Dartnell, LR

Fallaize, DR

Ward, JM

Whiting, SJ

English

UCL Discovery

http://journals.cambridge.org Downloaded: 22 Nov 2013 IP address: 144.82.107.56Antarctic Science 22(2), 171–172 (2010) & Antarctic Science Ltd 2010 doi:10.1017/S0954102009990745Short NoteDesiccation resistance of Antarctic Dry Valley bacteria isolatedfrom contrasting locationsLEWIS R. DARTNELL1,2*, DAVID R. FALLAIZE1,2, SAMANTHA J. WHITING2 and JOHN M. WARD21Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX), University College London,Gower Street, London WC1E 6BT, UK2Research Department of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK*l.dartnell@ucl.ac.ukReceived 3 July 2009, accepted 5 October 2009IntroductionThe McMurdo Dry Valleys of Antarctica exhibit extensiveareas completely devoid of ice and are extremelyinhospitable to life (Cowan et al. 2002), yet manyorganisms are known to survive loss of virtually all theircellular water (Billi & Potts 2002). This study aims toinvestigate the link between desiccation resistance and themicrohabitat of Antarctic bacteria testing if bacteria froma more sheltered habitat will exhibit lower desiccationresistance than bacteria from an exposed location. Inaddition, Deinococcus radiodurans Brooks and Murray isused as model desiccation-resistant organism, in contrastto Escherichia coli, a human enterobacterium with noadaptive requirement for desiccation-resistance.Materials and methodsSamples of Miers Valley soil were collected in February 2002from beneath a dead algal mat surrounding a small frozen lakeand the desiccated mineral soil from the valley ﬂoorrepresenting a sheltered and organic nutrient-rich niche, anda more exposed oligotrophic environment, respectively.Samples were below 08C until 1–2g of soil were vortexedwith an equal volume of sterile water and 50mlv o l u m e st h e nspread on quarter-strength nutrient broth agar (14NB; CM0001Nutrient Broth, Oxoid, 3.25g and 20g agar per 1l deionisedwater), starch agar (5g per l potato starch; Sigma and 20g perl agar), quarter-strength Czapek-Dox agar (8.75gl-1 CZD,Difco and 20g per l agar), and plates incubated at 48C. Pureisolates were obtained by subculturing individual colonies twofurther times. Subsequent investigation found that isolates alsogrew well at room temperature.Identiﬁcation of isolated bacteriaSingle colonies were picked by sterile loop, resuspended in20ml of sterile water, heated to 958C in a thermocycler,then centrifuged. A 1ml volume of the supernatant wasused as the template for a polymerase chain reaction (PCR)with Taq polymerase. Primer set CC/CD (Rudi 1997) wasselected to amplify a ,600bp region of the bacterial 16SrRNA gene. PCR products were puriﬁed using the QIAquickPCR Puriﬁcation Kit (Qiagen) and ligated into the pCR2.1vector with the TOPO R TA cloning kit (Invitrogen). A vialof OneShot R chemically competent E. coli cells (Invitrogen)were transformed and then plated onto selective media.Recombinant colonies were picked and grown in liquidmedium, then plasmids extracted using QIAprep SpinMiniprep Kit (QIAGEN). Recombinant pCR2.1 containingthe 16S rRNA gene inserts were sequenced using vector-speciﬁc primers M13F and M13R (Pearce et al. 2003).Identiﬁcation of each isolate based on its 16S rRNA genesequence was conﬁrmed using both the Ribosomal DatabaseProject, RDP, (http://rdp.cme.msu.edu) and BLASTn (NCBI)sequence search utility (www.ncbi.nlm.nih.gov/BLAST).Desiccation survivalPseudomonas sp.a n dPsychrobacter sp., from beneath the algalmat, Bacillus sp. and Streptomyces sp., from the exposed valleyﬂoor, and D. radiodurans and E. coli Top10 were streaked ontosterile agar plates. A single colony was picked to inoculate14 NB (Oxoid) liquid cultures, and incubated with constantagitation at room temperature for three days. 100ml samples ofeach liquid culture were pipetted into ten separate 35mmFalcon tissue culture dishes, and s t a c k e di ns t e r ilized air-tightNalgene tubs over 2cm of granulated anhydrous calciumchloride as a desiccant. In addition, a control dish containing100mlo fs t e r i l e14N Bw a sp r e p a r e di nt h es a m ew a ya sac h e c kfor contamination. After set time periods, culture dishes wereremoved and