Heterotrophic Planktonic Microbes: Virus, Bacteria, Archaea, and Protozoa

34 Pages·2015·1.44 MB·English

Heterotrophic Planktonic Microbes: Virus,
Bacteria, Archaea, and Protozoa
JEDA.FUHRMANANDDAVIDA.CARON
4.2.2
BACKGROUNDANDHISTORICAL TheMicrobialLoopRevolution
DEVELOPMENT Pomeroy’sprescientanalysis,“Theocean’sfoodweb,achang-
ing paradigm” (12) had a significant impact toward trans-
MarineMicrobialEcologyintothe1970s forming the field. This publication is often cited by many
Althoughheterotrophicmarinemicroorganismsintheopen microbialecologistsasaturningpointinourunderstanding
sea have been studied since the late 1800s and early 1900s ofthestructureandfunctionofmarineecosystems.Init,Pom-
by pioneers like Fischer, Haeckel, and Calkins (1–3), the eroypointedtolinesofevidencethatwerestartingtoemerge
contribution of these diminutive species to the food webs showingthatthesmallestmembersofthefoodweb,including
of oceanographic systems was not fully recognized until heterotrophic bacteria, cyanobacteria, and small protists
nearly a century later. Attention was drawn to the larger (algaeandprotozoa<20µm),wereprobablyresponsible for
and more conspicuoustaxa of photosynthetic protists (e.g., alargefractionofimportantsystemactivitiessuchasoverall
diatoms and many dinoflagellates) early in the history of respiration, photosynthesis, and organic matter turnover
biological oceanography. In contrast, little was known of (i.e.,theingestionoffoodparticlesbyprotozoaortheuptake
the abundances or activities of bacteria in the ocean until ofdissolvedsubstancesbybacteria).Earlystudiesofbacterial
the past 50 years, and most early studies of marine hetero- utilization of dissolved organic matter (e.g., 1), and subse-
trophic protists (the protozoa) focused on morphological quent studies of community metabolism into the 1980s
descriptions and natural history of larger species of these (e.g.,14)indicatedthatthisprocessaccountedforasurpris-
taxa rather than their functional roles in marine food inglyhighpercentageoftotalorganicmatterturnover.Sim-
webs.Improvementsinmicroscopymethodsusedtoobserve ilarly, Fenchel and Jorgensen (15) pointed out that
bacteria in seawater during the 1930s to 1950s indicated approximately 10–30% of primary productivity might be
that bacterial abundances were several orders of magni- released as dissolved organic matter, which was then pre-
tude greater than previously believed. However, confusion sumed to be taken up by bacteria who respired a portion
remained during regarding the ecological significance of and passed the rest on to the next trophic level, composed
marine bacteria because these early abundance estimates largely of protozoa. In this scenario, protozoa constituted a
were often hundreds of times greater than counts made by mechanismbywhichbacterialbiomassreenteredthe“classi-
cultivationtechniques(4). cal”foodwebincludingthemetazoanzooplanktonandnek-
During the 1960s and 1970s, however, the metabolic ton,andalsoservedasameansofremineralizingsomeofthe
activity of aquatic bacterial assemblages was demonstrated bacterial biomass back to inorganic nutrients and carbon
by the uptake of radioactive organic compounds in marine dioxide for subsequent utilization by primary producers.
andfreshwatersamples(e.g.,[5,6]andothers).Thesestudies Althoughtheseconclusionswerelaterfoundtobegenerally
demonstrated that organic compounds were readily turned correct,theconceptswerearadicaldeparturefromtheestab-
over by microorganisms in aquatic ecosystems, and that lishedgeneralbiologicaloceanographicthinkingofthetime.
microbialcommunities appeared tobe quite dynamic.Dur- Theintroductionsofepifluorescencemicroscopyoffluo-
ing that same period, abundant and diverse assemblages of rescentlystainedcells(16)andpolycarbonatefilterstoposi-
protozoaweredemonstratedfromawidearrayofaquaticeco- tionthecellsallinasingleopticalplane(17)weresignificant
systems using more quantitative approaches for the collec- technologicaladvancementsthatallowedmuchmoreaccu-
tion, observation, and enumeration of eukaryotes (7–10). rateestimatesofthetotalnumberofmicroorganismspresent
Together these observations implied the presence of an in natural water samples. Using this method, it was deter-
active and complex microbial community that might be minedthatbacteriaaretypicallypresentatabundancesof1
responsible for much of the metabolic activity in marine millioncellspermlinnear-surfaceseawater.Thisnumberis
ecosystems. This hypothesis stood in contrast to broad oce- surprisinglyconstantaroundtheworld,withmostvariation
anographic models of the time that included the bacteria fallingwithinafactorof10worldwide.Appreciationofthe
onlyasasink for nonliving organic matteronthe seafloor importance of medium to large protozoa (i.e., >20µm) in
andlargelyignoredthepotentiallyimportantrolesofheter- oceanic food webs wasmade possiblein the late 1960s and
otrophic protists (11). early 1970s largely through the pioneering work of Beers
doi:10.1128/9781555818821.ch4.2.2
Downloaded from4 w.2w.w2-.1asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
4.2.2-2 ▪ AQUATICENVIRONMENTS
etal.(7,18).However,aswithmarinebacteria,epifluores- Algae: protists that exhibit phototrophic nutrition. Like
cencemicroscopyfacilitatedtheobservationofsmallsingle- protozoa, algae span a wide size range (<1 to >200
celled eukaryotes during the 1970s and 1980s and enabled µm),andhavegenerallybeenreferredtoasphototrophic
easy discrimination of protists without chloroplasts (proto- pico-,nano-,ormicroplankton.
zoa)fromthosewithchloroplasts(algae)basedontheauto- Amensalism: interaction where members of a species
fluorescence of photosynthetic pigments (19–21). The inflictsharmtoanotherspecieswithoutanycostsorben-
developmentandrefinementofthisapproachforeukaryotic efitsreceivedbytheother.
