Brevibacterium linens AE038-8 is an arsenic hyper-tolerant bacterial strain, previously isolated from well water in Tucumán, Argentina. The aim of this study is to investigate the resistance of this strain to different stress factors relevant for astrobiology studies. We found that B. linens AE038-8 is capable of tolerating high concentrations of heavy metals such as Cd(II), Cr(VI) and Cu(II), and a range of temperatures from 10 to 30°C. It is also able to grow in the range of pH values from 3 to 11, and when grown in the presence of NaCl, it can tolerate concentrations up to 5 M. We also study the ability of the strain to grow under combined stress conditions. In particular, we found that it is able to grow under high NaCl concentrations and low pH, conditions relevant to the habitability of brines in Mars.Fil: Maizel, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Alche, Laura Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Mauas, Pablo Jacobo David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; Argentin

Alche, Laura Edith

Maizel, Daniela

Mauas, Pablo Jacobo David

English

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Mem. S.A.It. Vol. 90, 658c© SAIt 2019 Memorie dellaPoly-extremophiles: exploring the limits ofhabitabilityD. Maizel1, L. E. Alché2, and P. J. D. Mauas11 Instituto de Astronomı́a y Fı́sica del Espacio, CONICET, UBA, Casilla de Correo 67 - Suc.28 (C1428ZAA), Buenos Aires, Argentina2 Departamento de Quı́mica Biológica, CONICET, UBA, 4 Piso, Pabellón II, CiudadUniversitaria (1428), Buenos Aires, ArgentinaAbstract. Brevibacterium linens AE038-8 is an arsenic hyper-tolerant bacterial strain, previ-ously isolated from well water in Tucumán, Argentina. The aim of this study is to investigatethe resistance of this strain to different stress factors relevant for astrobiology studies. We foundthat B. linens AE038-8 is capable of tolerating high concentrations of heavy metals such asCd(II), Cr(VI) and Cu(II), and a range of temperatures from 10 to 30◦C. It is also able to growin the range of pH values from 3 to 11, and when grown in the presence of NaCl, it can tolerateconcentrations up to 5 M. We also study the ability of the strain to grow under combined stressconditions. In particular, we found that it is able to grow under high NaCl concentrations andlow pH, conditions relevant to the habitability of brines in Mars.Key words. Brevibacterium linens – stress factors – poly-extremophiles – astrobiology1. IntroductionOne of the main areas of interest inAstrobiology is the study of extremophiles, or-ganisms that can adapt or even thrive in ex-treme conditions (Cavicchioli 2002). One ofthe main reasons for this interest is that theconcept of habitability, which refers to the po-tential of a celestial body to harbor life, is cen-tral to Astrobiology. Since the only life formswe know are the ones that can be found onEarth, it becomes fundamental to enlarge ourknowledge about extreme life forms inside ourown planet, in order to accurately understandwhich are the physical or geochemical condi-tions that can limit the presence of life. In par-ticular, life originated on Earth under very ex-treme conditions!On the other hand, the study of some typesof extremophiles is central to the hypothesisof panspermia, which proposes that certain lifeforms can survive space travel, and propagatelife across different planets. These organismsshould be able to survive to high-vacuum con-ditions, high levels of radiation and extremetemperatures (Abrevaya et al. 2011). Thesestudies are also central to prevent contamina-tion of extraterrestrial environments with ter-restrial microorganisms during space missions(Martı́n-Torres & Zorzano 2017).Studies of extremophiles cover organismsthat can survive in very high concentrationsof salt (halophiles) or heavy metals, in veryhigh or low temperatures (thermophiles or psy-chrophiles), in acid or alkaline environments(acidophiles or alkaliphiles), in extremely dryMaizel et al.: Poly-extremophiles 659conditions (xerophiles) or under high level ofradiation (radioresistant), among