing cytosolic NADH oxidation capacityandhencehaspotentialtobeusedinhigh-throughput screening for selecting improvedvariants basedon single cell fluorescence.
Microbialstressnetworksandcross-talkwithhigher organisms
Regulatory networks allow complex physiological responses mediated by coordinated changes at both the molecular and cellular levels, enabling pathogens to cope with the stressful conditionsimposedbythehostorganism.ThesigmafactorSigEof Mycobacteriumsmegmatis,togetherwithitsanti-sigmafactorRseA and the MprA/MprB two-component system, was described by FedoraBabic(UniversityofPadova, Italy)asadynamic network whoseperturbationaffectsbacterialsurvivalundervariousstress conditions (unpublisheddata).GrzegorzWegrzyn(University of Gdansk,Poland)showedthatoxidativestress,usuallyoccurringin thehumanintestine,inducestheShigatoxin-convertingprophage in enterohaemorrhagicE. colistrainsthrough theOxyR protein [17]. Prophage induction was also evoked by human stress hormones; onthe otherhand, starvationstressinhibited Shiga toxin production, suggesting that this mechanism is tightly regulated bythecellularresponsetoenvironmental conditions. Theinfluenceofhost-imposedstressesonmicrobialresponsewas alsohighlightedbyAlistair J.P.Brown(University ofAberdeen, UnitedKingdom),whoillustratedthesignificanceofcombinatorial cationicplusoxidativestressesinhostdefencesagainstCandida albicans.Heproposedamechanisminwhichcationsinterferewith the intracellular response to H2O2 exposure, leading to ROS accumulationandkillingofthepathogensbyneutrophils[18].
Roy Parker (University of Colorado Boulder, USA) illustrated eukaryotic stress granules (SG) as heterogeneous and dynamic mRNA-proteincomplexesactinginthecontroloftranslationand mRNAdegradationwhentranslationisinhibited.Super-resolution microscopy reveals that SG contain stable core substructures surroundedbyaphase-separatedshell[19].Proteomicanalysisof SG cores indicated that the assembly of these granules occurs throughadensenetworkofprotein–proteininteractions,inwhich differentATP-drivenmachinesarerequired.Interestingly, neuro-nalRNAgranules,involvedinsynapticplasticity,habituationand memory, share several common features with SG and various neurodegenerative diseases are characterised by pathological granules, which may be linked to defects in ATP hydrolysis. Therefore, mechanisms that are revealed and generally are investigated under stressfulconditions mayalso havea crucial roleinphysiologicalfunctions.
The complexity of biological responses was also shown by Hitoshi Nakamoto(Saitama University,Japan): opposite modu-lationsofasingleproteinmayunexpectedlydeterminethesame effect. He focused attention on the ATP dependent molecular chaperone Hsp90, whose activity is essential for cancer cell survivalasmanycancer-relatedproteinsaresubstratesforHsp90. His research highlighted that, in addition to inactivation, the hyperactivationoftheHsp90ATPaseactivity isalsoharmfulto cancer cells [20]. These outcomes are promising for the development of novel therapeutic strategies in killing cancer cells.
Acknowledgements
WethankMichaelSauerandJeffColeforcriticallyreadingthe manuscript and providing helpful comments. Grants for atten-dancebystudentsandyoungscientistsweresponsoredbyFEMS. EPPENDORFis alsogratefullyacknowledgedforproviding spon-sorship including cash prizes for the best posters by young scientists.
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