microorganismswasinstrumentalinestablishingthestanding
Autotroph: an organism that uses carbon dioxide as its
stocksofsmallprotozoa,whichtypicallyoccuratabundances
sourceofstructuralcarbon.
oftenstothousandspermlinmostmarineecosystems.The
Biomass:themassoflivingorganismswithinapopulation,
development of microscopy approaches for larger protozoa
community,orecosystem.
such as heterotrophic dinoflagellates and ciliates (10, 22)
has been equally important in documenting abundances of Chemolithotroph:anorganismthatusesreducedinorganic
thesetaxathatrangeuptotenspermlinmuchoftheworld’s moleculesasitsenergysource.
oceans. Chemoorganotroph:anorganismthatusesreducedorganic
Estimates of the overall biomass of various microbial carbonasitsenergysource.
assemblages,andtechniquestomeasurerateprocesses(e.g., Commensalism: interaction between species where one
rates of growth, substrate uptake, prey consumption) began benefitsfromtheotherbuttheotherisnotaffected.
to appear in the late 1970s and 1980s, and the refinement
Competition:species-speciesinteractionsthathaveanega-
oftheseestimatesandmeasurementscontinuetothepresent
tiveeffectonbothspecies.
day.Earlyattemptstomeasurebacterialgrowthratesinsea-
water involved “indirect” methods such as relating the fre- Cyanobacteria: Prokaryotic photosynthetic organisms that
containchlorophyllaandgenerateoxygenduringphoto-
quency of dividing cells to rates of division in cultured
synthesis.Thefree-livingancestorsofprimarychloroplasts.
strains (23). Isotope-uptake based approaches, specifically
the incorporation of radioactively labeled thymidine into Exploitation:species-speciesinteractionsthathaveanega-
DNA(24,25)and/ortheincorporationofleucineintopro- tiveeffectononespecieswhilebenefitingtheother—
tein (26) have become the most commonly used methods canincludebothparasitismandpredation.
(see “Estimating ‘Bacterial’ Biomass and ‘Bacterial Produc- Heterotroph:anorganismthatusespreformedorganiccar-
tion’”). These methods have indicated that bacterial dou- bonasitssourceofstructuralcarbon,alsoheterotrophic
bling times can be on the order of one day in coastal orheterotrophywhenappliedtometabolism.
temperatewaters.Combinedwithestimatesofbacterialbio- Mixotroph:anyofanumberoftypesoforganismsthatcom-
mass, these results led to the conclusion that bacteria must bine (in one organism) multiple metabolic types as
be consuming a substantial proportion—on the order of describedabove.Forexample,aprotistthatconsumesbac-
50%—of the total system primary productivity. A similar teriaasprey(heterotroph)butalsocontainsfunctioning
conclusionwasreachedusingdirectestimationofmicrobial chloroplasts (phototroph) will often be referred to as a
respirationbycarefulmeasurementsofoxygenconcentration mixotroph.Similarlyanarchaeonthatoxidizesammonia
changes(micro-Winklermethod)inseawaterthathadbeen forenergy(chemolithotroph)butusesaminoacidstobuild
prefiltered through5µm porefiltersto removeanimalsand proteins(heterotroph)couldbeconsideredamixotroph.
manyoftheprotists(14).
Mutualism:interactionbetweenspecieswherebothbenefit
Duringthissameperiod,smallprotozoa(primarilyflagel-
fromeachother.
lates and ciliates) were gaining recognition as important
Phototroph:anorganismthatuseslightasitsenergysource
consumers of bacteria in the marine plankton and benthos
(27–29).Anincreasingvolumeofexperimentalworkdem- forproductionofATP(ortoproduceprotongradientsin
the case of rhodoposin-based phototropy) and some-
onstrated a dominant role for small, bacterivorous protozoa
timesalsoreducingpowerfromwater(incyanobacteria).
as a mechanism for removing bacterial production and
repackagingbacteriaintolargerparticlesthatmightbecon- Phytoplankton: the photoautotrophic component of the
sumedbymetazoanzooplankton.Also,itbecamerecognized plankton including cyanobacteria and a large number
aroundthistimethatasignificantfractionofthephytoplank- ofeukaryoticphylathatcontainchloroplasts.
tonbiomassandproductionwasconsumeddirectlybyherbiv- Protists: eukaryotic species that can exist as a single cell
orousprotozoaratherthanbymetazoanzooplanktonsuchas other than a spore, gamete, or zygote (although there
copepods(30,31).Consequently,heterotrophicprotistswere aremanythatformcolonies).
acknowledged as an important food source for a variety of Protozoa:protiststhatexhibitheterotrophicnutrition.Pro-
metazoan zooplankton, and numerous experimental studies tozoaspanawidesizerange(≈2to>200µm),andhave
subsequentlydemonstratedthistrophicconnection(32,33). generallybeenreferredtoasnano-ormicrozooplankton.
These observations weresynthesized in a second bench-
Relativeabundance(andtherelatedterm,evenness):the
mark paper (34). The latter publication marked the begin-
contribution of each species or operational taxonomic
ning of the widespread recognition and use of the term
unittoacommunity.
“microbial loop” in marine planktonic systems, a concept
Speciesdiversity:acomplexconceptcomposedofspecies
that emphasizes the remarkable importance of the tiniest
richnessandrelativeabundance.
organismsaswellasdissolvedorganicmatterasanintermedi-
ateinmaterialandenergytransferinaquaticecosystems.An Speciesrichness:thenumberofdifferentspeciesoropera-
updatedillustrationofthisbasicconceptisshowninFig.1. tionaltaxonomicunitspresentinasample,habitat,or
environment.
DefinitionsandConcepts Stoichiometry:Studiesthatinvolvecalculationoftherela-
Abundance: the number of individuals in a sample or a tivequantitiesofelementsorcompounds,forexample,
population. C:N:Pratios.