others.However, conditions in extraterrestrialhabitats are most certainly extreme in many as-pects at the same time. For example, strongevidence of the existence of salt hydratesand brines on Mars has recently been found(Martı́n-Torres et al. 2015; Ojha et al. 2015),and it was proposed that these brines couldprovide a habitable environment (Leuko et al.2014). Of course, any form of life capable ofliving in brines should be halophilic, but a highconcentration of salt is not the only extremecondition in those environments. Such formsof life should also be able to endure at leastlow temperatures and high levels of radiation.Therefore, the next step to understand thereal limits of habitability should be to study or-ganisms that can survive under several extremeconditions at the same time, better known aspoly-extremophiles. In this paper, we studyBrevibacterium linens AE038-8, a bacterialstrain isolated from arsenic-rich groundwaterin Tucumán, Argentina. In our previous work,we determined resistance of this strain to ar-senicals and to the highest concentrations ofarsenic oxyanions reported so far in the liter-ature (Maizel et al. 2016).Here we investigate the tolerance of B.linens AE038-8 to multiple stress factors rel-evant for astrobiology, such as tolerance toheavy metals, to extreme pH values, differenttemperatures and to high salt concentrations,conditions which might be present in planetsof our Solar System and in exoplanets.2. Resistance of B. linens to differentextreme conditionsTemperature: Among the physical parametersimportant for life, temperature is probably oneof the most studied since extreme temperatureaffects the organisms in different ways, caus-ing destruction of biomolecules (Rothschild &Mancinelli 2001; Prieur 2007). We found thatB. linens grows at temperatures up to 55◦C, andthat the optimum growth temperature is 30◦C,in accordance with the value reported for otherstrains of Brevibacterium (Sharpe et al. 1977;Collins 2006).Most extremophiles are mesophiles likethis one. Approximately 60% of extremophilicbacteria reported in the literature have temper-ature optima of 40◦C or below: 25% of thesebacteria have temperature optima between 38and 40◦C, and approximately 50% between 32and 37◦C, like B. linens AE038-8.pH: Biological processes in normal condi-tions occur in the middle of the pH spectrum(between 5 and 8.5). Extreme concentrationsof hydrogen ions will have a determinant ef-fect on the ability of organisms to thrive in acertain environment (Oarga 2009). The bacte-rial isolates of the Brevibacterium genus re-ported so far grow at pH values between 3.5 to8.5 (Lukacs et al. 1995). In this case, B. linensAE038-8 shows a great versatility, being ableto tolerate pH values from 2.8 to 11. It cantherefore be considered as acidophilic, accord-ing to the definition of Norris et al. (2000).Heavy metals: It is well known that manymicroorganisms able to grow at low pH val-ues not only tolerate, but thrive in metal-richsolutions (Hallberg & Johnson 2001; Johnsonet al. 2001). We found that B. linens AE038-8 is resistant to high concentrations of Cd(II).In a previous study, we sequenced the com-plete genome of this strain, and we detecteda cadmium resistance operon which might ex-plain this resistance (Maizel et al. 2015). Sincebacteria from the Actinobacteria phylum areusually resistant to multiple heavy metals, westudied the resistance to Cr(VI) and Cu(II) aswell, and we observed that the bacterial strainis also highly resistant to these metals.NaCl: The ability of microorganisms tosurvive in high concentrations of salt is as-trobiologically relevant since it has been pre-viously proposed that sodium-chloride richbrines could be present in the surface ofJupiters moon Europa, as a consequence of hy-drothermal processes in its seafloor environ-ment (Kargel et al. 2000). We found that B.linens can survive in concentrations of NaClfrom 0.2 to 5.3 M. This fact means that it isan extreme halophile, according to the con-ventional classification. In fact, only a fewhalophiles can survive in the range 3 to 3.5 MNaCl (DasSarma & DasSarma 