rehydrated with 1ml phosphate buffer solution(PBS; Sigma), agitated for ten minutes on an oscillating tableat 20 oscillations/minute. A colony forming unit (CFU) assaywas performed in triplicate on the pre-desiccation cultures andeach recovered sample of rehydrated bacteria solution. Theratio of viable cells in the rehydrate to original culture givesthe population desiccation survival, corrected for the 90%efﬁciency of culture rehydration and recovery found here.Results and discussionOn the 14NB agar cultures, initial plate counts found a totalof 83103 cells.g-1 wet wt from beneath the algal mat and171http://journals.cambridge.org Downloaded: 22 Nov 2013 IP address: 144.82.107.5663102cellsg-1 wet wt from the exposed sampling location.Gram negative bacteria were dominant beneath the algal mat(9/12 isolates from all media types), with all isolates fromnon-spore-forming genera, and Gram positive bacteria weredominant in the exposed sample (11/12 isolates), with 5/12strains spore-forming. The strains used for this desiccationstudy were thus selected as characteristic species from eachsampling location. Bacillus, Pseudomonas,a n dStreptomycesare among the most commonly isolated bacterial genera fromthe McMurdo Dry Valley soils (Cowan & Tow 2004), andPsychrobacter aquatica Shivaji was originally isolated fromthe Dry Valleys (Shivaji et al. 2005).Figure 1 shows the proportion of surviving cells as afunction of desiccation time, colour-coded for the samplinglocations to make clear the correlation between microhabitatand desiccation survival. After a week of desiccation, viabilityof the two algal mat species is undetectable (, 10 viable cellsin the 100ml of desiccated culture). In contrast, isolates fromthe exposed location initially lose viability but soon plateau ataround one in 104 survival up to the full 28 days of desiccation.Deinococcus radiodurans exhibits the greatest resistance of allsix test strains, with roughly 60% survival at up to two weeksdesiccation, whilst E. coli shows very poor survival.These results support the prediction that Antarctic DryValley bacteria isolated from a more barren and exposedlocation exhibit much greater desiccation resistance thanthose adapted to more a more sheltered microenvironment.AcknowledgementsL.R. Dartnell and D. Fallaize were supported by an EPSRCDoctoral Training Centre studentship. S.J. Whiting wassupported by a BBSRC PhD studentship.ReferencesBILLI,D .&P OTTS, M. 2002. Life and death of dried prokaryotes. Researchin Microbiology, 153, 7–12.COWAN, D., RUSSELL, N., MAMAIS,A .&S HEPPARD, D. 2002. Antarctic DryValley mineral soils contain unexpectedly high levels of microbialbiomass. Extremophiles, 6, 431–436.COWAN,D .&T OW, L.A. 2004. Endangered Antarctic environments.Annual Review of Microbiology, 58, 649–690.PEARCE, D.A., GAST, C.J., LAWLEY,B .&E LLIS-EVANS, J.C. 2003.Bacterioplankton community diversity in a maritime Antarctic lake,determined by culture-dependent and culture-independent techniques.FEMS Microbiology Ecology, 45, 59–70.SHIVAJI, S., REDDY, G.S.N., SURESH, K., GUPTA, P., CHINTALAPATI, S.,SCHUMANN, P., STACKEBRANDT,E .&M ATSUMOTO, G.I. 2005.Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov.,from Antarctica. International Journal of Systematic and EvolutionaryMicrobiology, 55, 757–762.Fig. 1. The surviving fraction of bacterialpopulations against time spent desiccated.Sampling regions: dashed line5beneath analgal mat, dot-dash line5exposed valleyside. Double-exponential trend lines ﬁtted toStreptomyces and Bacillus, exponential trendsto all others.172 LEWIS R. DARTNELL et al.

Antarctic Dry Valley mineral soils contain unexpectedly high levels of microbial biomass.

Bacterioplankton community diversity in a maritime Antarctic lake, determined by culture-dependent and culture-independent techniques.

Endangered Antarctic environments.

Life and death of dried prokaryotes.

Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov., from Antarctica.

Desiccation resistance of Antarctic Dry Valley bacteria isolated from contrasting locations

Desiccation resistance of Antarctic Dry Valley bacteria isolated from contrasting locations

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