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
HeterotrophicPlanktonicMicrobes:Virus,Bacteria,Archaea,andProtozoa ▪ 4.2.2-3
FIGURE1 Anearlyvisionofthe“microbialloop”anditsconnectionstotheclassicalgrazingfoodchainviadissolvedorganicmatter(DOM)
fluxandparticulatetrophictransfer,withvirusesincludedasasideloop.Modifiedfrom(34).Largegrayarrowsindicatetheflowoforganic
carbonandenergyintohighertrophiclevelsofthefoodweb,withrecognitionoftheimportantrolesforheterotrophicmicrobes(bacteria
andprotozoa)inthisprocess.LargestippledarrowsindicatetheproductionofDOMviaexcretionandtrophicinteractions(notallgroups
arerepresented).Thin,dottedarrowsindicatemineralizationofmajornutrientscontainedinorganicmatterrespiredbyconsumers.White
arrowsindicatebacterialysisbyvirusesandDOMreleasedbythatprocess.doi:10.1128/9781555818821.ch4.2.2.f1
Symbiosis: the intimate living together of two kinds of productionofnonphotosyntheticbacterialbiomassbasedon
organisms,especiallyifsuchanassociationisofmutual theheterotrophicconsumptionofpreformedorganicmatter
advantage—too vague to be of use in quantitative (i.e.,organicmatterinvariousformsthathasbeenproduced
descriptions of population interactions but very useful primarilybyphytoplankton).
inindicatingacloseassociationamongorganisms. Bacterial biomass is usually determined by converting
Syntrophy:ametabolicmutualismwhereonespeciesuses directcountsofbacteriausinganestimateoftheamountof
thewasteproductproducedbytheother,andinsodoing carbon percell. Direct counts are most commonly done by
allows both metabolic pathways to be energetically epifluorescence microscopy with stains such as acridine
feasible. orange, 40,6-diamidino-2-phenylindole or SYBR green I
(17,38,39).Specialproceduresareusuallyappliedforsedi-
Zooplankton: Planktonic eukaryotes that consume other
mentsamplesandsamplescontaininglargenumbersofbacte-
plankton. Includes single-celled organisms (protozoa
riaattachedtoparticles(40).SYBRgreenIalsopermitsdirect
orprotists)andmetazoans,andsomethatareplanktonic
visualization and counts of viruses in the same preparation.
onlyaslarvae.
Increasingly, direct bacterial counts in seawater samples
have been performed by flow cytometry of fluorochrome-
Estimating“Bacterial”Biomassand“Bacterial stainedcells(41,42),amethodthatallowsseparatecounts
Production”:DefinitionsandMethods ofcyanobacteria,suchasSynechococcusandProchlorococcus,
Aquaticmicrobiologiststendtousetheterm“bacteria”witha whichhaveuniquefluorescentsignaturesduetotheirphoto-
lowercase“b”todescribeorganismsthatappeartobeprokary- syntheticpigments,andwhichcansometimesmakeupasub-
otic by microscopy—that is, organisms with no membrane- stantial fraction of the total number of bacteria (43). Flow
bound nucleus. They include members of the taxonomic cytometryisrapidandhasastatisticaladvantageinthatittyp-
domainsBacteriaandArchaea(see“BacteriaandArchaea”). icallyobservesthousands of prokaryotic and minutephoto-
Organismswithinandbetweenthesedomainsdifferinmany syntheticeukaryoticcellsratherthanthehundredscounted
biochemicalandgeneticaspects,buttheytendtolooksimilar microscopically;drawbacksincludethecostoftheinstrument
bytraditionalepifluorescencemicroscopy.Specialmethods, andthefactthatcellsattachedtoeachotherortootherpar-
suchasdifferentversionsoffluorescenceinsituhybridization ticlesarecountedasone.Bacterialcarbonpercellhasbeen
(FISH) are required to distinguish individual members of estimatedinavarietyofways,mostcommonlyfromadeter-
thesedomainsmicroscopically(35–37). minationofcellvolumeandcarbondensityperunitvolume.
Theterm“bacterialproduction”herereferstoheterotro- These numbers are difficult to obtain accurately for native
phicproductionofbiomassbybacteria.Itismeanttoinclude marine bacteria, which are very small, typically 0.5μm in
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
4.2.2-4 ▪ AQUATICENVIRONMENTS
diameter (range is about 0.2–1μm for free-living unicells). environments at approximately 106 cells per ml. Samples
Published estimates of bacterial carbon percell vary widely from around the world rarely vary by more than threefold
andprobablyconstitutethegreatestuncertaintywithestimat- from this typical value (i.e., rarely <3×105 or >3×106),
ingbacterialbiomassinnaturalsamples.Typicalestimatesof whichisextraordinarycomparedtophytoplanktonandzoo-
the carbon content of a bacterium range from 7 to 50fg C plankton,whichmayvarybyseveralordersofmagnitudeover
(1fg is 10−15g), with most open ocean estimates near 10– thesamespatialscales.However,despitethisremarkablegen-
20fg C per cell and coastal ones about double that (44). eral predictability, there is significant variation across both
Thus, in a typical mesotrophic ocean environment with spaceandtime.Morenutrient-rich,eutrophicenvironments
109bacteria per liter, and an average per cell C content tendtohavemorebacteria(sometimes>107perml;47),and
of 15fg, bacterial biomass would be approximately 109× oligotrophic open ocean environments have less (summar-
1.5×10−14=1.5×10−5gCperliter,or15μgCperliter. izedbelow).Althoughbacterialassemblagesinwarmtemper-
Bacterialproductionismostoftenmeasuredbyincorpora- atecoastalwatersmayhavedoublingtimesasshortas1day,
tionoftritiatedthymidineintoDNA(24,45)ortritiatedleu- thisisattherapidendofthespectrumofinsitugrowthrates.
