Model organisms for genetics in the domain Archaea: methanogens

32 Pages·2011·0.42 MB·English

REVIEW ARTICLE
ModelorganismsforgeneticsinthedomainArchaea:methanogens,
halophiles,ThermococcalesandSulfolobales
JohnA.Leigh1,Sonja-VerenaAlbers2,HaruyukiAtomi3&ThorstenAllers4
1DepartmentofMicrobiology,UniversityofWashington,Seattle,WA,USA;2Max-Planck-InstituteforTerrestrialMicrobiology,Marburg,Germany;
3DepartmentofSyntheticChemistryandBiologicalChemistry,GraduateSchoolofEngineering,KyotoUniversity,Nishikyo-ku,Kyoto,Japan;and
4SchoolofBiology,Queen’sMedicalCentre,UniversityofNottingham,Nottingham,UK
Correspondence:ThorstenAllers,Schoolof Abstract
Biology,Queen’sMedicalCentre,Universityof
Thetreeoflifeissplitintothreemainbranches:eukaryotes,bacteria,andarchaea.
Nottingham,NottinghamNG72UH,UK.Tel.:
Our knowledge of eukaryotic and bacteria cell biology has been built on a
1441158230304;fax:[phone];
e-mail:[email protected] foundationofstudiesinmodelorganisms,usingthecomplementaryapproaches
ofgeneticsandbiochemistry.Archaeahaveledtosomeexcitingdiscoveriesinthe
Received31March2010;revised22December fieldofbiochemistry,butarchaealgeneticshasbeenslowtogetofftheground,not
2010;accepted22December2010. least because these organisms inhabit some of the more inhospitable places on
Finalversionpublishedonline7March2011. earthandarethereforebelievedtobedifficulttoculture.Infact,manyspeciescan
be cultivated with relative ease and there has been tremendous progress in the
DOI:10.1111/j.1574-6976.2011.00265.x
developmentofgenetictoolsforbothmajorarchaealphyla,theEuryarchaeotaand
theCrenarchaeota.Thereareseveralmodelorganismsavailableformethanogens,
Editor:MeckyPohlschroder
halophiles, and thermophiles; in the latter group, there are genetic systems for
Sulfolobales and Thermococcales. In this review, we present the advantages and
Keywords
disadvantages of working with each archaeal group, give an overview of their
archaea;genetics;methanogens;Sulfolobales;
Thermococcales;halophiles. different genetic systems, and direct the neophyte archaeologist to the most
appropriatemodelorganism.
S
W
E
amenable to experimental manipulation. For microbial
I Introduction
V geneticists, the minimal specification is the ability to grow
E In his ‘An Essay on Man’, the English poet Alexander Pope in isolation on solid media. Robert Koch first recognized
R exhortsusto‘Knowthenthyself,presumenotGodtoscan; thatacolonyformedonanagarplaterepresentstheclonal
The proper study of Mankind is Man’. The resounding expansion of a single cell, and this unassuming mound of
Y
successofbiomedicalresearchusingmodelorganismsgives cells has always been the cornerstone of microbiology. The
G
usreasontodoubtthewisdomofPope’swords.Mostofour abilitytogeneratemutantsisanotherpartofthefoundation
O knowledge of fundamental biological processes has come of microbial genetics. Traditionally this was carried out by
from work on simple and experimentally tractable species random mutagenesis (forward genetics), but since the
L
O such as Escherichia coli, Saccharomyces cerevisiae, and Cae- molecularbiologyrevolutionthepreferredmethodhasbeen
norhabditis elegans. Over time, these basic principles have targetedmutationofaspecificgene(reversegenetics).The
I
B been verified in complex species such as man, but simple latter wouldnotbepossiblewithout methodsfor transfor-
O modelorganismsremainvitaltofurthermedicaldiscoveries mation,selectablemarkers,plasmidvectors,andsystemsfor
(Fields & Johnston, 2005). Nevertheless, there is danger in gene knockout by homologous or site-specific recombina-
R
suchareductionistapproach–noteverythinginbiologycan tion. Of late, reverse genetics has been made significantly
C
belearnedfromE.coli.Inordertowitnessthe‘grandeurin easier by whole genome sequencing, and is now taken for
I
M this view of life’ (apologies to Charles Darwin), we must grantedinthegenetictoolbox.
expand our repertoire of model organisms to include Archaea makeup one ofthe three main branches of the
representativesofthedomainArchaea. evolutionary tree; they are as different from eukaryotes as
The choice of model organism is critically important. It theyarefrombacteria(Garrett&Klenk,2007).Thedistinct
shouldbeeasytogrow,haveashortgenerationtime,andbe status of archaeawas revealed in the late 1970s, when Carl
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578 J.A.Leighetal.
Woeseandcolleaguesseizedupontheemergingtechnology protocols. In addition, haloarchaea are easy to cultivate
ofnucleicacidsequencingtotackletheproblemofprokar- becausetheygrowatmoderatetemperatures.Methanogenic
yotic phylogeny. Woese chose small-subunit rRNA as a archaea are also mesophilic, but unlike haloarchaea, their
molecularchronometer;rRNAisanessentialcomponentof cytoplasmisnothypersaline.Thishaspermittedthedirect
all self-replicating organisms and shows remarkable se- adaptationofmanytoolsfrombacterialgeneticstometha-
quence conservation. The tree he constructed showed that nogens; bacterial antibiotics remain the exception, their
an unusual group of methane-producing microorganisms targets are generally not found in archaea. Thermophilic
were not bacteria, but formed a separate domain. Woese archaeaofkingdomsEuryarchaeotaandCrenarchaeotahave
termed these organisms ‘archaebacteria’, but later changed long been of interest to biochemists and structural biolo-
this name to archaea (Woese & Fox, 1977; Woese et al., gists, owing to their thermostable enzymes. They offer
1990). The tree suggested a closer relationship between significant potential for biotechnology, and for researchers
archaea and eukaryotes, compared with bacteria, and re- wishing to usea multidisciplinaryapproach that combines
flected the common heritage of information-processing geneticswithbiochemistry.
systems found in the archaeo-eukaryal lineage. This had Inthisreview,weofferguidancetomicrobiologistswho
been noted by Wolfram Zillig and colleagues in the 1980s, wishtoconverttothethirddomain,andreassurethemthat
whentheyfoundthatarchaealDNA-dependentRNApoly- archaealgeneticsisnotdifficultorunusual.Thefirststepon
meraseisstrikinglysimilartoitseukaryoticcounterpart,in this road is to choose the most appropriate model organ-
terms of both complexity and subunit composition (Huet isms.Wedealinturnwitheacharchaealgroup,highlighting
et al., 1983). Genome sequencing in the 1990s confirmed theiradvantagesanddisadvantagesintermsofthescientific
that archaea are a genetic mosaic – their information questions that can be addressed, and the tools available to
processingsystemsshowsignificanthomologytoeukaryotic answerthesequestions.Ourhopeisthatmoremicrobiolo-
counterparts,whilemostoperational(housekeeping)func- gistswillworkonarchaea,andthosewhoalreadydosowill
tions have a bacterial aspect (Olsen & Woese, 1996; Rivera venture beyond the safe environs of biochemistry and
etal.,1998;Yutinetal.,2008). structuralbiology.Onlywhengeneticshasfoundaplacein
Over time, many halophilic and thermophilic microor- everyarchaeallaboratorywillthethirddomainofliferank
ganisms have found their home in the archaeal domain. alongsideitseukaryoticandbacterialcousins.