2017).660 Maizel et al.: Poly-extremophilesTable 1. Growth of B. linens AE038-8 in increasing concentrations of NaCl and NaCl plusarsenic compounds. Growth in each condition was qualitatively determined as +: growth or -:absence of growth.NaCl concentration0.2 M 0.5 M 1 M 2 M 3 M 4 M 5 M 5.3 MNaCl + + + + + + + -NaCl + As(III) 3 mM + + + + + - - -NaCl + As(V) 10 mM + + + + + + + -Many of the extreme halophiles reported sofar belong to the Archaea domain. However,some extremely halophilic bacteria have beendescribed, in particular from the phylumActinobacteria, to which B. linens belongs(Bowers et al. 2009). Interestingly, most of theextreme halophiles reported so far were iso-lated from hypersaline natural environmentssuch as the Great Salt Lake in the UnitedStates, the Dead Sea or evaporite sites inthe Atacama Desert and Tunisian sabkhas(Stivaletta et al. 2009; Barbieri & Stivaletta2011; DasSarma & DasSarma 2017). However,the case of B. linens AE038-8 is very different,since it was not isolated from a saline envi-ronment. Only a few similar cases have beenfound. For example, Purdy et al. (2004) re-ported three haloarchaea isolated from estu-arine sediments at Essex, UK (with a salinitylevel similar to that found in sea water), whichwere able to grow at 2.5% NaCl, a salinitymuch lower than the one reported here.Combined effect of NaCl and arsenic:It is important to study the effect of differ-ent stress factors combined, since the environ-ments in planets different from Earth will mostcertainly be extreme in many aspects. We haveshown that B. linens can tolerate up to the veryextreme concentrations of 75 mM and 1 M ofAs(III) and As(V) respectively (Maizel et al.2016). Here we investigated its growth in pres-ence of NaCl and arsenic simultaneously. Theresults are summarized in Table 1.We found that in a concentration of 3 mMAs(III), B. linens only grew up to 3 M NaCl,suggesting that both stress factors combinedexert a remarkable toxic effect on growth. Onthe other hand, in a concentration of As(V)10 mM the strain was able to grow up to 5 MNaCl, similar to what is found in absence of ar-senic, showing that arsenate is not significantlytoxic for B. linens. This result was expectedconsidering that arsenate has been proven to benotably less toxic than arsenite (Hughes 2002).We also studied the strain’s growth-curvesin different conditions (Fig. 1). We see that thecurve obtained with a concentration 3 mM ofAs(III) is essentially the same as the controlone, but that the presence of salt in a concentra-tion of 2 M introduces a delay in the growth ofa 22 hours (lag phase). If arsenite is added, thelag phase remains the same, but a strong differ-ence in the exponential phase can be seen, andgrowth is 7 times slower. Only after 10 hoursboth cultures stabilized, reaching the stationaryphase with almost the same cell density, notice-ably lower than the control one.Low pH and NaCl combined: It hasbeen suggested that different types of salts in-Fig. 1. Bacterial growth under different stres-sors. Diamonds: control. Squares: As(III) 3mM.Triangles: NaCl 2M; Circles: As(III) 3mM + NaCl2MMaizel et al.: Poly-extremophiles 661cluding chlorides are abundant on Mars, andthat its surface is also acidic (Oren 2014).Although neither liquid water nor brines arecurrently stable on the red planet, it has beenstated that liquid brines could temporarily bepresent in certain areas as a result of deliques-cence (Rennó et al. 2009; Davila et al. 2010).Therefore, microorganisms not only able totolerate high salinity, but also able to thriveat the lowest end of the pH scale are relevantfrom an astrobiological perspective. We there-fore studied the ability of B. linens to grow atpH values of between 2.3 and 4, combined withNaCl concentrations up to 3 M.At pH 3.5 and 4 growth was observed at48 h after incubation at all NaCl concentra-tions tested, suggesting that NaCl does notplay a compelling role as a stress factor. Withlower pH values of 2.8 and 3, bacterial growthwas detected only at NaCl up to 2 M, after120 h of incubation. The pH value of 2.8 isfar lower