cine into protein (26, 46). Thymidine and leucine are Bacterialassemblagesintheopensea,especiallyinoligotro-
intracellularprecursorsofDNAandprotein,respectively,so phic environments, haveaverage generation timestypically
incorporation of these precursors can be used to estimate ofaweekorperhapsmore(seeTable1).Theseabundances
thetotalratesofsynthesisofthemacromolecules.DNAissyn- generallyapplytotheeuphoticzone,andbacteriaincolder,
thesizedforcell divisionandproteinsynthesized roughlyin darkerwatershavesubstantiallylowerabundancesandslower
proportiontototalbiomass,someasuringtheirratesofsynthe- growthratesthanthoseofsurfacewaters(48).Benthicbacte-
sisispresumedtotrackproduction.Bothmethodshavebeen ria also exhibit fairly constant abundances across wide geo-
calibratedonthebasisoftheoreticalconsiderationsaswellas graphic ranges, but benthic bacteria occur at much higher
purelyempiricalapproaches,andbothmethodsyieldsimilar densities because of the organic-rich and particle-laden
results(44).Leucinehasalowerdetectionlimit,soitispre- natureoftheenvironment (anaverageof≈109permlfluid
ferredforslowerrates.Theresultsofthymidineincorporation volumeistypical;49).Thisvalueisthreeordersofmagnitude
aremostoftenpresentedascellsproducedperliterperhour, greaterthanabundancesinthewatercolumn,implyingthat
whichcanbeconvertedtoacarbonproductionrateviaesti- theabundancesofbenthicbacterialassemblagesareregulated
matesofcarbonpercellasnoted.Leucineincorporationmay byadifferentsetofparametersthanplanktonicassemblages.
beusedtocalculatecellproductionaswellasbiomassproduc- Bacterial biomass and productivity vary temporallyon a
tiondirectly,becauseproteinisamajorbiomassconstituent. numberof scales ranging from diel(50)toseasonal(51)or
interannual (52). On time scales of hours, bacterial abun-
dance and production have been shown to often peak in
GeographicandTemporalDistributionsof themiddleofthedayandbelowinthemiddleofthenight
MicrobialBiomassandActivity (50,53–55).Thispatternhasbeeninterpretedasatightcou-
Bacterial abundances by epifluorescence microscopy show pling between the production of labile organic compounds
that bacteria are present in most marine euphotic zone via photosynthesis and bacterial growth on one hand, and
TABLE1 Bacterioplanktonpropertiesinrelationtophytoplanktonintheopensea,ascompiledbyDucklow(44)
Property NAtlantica EqPac-Sprb EqPac-Fallc SubNPacd Arabiane Hawaiif Bermudag RossSeah
Euphoticzonem 50 120 120 80 74 175 140 45
Biomass(mgCm−2)
Bacteria 1000 1200 1,467 1,142 1,448 1,500 1,317 217
Phytoplankton 4,500 1,700 1,940 1,274 1,248 447 573 11450
B:P 0.2 0.7 0.75 0.9 1.2 3.6 2.7 0.02
Production(mgCm−2d−1)
Bacteria 275 285 176 56 257 Nd 70 5.5
Phytoplankton 1,083 1,083 1,548 629 1,165 486i 465 1248
B:P 0.25 0.26 0.11 0.09 0.22 Nd 0.18 0.04
Growthrates(d−1)
Bacteria 0.3 0.13 0.12 0.05 0.18 Nd 0.05 0.25
Phytoplankton 0.3 0.64 0.8 0.5 0.93 1.1 0.81 0.11
B:P 1 0.2 0.15 0.1 0.19 Nd 0.06 2.3
Notes:Allbacterialbiomassestimatesbasedon20fgCpercell.DatamayoverestimateheterotrophicbacterialbiomassasaconsequenceoflowerCpercellor
interferencebyProchlorococcusandArchaea.Productionestimatedfrom3,000gCpermoleleucineincorporated.
aEasternNorthAtlanticspringphytoplanktonbloom,47N20W,May1989,n=13.
bEquatorialPacific,0N140W,March–April1992,n=8.
cEquatorialPacific0N,140W,September–October1992,n=19.
dSubarcticNorthPacific,45N.
eNorthwestArabianSea10–20N,165E,January–December1995,n=21.
fHawaiiOceanTimeSeries(HOT);1995–1997;n=21(http://hahana.soest.hawaii.edu/hot/hot_jgofs.html).
gBermudaAtlanticTimeSeries(BATS);1991–1998,n=106pairedcomparisons.Theratiosaremeansoftheratios,notratiosofthemeans.BPcalculated
fromthymidineincorporation(1.6×1018cellspermoleincorporated).
hRossSea,Antarctica,76S180W,1994–1997.
i1989–1996;n=64.
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
HeterotrophicPlanktonicMicrobes:Virus,Bacteria,Archaea,andProtozoa ▪ 4.2.2-5
bacterialmortalityviagrazersorvirallysisontheotherhand. isnophotosynthesis,thissituationisalsooftentrueinoligo-
It is consistent with some measurements of extremely rapid trophic surface waters. Measurements made in oligotrophic
turnover, sometimes several times per hour in rich coastal waters bear out the high bacterial contribution to total
waters, of labile dissolved organic compounds such as dis- biomass(64,65).Moreover,ChoandAzam(66)confirmed
solvedfreeaminoacids(56)andmightalsoindicategreater alinearrelationshipbetweenthelogofchlorophyllandlog
predation pressure by protozoa during the night. On longer of bacterial abundance, but only at chlorophyll concen-
time scales of weeks to months, bacteria show distinct sea- trations above approximately 0.5μg per liter. Below that
sonalpatterns.Forexample,intemperatecoastalwaters,bac- concentration,bacterialabundancedidnotcorrelatesignifi-
terial biomass and production increase considerably in cantlywithchlorophyll.Itshouldbenotedthatsubsequent
summer months compared with winter. However, bacteria analyses have revealed that early epifluorescence measure-
do not typically show a significant increase during early mentsofbacterialbiomassincludedthecommoncyanobacte-
“spring”bloomsintemperatewaterswhenwaterisstillvery rium Prochlorococcus, which can make up to 20% of total
cold (51). It has been hypothesized that this phenomenon bacterialnumbers(67).Nonetheless,heterotrophicbacterial
istheresultofthesuppressionoftherateofsubstrateuptake biomassisamajorfractionofthelivingbiomassofallplank-
bytemperatebacterialassemblagesatlowtemperature(57). tonicecosystems.