Several of these species had been studied long before the
thirddomainoflifewasproposed.Forexample,bacterior-
Methanogens
hodopsin had been discoveredin Halobacterium salinarum
in 1971 (Oesterhelt & Stoeckenius, 1971), Thomas Brock
Introduction to methanogens,an ecologically
had isolated Sulfolobus acidocaldarius from acidic mud
and biochemicallydistinctive group
ponds in Yellowstone National Park in 1972 (Brock et al.,
1972),andinthe18thcenturyAlessandroVoltahadunwit- In 1977, a collaboration between the laboratories of Carl
tingly unearthed methanogenic archaea in the swamps of Woese and Ralph Wolfe resulted in the finding that the
northern Italy. What appeared to bind together all these methanogens were ‘only distantly related to typical bacteria’
exotic microorganisms was their love of habitats that had (Fox et al., 1977). Thus, the methanogens became the first
previously been considered uninhabitable. However, culti- knownArchaea.Theyarenowknowntocomprisefiveorders
vation-independent analysesof microbial biodiversity have of the Euryarchaeota: Methanococcales, Methanosarcinales,
since revealed that archaea are surprisingly abundant in Methanobacteriales, Methanomicrobiales, and Methanopyrales
‘normal’ environments (DeLong, 1998; Karner et al., 2001; (Liu&Whitman,2008).Genetictoolsareavailableforcertain
Robertsonetal.,2005).Unfortunatelythesearchaeaarenot speciesofthefirsttwoorders.
fit for genetics, because with the exception of the recently The methanogens are those organisms that generate
isolated Nitrosopumilus maritimus (Konneke et al., 2005), methaneasacatabolicend-product(Wolfe,1996).Biological
theycannot yetbeculturedinisolation(Hugenholtzetal., methanogenesis occurs in a variety of anaerobic habitats,
1998;Schleperetal.,2005).Wearethereforeleftwithfour including marine and freshwater sediments, rice paddies,
groups of archaea for which genetic systems have been bioreactors and sewage sludge digesters, landfills, animal
developed:methanogensandhalophiles(botheuryarchaea), digestivetracts,andhydrothermalvents(Wolfe,1996).Most
as well as thermophilic euryarchaea (Thermococcales) and of these habitats contain an anaerobic ecosystem in which
crenarchaea(Sulfolobales)(Fig.1). methanogenesis is the final step in the decomposition of
Eachofthesearchaealgroupshasitsownuniqueselling organic matter. However, in habitats such as hydrothermal
point. Haloarchaea are renowned for the comparative vents the substrates for methanogenesis, H and CO , are
2 2
sophistication of their genetic systems, the development of presumablyofgeochemicalorigin.Muchofthemethanethat
whichwas made possible by early workon transformation is generated is reoxidized to CO or becomes sequestered in
2
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Archaealmodelorganisms 579
Halorubrum lacusprofundi
0.02 Halogeometricum borinquense
Haloferax volcanii
Haloquadratum walsbyi
Halomicrobium mukohataei Halobacteriales
Natronomonas pharaonis
Haloarcula marismortui
Halorhabdus utahensis
Halobacterium sp. NRC1
Halobacterium salinarum
Methanospirillum hungatei
Methanocorpusculum labreanum Methanomicrobiales
Methanosphaerula palustris
Methanoculleus marisnigri
Methanocella paludicola Methanocellales
Methanosaeta thermophila
Methanosarcina mazei
Methanosarcina barkeri Methanosarcinales
Methanosarcina acetivorans E
u
Methanococcoides burtonii ry
MethanotherMmeothbaanctoesrp thhaeermraa suttaodtrtompahnicaues Methanobacteriales arch
a
Methanobrevibacter smithii e
o
Picrophilus torridus ta
Thermoplasma volcanium Thermoplasmatales
Thermoplasma acidophilum
Aciduliprofundum boonei
Methanococcus aeolicus
Methanococcus maripaludis
Methanococcus voltae Methanococcales
Methanococcus vannielii
Methanocaldococcus jannaschii
Thermococcus onnurineus
Thermococcus gammatolerans
Thermococcus kodakaraensis
Thermococcus barophilus
Thermococcales
Thermococcus sibiricus
Pyrococcus furiosus
Pyrococcus horikoshii
Pyrococcus abyssi
Archaeoglobus profundus
Ferroglobus placidus Archaeoglobales
Archaeoglobus fulgidus
Methanopyrus kandleri Methanopyrales
Pyrobaculum islandicum
Pyrobaculum calidifontis
Pyrobaculum aerophilum
Thermoproteus neutrophilus Thermoproteales
Pyrobaculum arsenaticum
C
Caldivirga maquilingensis re
Thermofilum pendens n
a
Metallosphaera sedula rc
Sulfolobus acidocaldarius ha
Sulfolobus tokodaii Sulfolobales eo
Sulfolobus islandicus ta
Sulfolobus solfataricus
Ignicoccus hospitalis
Desulfurococcus kamchatkensis
Staphylothermus marinus Desulfurococcales
Aeropyrum pernix
Hyperthermus butylicus
Fig.1. Phylogenetictreeshowingkeyarchaealspecieswithgeneticsystems.Phylogenetictreebasedon16SrRNAgenesequencesofselectedarchaeal
species whose genome sequences are available. Organisms indicated with solid red stars are key species that have been the focus of genetic
development; open stars indicate species where genetics has been applied or where there is potential for genetics. Sequence alignments were
performedusingClustalWandthetreewasconstructedbyneighborjoining;brancheswithbootstrapvaluesof o50%arenotshown.