than the minimum pH reported forgrowth of strains of the Brevibacterium genus.We did not observe any growth at pH 2.3 af-ter 300 h of incubation, even in the absence ofNaCl. Since acidic hypersaline environmentshave been scarcely studied in comparison toprevalent alkaline to neutral hypersaline en-vironments, few halo-tolerant acidophilic mi-croorganisms able to thrive in those conditionshave been reported (Seckbach 2013).3. DiscussionProkaryotic microorganisms are known to behighly adaptable to different stresses and tothrive in harsh environments. This is proba-bly a consequence of the flexibility of theirgenomes, which allows life to adapt to a widespectrum of environmental constraints. As thistype of studies evolve, it becomes more evi-dent that resistance to different stressors mightbe connected. For example, we found that B.linens AE038-8 is, on the one hand, toler-ant to the highest concentrations of arseniccompounds known so far, and, on the otherhand, can grow at high concentrations of heavymetals. This two properties might be related,since it has been shown that an in vitro pre-treatment of cell cultures with arsenic fre-quently provides crossed protection againsttoxicity caused by other metals, such as cad-mium (Carrasco et al. 2005).Metal resistance mechanisms of aci-dophiles were extensively studied, in particu-lar for copper and cadmium. Dew et al. (1999)reported an example of acidophilic bacteria re-sistant to toxic concentrations of Cu(II) andCd(II). Other acidophilic bacteria resistant toCd(II) were reported by Dopson et al. (2003)and Pikuta et al. (2007).Additionally, we observe biofilm formationat pH between 3 and 11. The formation ofbiofilms is generally considered as a cellulardefense mechanism from several types of en-vironmental stresses, through physical protec-tion of the bacterial cells and nutrient seques-tration (Bollinger et al. 2001; Stewart 2002;Castonguay et al. 2006; Harrison et al. 2007;Koerdt et al. 2010). Thus, the ability of thisbacterial strain to form biofilms during growthcould partially explain its adaptation to differ-ent pH values and temperatures, and the highresistance observed to the many stress factorstested in this study.In summary, B. linens AE038-8 is a bacte-rial strain isolated from arsenic-rich well wa-ter (Maizel et al. 2016). Apart from the ar-senic content, the physico-chemical character-istics of this natural environment are not re-ally considered ”extreme”. Even though thisstrain grows optimally at normal conditions(i.e. 30◦C and pH 7), it is able to grow underthe many stress factors evaluated in the presentwork. What makes this bacterial strain highlyresistant to different stressors still remains un-known. In this context, we suggest that the hab-itability range of extremely tolerant bacteriasuch as B. linens AE038-8 is actually muchwider than what was assumed until now.ReferencesAbrevaya, X. C., et al. 2011, Astrobiology, 11,1034Barbieri R. & Stivaletta N. 2011, Geol. J. 46,513Bollinger, N., et al. 2001, J. Bacteriol., 183,1990Bowers, K., et al. 2009, Saline systems, 5, 9662 Maizel et al.: Poly-extremophilesCarrasco, J. A., et al. 2005, Soil. Biol.Biochem., 37, 1131Castonguay, M. H., et al. 2006, Res.Microbiol., 157, 471Cavicchioli, R. 2002, Astrobiology, 2, 281Collins, M. 2006, The genus Brevibacterium.In: The Prokaryotes. Springer, p. 1013DasSarma, S. & DasSarma, P. 2017, Halophi-les. In eLS, John Wiley & Sons, Ltd.Davila, A. F., et al. 2010, Astrobiology,10, 617Dew, D. W., et al. 1999, In Alta Copper 99.Brisbane, AustraliaDopson, M., et al. 2003, Microbiol., 149, 1959Hallberg, K. B. & Johnson, D. B. 2001, Adv.Appl. Microbiol., 49, 37Harrison, J., et al. 2007, Nat Rev. Microbiol.,5, 928Hughes, M. F. 2002, Toxicol. Lett., 133, 1Johnson, D. B., et al. 2001, EnvironmentalMicrobiol., 3, 630Kargel, J. 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Poly-extremophiles: exploring the limits of habitability

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Poly-extremophiles: exploring the limits of habitability

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