However,whiletemperatureprobablyhastheeffectofsetting The geographical and temporal distributions of marine
alimitonmaximal growthrates (asfor phytoplankton [58] protozoa are much more varied than those of the bacteria.
andprotozoa[59]),temperaturealonedoesnotappeartobe Asanall-inclusivegroup,protozoagenerallyoccurinplank-
themainfactorcontrollinggrowthofmarinebacteriaunder tonicecosystemsatabundancesrangingfrom10sto1,000s
mostcircumstances(see“Light,Temperature,andPressure”). per ml. Abundances in benthic ecosystems can be one to
Itwouldbeoverlysimplistictothinkthatallthebacteria three orders of magnitude higher, commensurate with the
and archaea in a sample or habitat have the same level of higherabundancesofbacteriainthoseecosystems.However,
activitypercell,butitisalsoeasytothinkofmeasuredactiv- itisimportanttorememberthatliketheterm“‘bacteria,”the
ities as characteristic of all members of a microbial assem- term“protozoa”isaratherartificialconglomerationofevolu-
blage. So the question arises: are most of the cells active at tionarilyandecologicallydivergenttaxa(see“TheChanging
asimilarlevel,oraresomehyperactivewhileothersarecom- and Complex World of Eukaryote Phylogeny”). Thus, the
pletelydeadormoribund?Thisquestionhasbeenaddressed abundances of specific lineages of bacteria or protozoa may
severalways,includingmicroautoradiography,selectivestain- show spatial (or temporal) variability that is considerably
ing,“directviablecounts”(wherenutrientsareaddedtosee greaterthanthevariabilitycharacteristicoftheseoverarching
whatpartofthecommunitygrows),andinsituhybridization groupings.
(e.g.,60).Basedonthesecontrastingapproaches,itappears
that acontinuum ofactivityexists within bacterialassemb-
lagesfromtrulydead(cannotberevived)toextremelyactive. TheChangingandComplexWorldof
Areasonableinterpretationoftheexistingdataisthatunder EukaryotePhylogeny
typicalconditions,asmallpercentageofthemarinebacterial Notthatlongago,textbooksstilldividedeukaryoticorgan-
cells,perhaps10–20%,aregenerallyinactiveordead;theplu- isms into four major kingdoms (Animalia, Plantae, Fungi,
rality or majority of cells, perhaps 25–75%, are intact and and Protista) while prokaryotic organisms were placed into
havesomemoderatelevelofactivity;andasmallpercentage, asinglekingdom,theMonera(68).Withinthisscheme,pro-
perhaps5–20%,arehighlyactive.Itisusefultoconsiderthis tists(eukaryoticorganismsthatcanexistassinglecells)were
spectrum, conceptually and numerically, when modeling dividedintotwosubkingdoms(algaeandprotozoa)basedon
microbialprocesses. theirbasicnutritionalmode,acarryoverfromthehistorical
Comparisons of bacterial and phytoplankton biomass distinctionbetweensinglecellswith“animal-like”or“plant-
withinplanktonicecosystemsshowthatthesearepositively like” nutrition. This distinction presupposed a basic evolu-
correlatedacrossbroadscales.Analysesofmarineandfresh- tionarydivergenceamongprotistsintospeciesthatretained
watersamplesfromseveralstudies(61,62)haveshownthat a heterotrophic, phagocytotic mode of life (protozoa) and
bacterial abundance increases with chlorophyll concentra- those that abandoned phagocytosis for a photosynthetic
tion,atleastatthelevelofalog-logrelationship.Similarly, modeoflife(algae).Moreover,thepresence/absenceofchlor-
bacterialabundancesandtheabundancesofsmallprotozoa oplastswasafeaturethatcouldbeeasilydistinguishedbyearly
correlateoverbroadspatialandtemporalscales(63).These microscopists.
relationshipsaresensibleinthatonthelargestscale,primary Thefive-kingdomclassificationsystemofWhittakerwas
productionisthesourceoforganicmaterialthatfuelshetero- recognized as an improvement over previous classification
trophicbacterialactivity,andbacteriaconstitutethepreyof schemes, but it posed a number of problems relating to
manysmallprotozoa.Individualdatasetsalsohavesometimes protists. For example, the distinction between single-celled
shownstrongcorrelationsbetweenbacterialabundanceand and multicellular eukaryotes was somewhat arbitrary. More
chlorophyll (e.g., 45), but variability in this relationship important, the division of protists based on whether they
overshorttemporalorspatialscalesistobeexpected.Itwould were heterotrophic or photosynthetic was clearly not an
presumablybeaconsequenceofrapid,short-termchangesin appropriate feature if the classification was to recapitulate
therateofsubstratesupplyaswellasthenormal,oscillatory evolutionary relationships. We now know that chloroplast
nature of predator-prey relationships between bacteria and acquisition and loss has occurred several times in the bio-
theirconsumers. logicalhistoryofourplanet (69),givingrisetosomeclosely
Interestingly,theextrapolationofthepositivelog-logrela- related protistan taxa that differ largely in the presence or
tionship between bacterial and phytoplankton biomass absence of a chloroplast. Further complicating the matter,
to environments with very low chlorophyll concentrations withinmanyprotistanlineagestherearespeciesthatpossess
(e.g.,ultra-oligotrophicoceans)indicatesthatbacterialbio- chloroplasts and carry out photosynthesis (phototrophy)
massmayexceedphytoplanktonbiomassinthesesituations. butalsopossesstheabilitytoingestanddigestprey(hetero-
Whilethisconclusionisobviousforthedeepsea,wherethere trophy; 70–72). Some heterotrophic protists even ingest
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
4.2.2-6 ▪ AQUATICENVIRONMENTS
phytoplanktonpreyandretainthechloroplastsoftheirpreyin microplankton; 0.2–2mm=mesoplankton). Most protozoa
afunctionalstateforalimitedamountoftime(kleptidoplas- fall into the nanoplankton or microplankton size classes.
tidy;73). Various forms and degrees of mixotrophy (mixed Modeling microbial trophodynamics using this convention
phototrophic and heterotrophic nutrition) are common assumesthatprotozoainonesizecategorygenerallyconsume
amonganumberofalgal/protozoanlineages(74–77).Under prey one order of magnitude smaller in size (34, 85).