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580 J.A.Leighetal.
methanehydrates.However,asignificantamountofmethane beguntofillsomegapsinourknowledgeleftfrombiochem-
is emitted into the atmosphere where it becomes a major ical approaches, as mentioned below. In addition, methano-
greenhouse gas (Liu & Whitman, 2008). The product of gens have been chosen for many studies of the molecular
methanogenesisisalsoofobviousimportanceasafuel. biology and physiology of Archaea. Methanocaldococcus jan-
Methanogenesis is a kind of anaerobic respiration where naschiibecamethefirstspeciesofArchaeatobesubjectedto
single-carbon (C-1) units, most notably CO , serve as the genome sequencing in 1996 (Bult et al., 1996), and many
2
electronacceptor.Thermodynamicsdictatesthatmethanogen- studiesfollowed.GenetictoolshavenotbeendevelopedforM.
esiswilloccuronlywhenmorefavorableelectronacceptorsare jannaschii, but many questions can be addressed using the
absent.Hence,methanogenesisismostprevalentwhensulfate, genetic tools for its relatives in the genus Methanococcus
nitrate, oxidized metals, and, especially, oxygen are absent. (Tumbula & Whitman, 1999). Methanogens are models for
BecauseCO istheonlyelectronacceptorthatdoesnotoweits archaeal replication (Walters & Chong, 2009), transcription,
2
abundancetophotosynthesis,methanogenesisisfavoredasan regulation(Geiduschek&Ouhammouch,2005),osmoregula-
early metabolism on earth, predating photosynthesis and tion(Spanheimer&Muller,2008),andproteinstructure.The
otherformsofrespiration(Kasting&Siefert,2002). role of methanogens in nature leads directly to questions of
Substrates for methanogenesis are relatively restricted syntrophy, the associations between organisms that facilitate
(Whitman et al., 2001). Nearly all species in the orders the transfer of nutrients (Shimoyama et al., 2009). The
Methanococcales, Methanobacteriales, Methanomicrobiales, discoverythatcloserelativesoftheMethanosarcinalesaswell
andMethanopyralesarehydrogenotrophic,usingH andCO. assulfatereducersareinvolvedinanaerobicmethaneoxida-
2 2
Many of these species can also use formate. In contrast, tionhasbroadenedtheimportanceoftheseorganismsinthe
theMethanosarcinalesiscomprisedofmethylotrophicspecies, globalcarbonandsulfurcycles(Knittel&Boetius,2009).
whichusemethylcompoundssuchasmethanolandmethyla- Methanogens are strict anaerobes that require special
mines; some can use H and CO as well. In addition, measures for their growth in the lab. However, in the late
2 2
MethanosarcinaandMethanosaeta,membersoftheMethano- 1970s the relatively tricky Hungate technique was replaced
sarcinales,useacetate.Mostrecognizedspeciesofmethanogens withthetechniqueofBalchandWolfe(Balchetal.,1979),and
are mesophilic, but hyperthermophilic and psychrotolerant the requirements for anaerobiosis are easily achieved with a
species are alsowell known. To date, genetic tools have been modestexpenditureonequipmentandminimaltraining.
developedonlyforcertainmesophilicspecies.
The methanogens are biochemically distinctive. The en-
Keyspecies of methanogensthat have genetic
zymes and unique coenzymes of methanogenesis are known
systems
thanks largely to the work of Ralph Wolfe, Rolf Thauer,
GodfriedVogels,andGerhardGottschalk.Ourunderstanding Speciesforwhichgenetictoolshavebeendevelopedbelong
ofhowmethanogenesisiscoupledtoenergyconservationhas to the genera Methanococcus and Methanosarcina. Thus,
beenslowertodevelop.Asforallrespirers,energyconserva- there are genetic systems for representatives of the two
tion is fundamentally chemiosmotic. A methyl transfer step metabolic types, hydrogenotrophic and methylotrophic
plays a central role in most methanogenic pathways and methanogens. Both genera have well-developed tools, but
directlydrivestheexportofsodiumions.Othercomponents eachhasitsintrinsicadvantages.
of the energy conservation apparatus appear to differ in the Methanococcus species grow relatively fast (doubling
methylotrophicandhydrogenotrophicmethanogens.Methy- timesaround2h)andliquidculturesgrowtohighdensities
lotrophic methanogens have cytochromes and a proton- overnight.ForMethanococcus maripaludis, colonies ofuse-
translocating electron transport chain, which they use to fulsizeoftenformin2daysafterinoculationonagarplates.
conserveenergyinthelast,exergonicstepinmethanogenesis. Inaddition,forM.maripaludisarobustsystemforcontin-
Thesecomponentsarelackinginhydrogenotrophicmethano- uous culture in chemostats has been established and used
gens, making it unclear how these organisms achieve a net effectively in studies of global regulation (Haydock et al.,
positivegaininenergyconservation,becausethefirststepin 2004;Hendricksonetal.,2008).ThegenomesofMethano-
methanogenesisfromCO isendergonic.Arecentlyproposed coccusspeciesaresmall(1.6–1.8Mbp),streamliningannota-
2
mechanism involving electron bifurcation, where exergonic tion and transcriptomic and proteomic analyses. Genome
electron flow directly drives endergonic electron flow, could sequences for four species [M. maripaludis (four strains),
explainthisconundrum(Thaueretal.,2008). Methanococcus voltae, Methanococcus vannielii, and Metha-
nococcus aeolicus] are available currently. The relatively
restrictedsubstraterangeformethanogenesisinhydrogeno-
Whystudymethanogens?
trophicspecieslimitstheutilityofgeneticsinMethanococcus
Themethanogenicpathwayitselfhascapturedthecuriosity to study methanogenesis itself. Nevertheless, genetics de-
of many for decades. Recently genetic approaches have monstratedtheroleinvivoofanalternativepathwayforthe
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Archaealmodelorganisms 581
Table1. Genetictoolsformethanogens
Methanococcus Methanosarcina
Negativeenrichment Enrichmentofauxotrophicmutants(Ladapo&Whitman,1990)
DNAdelivery PEG-mediatedtransformation(Tumbulaetal.,1994), Liposome-mediatedtransformation
conjugation(Dodsworthetal.,2010) (Metcalfetal.,1997)
Replicativeshuttlevectors Gardner&Whitman(1999) Metcalfetal.(1997)
Positiveselection Puromycin(Gernhardtetal.,1990),neomycin Puromycin(Gernhardtetal.,1990),
(Argyleetal.,1996) pseudomonicacid(Boccazzietal.,2000)
Counterselection hpt(8-azahypoxanthine),upt(6-azauracil)(Moore&Leigh,2005) hpt(8-aza-2,6-diaminopurine)
(Pritchettetal.,2004)
Markerlessgeneticexchange Moore&Leigh(2005) (Pritchettetal.,2004)
(pop-in/pop-outgene
replacement)
Ectopicintegration Intohptorupt(Moore&Leigh,2005) EnhancedwithfC31site-specific
recombinationsystem(Gussetal.,2008)
Transposoninsertion Invitro(Porat&Whitman,2009) Invivo(Zhangetal.,2000)
Reportergenes lacZ(b-galactosidase)(Lie&Leigh,2002),uidA uidA(b-glucuronidase)(Pritchettetal.,2004)
(b-glucuronidase)(Benekeetal.,1995)
Regulatedgeneexpression nifpromoter(Lie&Leigh,2002;Chabanetal.,2007) Tetracycline-responsivepromoters
(Gussetal.,2008)
reductionoftheelectroncarriercoenzymeF (Hendrick- then,thegenetictoolsformethanogenshavebeenexpanded
420
son & Leigh, 2008), and the role of the energy-conserving andimproved.GeneticsbecamefeasibleinMethanosarcina,
hydrogenaseEhbincarbonfixation(Poratetal.,2006). whichnormallygrowsinmulticellularpackets,whencondi-
Methanosarcina species grow more slowly (doubling tions for growth as single cells were found (Sowers et al.,
times around 8h), and the formation of colonies requires 1993), and W. Metcalf documented the high-efficiency
about14daysofincubation.ThegenomesofMethanosarci- transformationofMethanosarcinausingliposomes(Metcalf
naspeciesarerelativelylarge,rangingfrom4.1to5.8Mbp. etal.,1997).Table1outlinesthegenetictoolsavailablefor
Genome sequences for three species (Methanosarcina acet- methanogens.Mostofthesetechniqueswereworkedoutfor
ivorans,Methanosarcinabarkeri,andMethanosarcinamazei) M.maripaludisandM.acetivorans,butmanyofthemhave
are available currently. Despite their slower growth, the also beenapplied successfully in M. voltae, M. barkeri, and
metabolic versatility of Methanosarcina allows more possi- M. mazei. It has been possible to adapt many tools from
bilities for the study of the methanogenic pathway. For standardbacterialgeneticstothemethanogensbecausethey
example,mutantsinoxidation/reductionstepsbetweenthe growatmoderatetemperaturesandsaltconcentrations.
formylandmethyllevelslosttheabilitytogrowonH and
2
CO ormethanolalone,butgrewwellonH andmethanol
2 2 DNA delivery, positive selection, shuttle vectors,
(Welander&Metcalf,2008).
and insertional gene disruption
DNAisintroducedintoMethanococcusandMethanosarcina
Genetic toolsfor methanogens
species by transformation and plating under anaerobic
Thebasicelementsofthegenetictoolboxconsistofameans conditions. In M. maripaludis the polyethylene glycol
ofDNAdelivery,selectionforthatDNA,andawayforthe (PEG)-mediated transformation of spheroplasts results in
DNAtoreplicate.Reliableplatingofsinglecells,whichgrow frequencies near 105transformantsmg(cid:3)1 DNA and
intoclonalcolonies,isalsoneededformutantscreening.In 10(cid:3)5transformantsCFU(cid:3)1. In M. acetivorans a liposome-
methanogens a genetic manipulation of this kind was first mediated method achieves higher frequencies, as high as
achieved in 1987 when Bertani (of Luria–Bertani medium 108transformantsmg(cid:3)1 DNAand 20% of the CFU. In both
fame) and Baresi transformed auxotrophs of M. voltae to cases, one easily obtains thousands of colonies in a single
prototrophy(Bertani&Baresi,1987).Selectionbyantibiotic experiment. Puromycin transacetylase from Streptomyces
resistancewasinitiatedwhenA.KleintransformedM.voltae works well (Gernhardt et al., 1990), and selection in both
byexpressingapuromycinresistancemarkerfromStrepto- generaismostcommonlyachievedusingpuromycin.InM.
myces (Gernhardt et al., 1990). In addition, W. Whitman maripaludis, neomycin resistance is also achieved using
devised a strategy for the enrichment of auxotrophic mu- aminoglycoside phosphotransferase genes (Argyle et al.,
tants in M. maripaludis (Ladapo & Whitman, 1990). Since 1996).Replicativeshuttlevectorshavebeendevisedforboth
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582 J.A.Leighetal.
(b) (c) (d) (e)
(a)
Fig.2. Geneknockoutmethodsusedinarchaealgenetics.Specificdetails(suchasselectablemarkers)areillustrativeanddonotnecessarilyapplyforall
archaealgroups.Seetextforfurtherdetails.(a)Genereplacementwithselectablemarker(inthiscasetrp),byrecombinationbetweenflankingregions
ofthegeneandachromosomaltarget,useslinearDNA.(b)Pop-in/pop-outdeletionmethodusescircularDNA.Integrationofthedeletionconstruct
(pop-in)isselectedbytransformationtouracilprototrophy.Intramolecularrecombinantsthathavelosttheplasmid(pop-out)arecounterselectedusing
5-FOA.Inmethanogens,twodifferentmarkersareusedforselectionandcounterselection,respectively.(c)Variantofthepop-in/pop-outmethodfor
genedeletion,wherethegeneisreplacedwithamarkerallowingdirectselection(inthiscasetrp).Usedfordeletionofgenesthatareimportantforcell
viability.(d)Refinementofthepop-in/pop-outgenereplacementmethod,wherethegenefunctioniscomplementedintransfromashuttlevector.Loss
oftheshuttlevector(plasmidshuffling)andgenedeletionisensuredbycounterselectionwith5-FOA.(e)Ectopicintegrationattheuralocus.Pop-inofa
constructbearingthepointmutation(ofexperimentalgene,notura)isselectedbytransformationtothymidineprototrophy.Counterselectionwith5-
FOAensuresthattheurageneisreplacedwiththepointmutation.
genera, using replicative elements from naturally occurring selectionresultsinamerodiploidinwhichintegrationofthe
plasmidsofstrainsofeachgenus.DNAlackingautonomous entireplasmidhasoccurredinasinglerecombinationevent.
replication can integrate into the chromosome via homolo- Counterselection results in a second recombination event,
gousrecombination,whichappearstooccurataparticularly removingthevector.Becausethesecondrecombinationevent
highfrequencyinM.maripaludis(Tumbulaetal.,1997).This canoccuronthesameortheoppositesidefromthefirst,the
allowsforgenereplacementanddisruptionbyinsertionofthe resultiseitherawildtypeoramutantlocus,whichmustbe
selectable marker (Fig. 2a), and other genetic manipulations distinguishedbyscreening.ForMethanosarcina,vectorshave
describedbelow.Inadditiontotransformation,aconjugation beenequippedwithrecognitionsitesfortheFlpsite-specific
system from E. coli to M. maripaludis has been described recombination system, allowing more expedient removal of
recently (Dodsworth et al., 2010). It is less laborious than themarker(Rother&Metcalf,2005).
transformationandmaybeusefulforroutinegeneticmanip-
ulationofthismethanogen.