Whittaker’sscheme,phytoplanktonecologistsstudyingalin- Althoughthisapproachmissesmuchofthedetailanddiver-
eage of microalgae might have had little familiarity with sityofthetrophicactivitiesofindividualprotozoantaxa,itis
closely related heterotrophic species, while protozoologists anecessary,practicalcompromiseforexaminingcommunity-
studyingaparticularprotozoangroupmighthaveknownlittle scale flows of energyand elements. It also provides auseful
aboutcloselyrelatedphotosyntheticspecies. mechanismforsummarizingandcomparingtheabundances
One might expect, given these caveats, that the terms and biomasses of protozoafrom different environments and
“algae”and“protozoa”arenolongerused.Infact,theterm toothermicrobialassemblages.Protozoanabundance,sum-
“protozoa” is still commonly used (especially by ecologists) marized in this way, has been shown to contribute signifi-
torecognizethoseeukaryoticspeciesthatexistassinglecells, cantly to the living biomass of planktonic ecosystems
andwhosenutritionisdependentontheuptakeofpreformed throughouttheworldocean(Fig.3).
organicsubstances(primarilyviapreyingestion),whilepro- Estimatesofprotozoanbiomass,suchasthosedepictedin
tistspossessingchloroplastsarestillcommonlycalled“algae.” Fig.3,typicallydonotincludethecontributionofmixotro-
Similarly,althoughtheterm“protist”hasbeenabandonedas phicphytoflagellatestoheterotrophy.Thereispresently no
akingdomdesignation,itisstillwidelyemployedtodescribe easywaytodeterminetheabundancesofsmallphagotrophic
eukaryoticspeciesthatarecapableofexistenceassinglecells phytoflagellatesinnaturalsamples,sothesespeciesaretypi-
(i.e., algae and protozoa). The term “phagotrophic protist” cally counted as phytoplankton unless specific methods are
hasalsogainedpopularityinrecentyearsbecauseitrecognizes employedtoidentifythealgaeasconsumers,suchastheuse
thatmanyprotistanspeciesarecapableofphagocytosiseven of fluorescently labeled particles (87–90) or through the
thoughtheymayalsopossesstheirownchloroplastsandthus examination of food vacuole contents (91). On average
aretechnically“algae.” these species appear to constitute a modest percentage of
DespitetheshortcomingsofWhittaker’sscheme,itdomi- the phytoplankton assemblage (typically <25%), although
natedthehierarchicalorganizationoflifeforapproximatelya they mayat times dominate the phototroph assemblages of
quartercentury.During thepastfewdecades,however,this natural plankton communities. It is important to recognize
systemhasgivenwaytoaneworganizationalschemethatrec- that their inclusion as functional heterotrophs, rather than
ognizesthreedomainsoflife(Archaea,Bacteria,Eukarya[or phototrophs, could significantly shift the relative contribu-
Eucarya];78;Fig.2,upperpanel),andisbasedonwhatispres- tions of phototrophic and heterotrophic microbial biomass
entlybelievedtoreflectamorerealisticviewoftheevolution- tototalbiomasswithinmicrobialassemblages,andtheflow
arydistancesthathavedevelopedbetweenorganismsinthe ofenergywithinplanktoncommunities(92).
≈4 billion yearsthat life has existed on our planet. Within Heterotrophic protists that harbor photosynthetic pro-
the Eukarya of Woese’s scheme, hypotheses regarding the tists,ortheirchloroplasts,withintheircytoplasmconstitute
phylogenyof“protists”havechangedcontinuouslyandrap- another complexity for estimating the contribution of pro-
idly during the past two decades, reflecting new insights tozoato total microbial biomass. When bulk water samples
intoeukaryoteevolutionprovidedlargelybyDNAsequence are analyzed, the contribution of chlorophyll contained
information(Fig.2,lowerpanel;from[79]). within those protozoa is generally assumed to come from
TheformerprotistanphylaofWhittaker’ssystemhavenow free-living phytoplankton. However, studies have shown
been dispersed among candidate “supergroups” within the that chloroplast-bearing ciliates can contribute up to half
domainEukaryatobetterreflecthypothesizedphylogenetic the total biomass of planktonic ciliates in ecosystems, and
relationships.Forexample,thedinoflagellates(whichencom- chloroplast-retaining ciliates can sporadically dominate the
pass phototrophic, heterotrophic, and mixotrophic species) chlorophyllandprimaryproductionofsomeplanktoniceco-
nowformasinglegroupandhavebeenplacedtogetherwith systems (93–96). The environmental conditions promoting
theciliatesandapicomplexans(sporozoans)inthemonophy- the success of these ciliates are poorly known. Similarly,
letic Alveolata (Fig. 2, lower panel). On the other hand, many species of planktonic foraminifera, polycystine radio-
eukaryotic,heterotrophic,single-celledspeciesfallingwithin laria, and acantharia harbor large numbers (thousands per
thegeneraldescription“protozoa”arenowwidelydistributed protozoan) of endosymbiotic algae within their cytoplasm
amonganumberofprotistanlineages.Inshort,nutritionhas (97,98).Caronetal.(99)havedemonstratedthatprimary
beendemotedasaphylogeneticcharacter,andothercharac- productivitywithinthesespeciescancontributesignificantly
ters(presumablymoreindicativeofevolutionaryrelatedness) tototalprimaryproductivityinoceanicecosystemsandcanbe
haveascendedtoaddresssomelong-standingcontradictions, veryimportantlocallyintheconvergencesofLangmuircircu-
althoughthedebateovertherelationshipsamongsomeline- lationcells(100).
agesisstillveryactiveatthepresenttime;79).