Ectopic integration
Integration of constructs into the genome is desirable not
Markerless genetic exchange
onlyforgenedisruptionorformodificationbutalsoincases
InbothMethanococcusandMethanosarcina,systemshavealso whereartifactsduetomultiplecopiesonaplasmidaretobe
been devised for markerless genetic exchange. Using these avoided.InM.maripaludisthishasbeenachievedbyectopic
systems,mutations,includingin-framedeletions,canbemade incorporation of constructs into the sites of the counter-
inwhichtheselectablemarkerisremoved,allowingitsre-use selectablegeneshptorupt(Fig.2e)(Moore&Leigh,2005).
for subsequentmanipulations. Vectorsforthispurpose con- ForMethanosarcinaasystemhasbeendevisedthatusesthe
tain both selectable and counterselectable markers. hpt and fC31 site-specific recombination system to considerably
upt,encoding hypoxanthineanduracilphosphoribosyltrans- increasetheefficiencyofintegration(Gussetal.,2008).
ferase,respectively,allowforcounterselectioninthepresence
of nucleobase analogs in genetic backgrounds from which
Overexpression and controlled expression
thesegeneshavebeendeleted.Inthisapproach,oftentermed
pop-in/pop-out gene replacement, the construct to be ex- Replicative vectors for Methanococcus and Methanosarcina
changedintothegenomeisclonedwithhomologousflanking areequippedwithstrong promotersthatallowoverexpres-
DNA on both sides (Fig. 2b). After transformation, positive sion of genes. These vectors have been used to overexpress
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Archaealmodelorganisms 583
His-tagged proteins in their native species for subsequent sarcina. In another study, hydrogen cycling, a fundamental
purification (Dodsworth & Leigh, 2006). A tetracycline-in- strategyinchemiosmoticenergyconservation,wasshownto
duciblepromoterhasbeenconstructedforMethanosarcinaby occur(Kulkarnietal.,2009).AstrikingfeatureinMethano-
combining a strong promoter from M. barkeri with binding sarcina is that the methylamine methyltransferases contain
sitesforthebacterialTetRprotein,andusedinatestforgene pyrrolysine, the 22nd genetically encoded amino acid.
essentiality (Guss et al., 2008). Thissystem also has promise Genetic studies have helped determine the function of a
fortheinductionofgeneexpressionforthepurposeofprotein dedicated tRNA and aminoacyl-tRNA synthetase in pyrro-
production and purification. Attempts to adapt the tetracy- lysyl-tRNAsynthesis(Mahapatraetal.,2006,2007).
cline induction system for Methanococcus have not been Genetic studies in Methanococcus have addressed awide
successful.However,thenif(nitrogenfixation)promoterhas range of questions. A number of regulatory mechanisms
beenusedinM.maripaludisfordifferentialcontrolledexpres- havebeenstudied,andregulatoryfactorshavebeenidenti-
sion(Lie&Leigh,2002;Chabanetal.,2007)andhaspotential fied that govern the expression of genes for hydrogen
applicationintestsforgeneessentiality. metabolism (Sun& Klein, 2004) and nitrogen assimilation
(Lie&Leigh,2003;Lieetal.,2005).Anovelmechanismfor
the regulation of nitrogenase activity was discovered in M.
Transposon insertion
maripaludis,andevidentlyexistsinavarietyofdiazotrophic
In vivo transposon insertion has been devised for M. ArchaeaandBacteria(Dodsworthetal.,2005;Dodsworth&
acetivorans.Thetransposonsystemisderivedfromamini- Leigh,2006).GeneticstudiesinM.maripaludishaveledto
mariner element and inserts randomly into the genome at the identification of components of the archaeal flagellum
highfrequency.Thetransposoncontainsselectablemarkers system (Chaban et al., 2007), tRNA-dependent cysteine
forE.coliaswellasforMethanosarcina,andcontainsanE. biosynthesis (Stathopoulos et al., 2001; Sauerwald et al.,
colioriginofreplication,facilitatingcloningofthetranspo- 2005), and requirements for selenocysteine biosynthesis
soninsertionsites(Zhangetal.,2000).Thissystemworksin (Rotheretal.,2001;Yuanetal.,2006;Stocketal.,2010).
M. maripaludis only at low frequency. However, transposi- Numerous studies of global regulation at the transcrip-
tioninvitro(Porat&Whitman,2009)andintoanE.colil tomic and proteomic levels have been carried out with
lysate (Blank et al., 1995) have been used successfully to Methanococcus and Methanosarcina species, showing the
generateinsertionsinaclonedM.maripaludisgenecluster, global responses to alternative substrates, salt stress, and
followed by transformation into M. maripaludis. These availabilities of nutrients including hydrogen and nitrogen
approacheshavepotentialfordevelopmentintoanefficient (Hovey et al., 2005; Li et al., 2005a,b; Lessner et al., 2006;
transposoninsertionsystemforMethanococcus. Veit et al., 2006; Xia et al., 2006, 2009; Hendrickson et al.,
2007,2008;Pflugeretal.,2007;Jageretal.,2009).
Reporter genes
Halophiles
Genes used in reporter gene fusions are uidA (encoding b-
glucuronidase)forMethanosarcina(Pritchettetal.,2004)and Introduction to haloarchaea,the heterotrophic,
uidA (Beneke et al.,1995)and lacZ (b-galactosidase) (Lie & aerobic halophiles oftheEuryarchaeota
Leigh, 2002) for Methanococcus. Most applications measure
Halophilicarchaeainhabitthemostsalineenvironmentson
reporterenzyme activity in cell extracts. The use ofreporter
earth, including solar salterns and natural salt lakes. Like
genes for in vivo screening is more limited, because color
manyotherhabitatswherearchaeaarefound,saltlakeswere
developmentrequiresoxygen.However,M.maripaludiscolo-
once thought devoid of life. In 1936, Benjamin Elazari-
nieshavebeenexposedtoairandsprayedwithX-gal.Color
Volcani published the first report of microbial life in the
developmentoccursbeforelossofviability,andcoloniescan
DeadSea(Elazari-Volcani,1936).Hisworkwascommemo-
be returned to anaerobic conditions and picked. This ap-
rated by the naming of Haloferax volcanii, which was
proach was used to identify super-repressor variants of the
isolated from Dead Sea mud in 1977 (Mullakhanbhai &
transcriptionalrepressorNrpR(Lie&Leigh,2007).
Larsen, 1975). In fact, the discovery of halophilic archaea
predatestheproposalofthedomainArchaeabyCarlWoese
Discoveries and recent progress
in the late 1970s (Woese & Fox, 1977; Woese et al., 1990).
GeneticapproacheshavebeenparticularlyusefulinMetha- For instance, H. salinarum was unwittingly discovered in
nosarcinaforfillinggapsinourknowledgeofthemethano- 1922 as a red discoloration of salted fish (Harrison &
genic pathway. For example, the energy-conserving Kennedy, 1922). All halophilic archaea aremembers of the
hydrogenaseEchwasshowntoberequiredformethanogen- Euryarchaeota and somewhat confusingly, thefamily Halo-
esis and carbon fixation (Meuer et al., 2002) in Methano- bacteriaceae (Oren et al., 2009). In this review, we will use
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584 J.A.Leighetal.
instead the term haloarchaea unless when referring to the proteinssuchasbacteriorhodopsin.Itisunlikelythatthese
genusHalobacterium. pigments play a significant role in protection against UV.
Archaea are not alone in hypersaline environments; they WhileHbt.salinarumcanwithstandveryhighdosesofUV
sharethishabitatwithbacteria,fungiandalgae.Incontrastto (McCready&Marcello,2003),otherpigmentedspeciessuch
most halophilic bacteria and eukaryotes, haloarchaea main- as Hfx. volcanii are no more resistant than the model
tainanosmoticbalancewiththeirmediumbyaccumulating bacteriumE.coli(Delmasetal.,2009).
equimolar salt concentrationsin the cytoplasm (Christian & To date, 12 haloarchaeal genomes have been sequenced
Waltho, 1962; Oren, 2008). This ‘salt-in’ strategy predomi- [HalalkalicoccusjeotgaliB3(T),HaloarculamarismortuiATCC
nantly uses potassium because it attracts less water than 43049,Halobacteriumsp.NRC-1,Hfx.volcaniiDS2,Halogeo-
sodium.Theconverse‘salt-out’strategyfavoredbyhalotoler- metricum borinquense DSM 11551, Halomicrobium mukoha-
antbacteriaexcludessaltfromthecytoplasmandusesorganic taei DSM 12286, Haloquadratum walsbyi DSM 16790,
solutes such as glycerol or glycine betaine to maintain an HalorhabdusutahensisDSM12940,Halorubrumlacusprofundi
osmotic balance. The salt-out strategy is energetically costly ATCC49239,HaloterrigenaturkmenicaDSM5511,Natrono-
and less suitable at saturating salt concentrations, which is monaspharaonisDSM2160,NatrialbamagadiiATCC43099]
probably why haloarchaea predominate under hypersaline and many more are underway. They usually consist of one
conditions (Oren, 1999). There are notable exceptions: the main chromosome and a number of megaplasmids (Pfeiffer
bacteriumSalinibacterruberusesthearchaealsalt-instrategy etal.,2008a;Soppaetal.,2008).Structuraldifferencesintheir
andcoexistswithhaloarchaeaatnear-saturatingsaltconcen- respectivemegaplasmidsunderliethedistinctionbetweenHbt.
trations(Orenetal.,2002). salinarumandHalobacteriumsp.NRC-1,buttheyareessen-
Because of the salt-in strategy, haloarchaeal proteins are tiallythesamespecies(werefertobothasHbt.salinarum)(Ng
adapted to function in molar salt concentrations and etal.,2000;Pfeifferetal.,2008b).Polyploidyisasignatureof
commonly denature in low-salt solutions. The adaptation haloarchaea; there are 15–30 genome copies in Hfx. volcanii
to salt relies on several different strategies (Lanyi, 1974; and Hbt. salinarum (Breuert et al., 2006). Haloarchaeal
Mevarechetal.,2000). genomes are characterized by a high G1C content ((cid:5)65%)
(cid:4) Areductioninoverallhydrophobicity,byreplacinglarge (Soppaetal.,2008);theoneknownexceptionisHaloquadra-
hydrophobic residues on the protein surface with small tum walsbyi (45% G1C) (Bolhuis et al., 2006). It is often
hydrophilicresidues.Thisstrategyisusedbythedihydrofo- stated that the high G1C content is linked to the acidic
late reductase of Hfx. volcanii, which requires higher salt proteomeofhaloarchaea(averagepIof(cid:5)4.4),butitismore
concentrations for correct folding than the E. coli enzyme probablydue to evasion of insertion sequence (IS)elements
(Wrightetal.,2002). that target A1T-rich sequences (Pfeifer & Betlach, 1985;
(cid:4) An increase in acidic residues. A high density of negative Cohen et al., 1992; Hartman et al., 2010). Interestingly, IS
charges coordinates a network of hydrated cations, which elementsinH.walsbyihaveahigherGCcontentthantherest
maintaintheproteininsolution(Lanyi,1974).Forexample, ofthegenome(Bolhuisetal.,2006).Whileallhalophilesare
glucosedehydrogenaseofHaloferaxmediterraneiisveryacidic infestedwithISelements(Bruggeretal.,2002),theiractivity
(Brittonetal.,2006),andthemedianpIoftheHbt.salinarum varies between species. Halobacterium salinarum exhibits
proteome is predicted to be 4.9 (Kennedy et al., 2001). By genome instability due to frequent IS-mediated rearrange-
alteringthetotalcharge,itispossibletointerconverthalophi- ments(Sapienza etal., 1982; Simsek et al., 1982), but thisis
licandmesophilicformsofaprotein(Tadeoetal.,2009). muchlessofaprobleminHfx.volcanii,whereISelementsare
(cid:4) An additional domain that is not found in mesohalic confinedtononessentialregionsonthemegaplasmids(Cohen
proteins,asseenintheferredoxinofHbt.salinarum(Marg etal.,1992;Lopez-Garciaetal.,1995).
et al., 2005). The latter features a 30-amino acid insertion Incommonwithmethanogens,thegenomesofhaloarch-
nearitsN-terminus,whichisextremelyrichinacidicamino aeaencodemultipleisoformsofgenesthatarepresentasa
acidsandisessentialforcorrectproteinfoldingathighsalt single copy in other organisms. For instance, there are 16
concentrations. orc1/cdc6 genes for the DNA replication initiator in Hfx.