Individualprotozoancellsrangeinsizefromlessthan2µm
to greater than 1cm in diameter (>4 orders of magnitude) BACTERIAANDARCHAEA
(80, 81) with some colonial radiolaria forming cylindrical
gelatinous structures a centimeter in diameter and more “Culturable”versus“Nonculturable”Cells
than a meter in length (82, 83). Because they constitute Mostconventionalcultivationmethodscangrowonly1%or
suchalargesizerangeoforganisms,protozoaareoftendivided lessofthebacteriathatcanbevisualizedbydirectmicroscopy
intosizeclassesthatverycrudelycorrelatewiththeirgeneral techniques (e.g., 4). This is true even though most can be
nutritionalpreferences.Acommonlyusedconventionisthat showntobeactivebytechniquessuchasmicroautoradiogra-
ofSieburthetal.(84),whichgroupsplanktonicmicroorgan- phy(25).Thesereadilycultivableorganismsappeartorepre-
isms into order-of-magnitude size classes (0.2–2.0µm= sentagroupoffast-growingso-calledweedsthatareadapted
picoplankton; 2.0–20µm=nanoplankton; 20–200µm= totakeadvantageofrapidgrowthinrare,organicallyenriched
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
HeterotrophicPlanktonicMicrobes:Virus,Bacteria,Archaea,andProtozoa ▪ 4.2.2-7
FIGURE2 Thethreedomainsoflife(upperleft)asproposedbyWoeseetal.(78),andarecentoverviewofmodificationsthathavebeen
proposedbyAdletal.(79)tohigher-levelphylogenticgroupswithintheeukaryoticcomponentofthetree(lowerright).Domainsfigurefrom
Woeseetal.(78),eukaryotictreefigurefromAdletal.(79).doi:10.1128/9781555818821.ch4.2.2.f2
environments. This strategy contrasts with the numerically Becauseofthelowpercentageofmarinebacteriathatcan
dominant bacteria that are adapted specifically for growth be grown in standard media, organisms that until recently
in the dilute nutrient conditions that characterize the vast were called “nonculturable” make up the large majority of
majorityofthevolumeofthewatercolumn. bacteriaintheplankton.Onlyduringthepast∼20–25years
The most common taxa readily cultured from seawater havemolecularbiologicalmethodsbasedon16SrRNAgene
withstandardnutrientbrothmediaincludethegammapro- sequences been available to identify these organisms, and
teobacterial genera Vibrio, Alteromonas, Pseudoalteromonas, these powerful techniques have opened up a large area for
Marinomonas, Oceanospirillum, Shewanella (usually isolated exploration(seenextsection).Similarbutmorerecentstud-
fromsurfacessuchasshellfishandsediments),thealphapro- ies use 18S rRNA sequences for characterizing protistan
teobacterialgeneraRoseobacter,Sphingomonas,membersofthe diversity,aswillbenotedbelow.
familyFlavobacteriaceae,andPlanctomycetes,assummarized
inGiovannoniandRappé(101)andFuhrmanandHagstrom
(102).ThecyanobacteriaSynechococcusandProchlorococcus MolecularPhylogenyandMetagenomics:
arealsonowreadilyculturable,butonlow-nutrientinorganic FieldApplications
mediatargetingphotosyntheticforms,asopposedtoorganic Modern phylogeny of microorganisms is based primarily
mediausedtocultivatetheotherslistedabove. ongeneticsequences,themostwell-studiedgenebeingthe
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
4.2.2-8 ▪ AQUATICENVIRONMENTS
FIGURE3 (a,b)PlanktonbiomassintheArabianSeaduringthe1995southwestmonsoon(a)andintermonsoonperiod(b).Areasofthe
boxesindicatetherelativemagnitudesofthebiomassineachcategory.Categorieswithinthedashedboxesina,b,carecomposedofprotozoa.
Arrows indicate the direction of energy/material flow in the food web, thicker arrows depicting greater flow. Redrawn from (86).
(c)Depth-integratedbiomass(mg/m2)intheupper100moftheSargassoSeanearBermuda,andintheupper200moftheequatorialPacific
at175°E.Thewidthofthebarsindicatesthebiomassineachsizecategory.Heterotrophshavebeenseparatedbysizeclass,whilephytoplankton
havenot.Sizeclassesdelineatedbythedottedboxarecomprisedofprotozoa.Redrawnfrom(65).doi:10.1128/9781555818821.ch4.2.2.f3
smallsubunitribosomalRNAgene(16SrRNAinBacteria relationships(ormakedistinctions)atthegenusorsometimes
and Archaea, and its larger homolog 18S rRNA in eukar- specieslevel.
yotes).Thismoleculeisstronglyconservedoverevolutionary The first phylogenetic studies based on 16S/18S rRNA
time,sothissinglemoleculehasbeenusedforconstructing genes used sequences derived from cultures. However, one
phylogenetictreesofalllivingorganisms(http://tolweb.org/ does not need culturesto obtain rRNA gene sequences (or
tree/phylogeny.html). Analysis of 16S/18S rRNA gene anyothersequences,forthatmatter).Anideadevelopedin
sequenceshasbeenusedtoevaluatedeepevolutionaryrela- thelabofNormanPaceinthemid-1980sinvolvedextraction
tionships among organisms and was instrumental in point- ofDNAdirectlyfromnaturalsamples,andthencloningand
ing out that Archaea, Bacteria, and Eukarya should be sequencingoftheDNAasameansofassayingthemicrobes
considered different Domains of equivalent phylogenetic present in the samples (103, 104). The original protocols
rank, above kingdoms (78). However, there are sufficient calledforcloningbycreatingwhatarecalled“phagelibraries”
differencesin16S/18SrRNAgenesequencestodemonstrate fromthenaturalDNA,butsince1986,PCRhasbeenapplied
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
HeterotrophicPlanktonicMicrobes:Virus,Bacteria,Archaea,andProtozoa ▪ 4.2.2-9
extensively for cloning and related studies. The target by advances in single-cell isolation which, when coupled
sequence can be almost instantly “identified” to its closest withhigh-throughputsequencingapproaches,reducethetre-
phylogenetic neighbor by what have come to be standard mendouscomplexitypresentinnatural,complexeukaryotic
onlinesequencecomparisons. communities to a manageable task (112). Such single-cell
Beyond the studyof targeted genes (like the 16S rRNA techniquesarealsoquitevaluableinstudiesofbacteriaand
gene), shotgun metagenomic studies have examined the archaea, though the amplification technique tends to be
entire genetic repertoire of the microbes in a given sample. veryunevenandtypicallygenerateslessthanhalfthegenome
The metagenome isthe collective genome of all organisms ofeachisolatedcellregardlessofdomain(113,114).