Besides their adaptation to salt, haloarchaea have other volcanii and 10 in Hbt. salinarum (Berquist et al., 2007;
characteristicfeatures.Theyareaerobicheterotrophs,some Noraisetal.,2007;Hartmanetal.,2010).Thismightbedue
ofwhichhavethepotentialforanaerobicgrowth(Falbetal., toarequirementforregulatoryandmetabolicflexibilityin
2008). They are slightly thermophilic with an optimum haloarchaea(Facciottietal.,2007),butitisalsopossiblethat
temperature of 40–501C, can withstand up to 601C, and these redundant homologs have accumulated as a result of
growreasonablywellat371C(Robinsonetal.,2005).Even lateral gene transfer (LGT). There is ample evidence for
Halorubrumlacusprofundi,whichwasisolatedinAntarctica, large-scale LGT, often with bacteria, and it has been pro-
grows best at 361C. Haloarchaea are generally pigmented posedthathaloarchaeaoriginallydescendedfrommethano-
withC-50bacterioruberinsandsomespeciescontainretinal gens that had acquired the genes for aerobic respiration
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Archaealmodelorganisms 585
frombacteria(Boucheretal.,2003).UnderpinningthisLGT endogenous function. The UvrABC complex, which is of
is a system for mating and gene transfer in Hfx. volcanii bacterial origin, is functional in the repair of UV-induced
(Rosenshineetal.,1989)andawidevarietyofhaloarchaeal DNA damage in both Hbt. salinarum and Hfx. volcanii
viruses(Dyall-Smithetal.,2003). (Crowleyetal.,2006;Lestinietal.,2010).
Whystudy halophiles? Keyspecies of halophilesthat have genetic
systems
Asignificant motivation for workingonhaloarchaeaisthe
sophistication of their genetic systems. They are easy to Therearetwo haloarchaeal model organisms: Hbt. salinar-
cultivate in the laboratory, have fast growth kinetics (Ro- um and Hfx. volcanii. Both species have mature genetic
binson et al., 2005), and are resistant to contamination by systems, but each has intrinsic advantages. Halobacterium
nonhalophilicmicroorganisms(ofcourse,cross-contamina- salinarum is the traditional choice for haloarchaeal cell
tionofhaloarchaealstrainswithinthelaboratoryremainsa biology;itwasisolatedmanyyearsagoandquicklybecame
constantthreat).Halophileswerethefirstarchaealgroupin a popular choice owing to its purple membrane protein,
whichroutinetransformationwithforeignDNAwaspossi- bacteriorhodopsin(Oesterhelt&Stoeckenius,1971;Marge-
ble (Charlebois et al., 1987; Cline et al., 1989). The ease, sin&Schinner,2001).ThegenomeofHbt.salinarumNRC-
efficiency, and broad applicability of PEG-mediated trans- 1 was published in 2000 (Ng et al., 2000), a second
formation has ensured that haloarchaea have remained at annotation with a different assembly of the megaplasmids
the forefront of genetic tool development (Soppa, 2006). waspublishedforHbt.salinarumR1in2008(Pfeifferetal.,
Perhapsthegreatesttestamenttothepopularityofhaloarch- 2008b).Thefirstarchaealmethodforgeneknockoutusinga
aea is the Halohandbook, an invaluable compendium of counterselectablemarkerwasalsopublishedin2000,using
methods for working with halophiles, which is diligently theura3geneofHbt.salinarum(Pecketal.,2000)(Fig.2).
curatedbyMikeDyall-Smith(Dyall-Smith,2009). Availability of the genome sequence coincided with the
Besides sophisticated genetics, there are many other growthoftranscriptomics,andconsequentlyHbt.salinarum
reasons for working on halophiles. Haloferax volcanii and has been a favorite model for systems biology (DasSarma
Hbt.salinarumcangrowoverarangeofsalinitiesandhave et al., 2006). DNA repair has been a fruitful topic, because
been exploited to uncover genes involvedin osmotic stress Hbt. salinarum is extremely resistant to UV and ionizing
(Bidle et al., 2007, 2008; Coker et al., 2007). Because radiation (Baliga et al., 2004; Whitehead et al., 2006).
halophilicproteinsfunctionunderconditionsoflowwater However,forthosewishingtocarryouttraditionalgenetics,
availability, they offer distinct advantages for structural Hbt. salinarum is perhaps not ideal. It grows slowly, its
biology and biotechnology. For example, the structure of genomeisunstableduetofrequentIS-mediatedrearrange-
the ribosome was solved using the complex from Har. ments (Sapienza et al., 1982; Simsek et al., 1982), and the
marismortui, leading to the Nobel Prize for Chemistry in rangeofselectablemarkersissomewhatlimited.
2009 (Ban et al., 2000). In biotechnology, there are few Haloferax volcanii is better suited to traditional genetics. It
success stories that can match bacteriorhodopsin. This has a generation time of (cid:5)2h (Robinson et al., 2005), its
purplemembrane protein from Hbt. salinarumwas identi- genomeisstable(Lopez-Garciaetal.,1995),anditgrowson
fied by Walther Stoeckenius in 1971 (Oesterhelt & Stoeck- synthetic media (Mevarech & Werczberger, 1985). PEG-
enius,1971)andhasbeenusedincountlessphotochemical mediated transformation protocols were originally developed
applications(Margesin&Schinner,2001;Oren,2010). forHfx.volcanii(Charleboisetal.,1987;Clineetal.,1989),and
Haloarchaea are an excellent choice to address the ‘pro- methods for gene knockout have incorporated additional
karyotic species question’: do prokaryotic organisms form selectablemarkers(Bitan-Baninetal.,2003;Allersetal.,2004),
genomicandphenomicclustersthataresufficientlycohesive thusfacilitatingtheconstructionofmultiply-mutatedcells(Fig.
that we might legitimately call them species (Doolittle & 2). A Gateway system for deletion construction (El Yacoubi
Zhaxybayeva,2009)?Haloarchaeaarephysiologicallydiverse et al., 2009), several reporter genes (Holmes & Dyall-Smith,
and inhabit distinct ecological niches (Oren, 2008), they 2000;Reuter&Maupin-Furlow,2004),aninduciblepromoter
havedynamicgenomeswithsystemsforgeneexchangeand (Large et al., 2007), and a system for protein overexpression
show evidence for LGT. Work on isolates of the genus (Allers et al., 2010) have been developed in the last decade.
Halorubrum from solar salterns and natural salt lakes has Haloferaxvolcaniihasalonghistoryofgenomeresearchgoing
shownthathaloarchaeaexchangegeneticinformationpro- back to 1991, when a physical map of overlapping genomic
miscuously, leading to the suggestion that there is no clones was published (Charleboiset al., 1991). Publication of
nonarbitrary way to define a prokaryotic species (Papke theannotatedgenomesequencein2010(Hartmanetal.,2010)
et al., 2004, 2007). Genetics has been used to answer the hasspurredthedevelopmentofwhole-genomemicroarrays(S.
questionofwhethergenesacquiredbyLGTcansupplantan Chimileski&T.Papke,pers.commun.),whichwillallowfaster
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586 J.A.Leighetal.
data analysis than the shotgun DNA microarrays available systems: one that targets unmethylated 50-CTAG-30 sites
previously(Zaigleretal.,2003). (Charlebois et al., 1987) (T. Allers, unpublished data) and
Rudimentary genetic systems are available for other ha- another that restricts methylated 50-GmeATC-30 DNA
loarchaea. Haloarcula marismortui can be transformed with (Holmes & Dyall-Smith, 1991). The latter results in a 102-
shuttleplasmidsfromHbt.salinarumandHfx.volcanii(Cline folddropintransformationefficiencyandhasbeencircum-
& Doolittle, 1992) and gene replacement studies have been vented by passaging DNA through an E. coli dam mutant,
used to investigate the Har. marismortui ribosome (Tu et al., which lacks the methylase that modifies 50-GATC-30 sites.
2005). However, this species harbors restriction/modification Thisisnolongernecessary,becauseanDmrrmutantofHfx.
systemsthatreducetheefficiencyoftransformationby104-fold volcanii was recently shown to lack the restriction enzyme
(Cline & Doolittle, 1992). Systems for Halorubrum are cur- thattargets50-GmeATC-30DNA(Allersetal.,2010).
rently under development (S. Chimileski & T. Papke, pers.
commun.). Haloferax mediterranei is closely related to Hfx.
Shuttle vectors
volcanii; they differ in that Hfx. mediterranei produces gas
vesicles. These gas-filled proteinaceous particles are used by Many replicative shuttle vectors have been developed, using
cellstoincreasetheirbuoyancyandfloattothesurfaceofthe originsofDNAreplicationtakenfromindigenoushaloarch-
brine;theyhavebeenstudiedintensivelybythelaboratoryof aealplasmids(Table2).ForHbt.salinarumthereareplasmids
FelicitasPfeifer,oftenusingHfx.volcaniiasaheterologoushost based on pGRB1, pHH1, and pNRC100 origins (Blaseio &
(Pfeiferetal.,2002;Hechler&Pfeifer,2009).Thisisoneofthe Pfeifer, 1990; Krebs et al., 1991; DasSarma, 1995), while for
great strengths of haloarchaeal genetics: because there are Hfx. volcanii there are vectors based on pHK2, pHV2, and
severalorganismswithmaturegeneticsystems,complementa- pHV1/4origins(Lam&Doolittle,1989;Holmesetal.,1994;
tion by genes from a related species can be used to identify Allersetal.,2004;Noraisetal.,2007).Someoftheseorigins
random mutations and thereby isolate novel enzymes. This arebroadrange:forexample,pHV2-basedvectorsreplicatein
approachwasusedtofindanovelthymidylatesynthaseandan both species (Blaseio & Pfeifer, 1990). Interestingly, pHV2-
alternative pathway for reduced folate biosynthesis in Hbt. based plasmids do not function in Hfx. volcanii mutants
salinarum,usinggenesfromHfx.volcanii(Giladietal.,2002; deficientintheRadArecombinase,wherepHK2-basedvectors
Levinetal.,2004).Morerecently,theessentialpitAgeneofHfx. are used instead (Woods & Dyall-Smith, 1997). For a com-
volcaniiwasreplacedbyanorthologfromthehaloalkaliphile prehensive list of plasmid vectors, see Allers & Mevarech
Natronomonas pharaonis; the latter lacks the histidine-rich (2005)orBerquistetal.(2006).
linkerregionfoundinHfx.volcaniiPitAanddoesnotcopurify
withHis-taggedrecombinantproteins(Allersetal.,2010).
Selectable markers
Bacterial antibiotics that are safe to use in eukaryotes are
Genetic toolsfor halophiles
largelyineffectiveagainstarchaea,becausethetargetsofthese
drugs (e.g. peptidoglycan cell walls) are not encountered in
Transformation
archaeal or eukaryotic cells. There are some exceptions
Moderngeneticstakesforgrantedamethodforintroducing (Hilpert et al., 1981); they have been exploited to develop
DNA into cells, and a means to select for cells that have selectablemarkersforhaloarchaealgenetics.Novobiocinisan
taken up the DNA. The development of transformation inhibitorofDNAgyrase(gyrB),anessentialenzymefoundin
protocolsisintimately linkedwith selectable markers.This both bacteria and archaea, and a resistant form of the gyrB
wasanacuteproblemintheearlydaysofarchaealgenetics, gene was isolated from Haloferax strain Aa2.2 (Holmes &
because bacterial antibiotics are largely ineffective against Dyall-Smith, 1991). Novobiocin has since become the most
archaea(Hilpertetal.,1981).ClineandDoolittleovercame widely used haloarchaeal antibiotic. An alternative is mevi-
this hurdle by assaying for transfection of Hbt. salinarum nolin(simvastatin),whichinhibitsHMG-CoAreductase.In
with naked DNA from halovirus FH, and scoring for humans,itisprescribedasacholesterol-loweringdrug,while
plaques on a lawn of cells (Cline & Doolittle, 1987). This inarchaeaitinhibitsmembranesynthesis.Amutantalleleof
allowed them to develop the PEG transformation protocol the Hfx. volcanii hmgA gene that leads to overexpression of
thatisusedtoday(Clineet al.,1989):theglycoprotein cell the enzyme has been harnessed as a mevinolin-resistant
surface layer, which depends on Mg21, is removed by markerforbothHfx.volcaniiandHbt.salinarum(Blaseio&
treatment with EDTA and DNA is introduced into spher- Pfeifer,1990;Lam&Doolittle,1992).
oplastsusingPEG600,afterwhichcellsrecoverinrichbroth Thelastdecadehasseenashiftawayfromantibioticsand
beforeplatingonselectivemedium.Thisprotocolyieldsup towards auxotrophic selectable markers, where genes in-
to 107transformantsmg(cid:3)1 DNA, depending on restriction/ volvedinaminoacidornucleotidebiosynthesisareusedto
modification systems. Haloferax volcanii has two such complementchromosomalmutations.Deletionofthegene
(cid:2)c2011FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev35(2011)577–608
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