in the sample. Initially these studies extracted DNA from Additionally, similar to DNA, mRNA is amenable to
allorganismsinasample(usuallyprefilteredthroughafilter extraction and sequencing although greater care must be
approximately 1µm to remove most organisms larger than taken during extraction and purification as RNA shows a
bacteria), sheared it to produce fragments, and cloned greatersusceptibilitytodegradationduringprocessing.Copy-
them into standard vectors, either as small (thousands of ingofmRNAbyreversetranscriptionofRNAintocDNA,
bases) or large (to hundreds of thousands of bases) inserts, followedbyDNAsequencing,hasallowedinsightsintothe
that is, fragments of DNA from the environment now metatranscriptomes of environmental samples. Metatran-
cloned into the vectors in a form suitable for sequencing. scriptomic studies provide information on gene expression
The best known early marine study of the former type is inanecosystem,andtherebyindicates“activity”ofthemicro-
the Global Ocean Survey, with initial results published by bialcommunityratherthansimply“potential”representedby
Venter et al. (105), which generated more than a billion thegenomicDNApresentinthesample,withmanyapplica-
basesofDNAsequenceandreported1.2millionpreviously tions, from showing which processes are being carried out
unsequenced genes, estimated to come from at least 1,800 by which organisms to fine-scaled diel studies (115–118).
differentgenomicspeciescumulativelyinthemanysamples Nevertheless, given variations in the lifetimes of different
theyanalyzed. transcripts and protein molecules, the transcriptome may
Withtheadventofnext-generationsequencingthatgen- not be fully representative of the current activity of an
eratesmillionsormoresequencesinarun(knownbyavariety organism.
ofacronymsincluding454,Illumina,SOLiD,etc.),cloning Limitations of these analyses include sequencing errors,
ofgeneshaslargelybeenreplacedbyclone-freesequencing. PCR mismatches or biases, clustering and bioinformatics
The extent and power of such sequencing has recently challenges, and chimeras generated during PCR. Also, the
been demonstrated by the ability to construct essentially phylogeneticresolutionof short sequences is limited, given
theentiregenomeofanuncultivatedmarineGroupIIEur- thehighconservationofrRNAsequences.Evenwithclone-
yarchaeon that constituted only approximately 2% of a freeshotgunmetagenomics,theremaybebiases,suchasnon-
microbial community sample, using SOLiD sequencing, random losses of DNA during extraction and preparation,
made possible by high coverage and the use of mate pair or biases (e.g., from G+C content or secondary structure)
sequencing of∼3,000bp fragments (106) (this length is in the sequencing procedures. Determination of species
neededtospanrepeatsandhighlyconservedgenesthatoth- diversity by these approaches provides an example of these
erwisemakegenomeconstructiondifficult). limitations.Shakyaetal.(119)workingwithsyntheticcom-
For16S rRNAgene studies, “tagsequencing”pioneered munities(purifiedgenomicDNAfrom16Archaearepresent-
by Sogin et al. (107) consists of amplifying a suitable sized ing3phylaand48Bacteriarepresenting16phyla,remixedto
part of the gene with broadly conserved primers (choice is simulateanenvironmentalDNAextract)appliedbothmeta-
important—fewaretrulyuniversalforthegroupsintended), genomic analysis (454 and Illumina platforms) and PCR
often“barcoded”toallowmultiplesamplestobecombined amplificationfollowedby454sequencingof16SrRNAgenes
intoasinglerun.Theamplifiedproductsarethensequenced, to determine both species richness and relativeabundance.
andsequencesprocessedenmasse.Manythousandsofpartial Theyfound thatPCR amplification/454 sequencing of 16S
SSUrRNAgenesequencespersampleareeconomicallyana- rRNAgenesyieldedanaccuratemeasureofspeciesrichness
lyzedthisway,thoughtheyareusuallyshort(currentlyafew (providing that appropriate data processing was applied)
hundredbaseseach,dependingonthesequencingplatform). butthattherelativeabundanceofupto94%ofthespecies
Thiswayofanalyzingthecompositionofmicrobialcommun- (depending on domain and variable region amplified) was
ities is now standard. These approaches provide so much over- or underestimated by at least 1.5-fold (values ranged
informationevenaboutveryraresequences,thattheresults fromnotdetectedto10.3-foldoverestimation).Incontrast,
have led to the important concept of the “rare biosphere,” both metagenomic approaches yielded relative abundances
organismsthatmaybeactiveordormantandconstitutinga that were within the authors’ 1.5-fold accuracy cutoff for
very small proportion of the community (e.g., often much ∼50ofthespecies.However,theyconcludedthataddressing
lessthan0.1%),butarepotentiallyimportantfordispersion, richness overestimation in metagenomic analyses, that is,
adaptationtochangingconditions,orevencriticalactivities distinguishing rare but real OTUs from experimental and
likenitrogenfixationorvitaminproduction(108).However, computational artifacts, awaits further computational and
due to the potential for artifacts like error sequences, this classification improvements. More recently, Parada et al.
approach requires rigorous application of quality filtering (120) used mock communities composed of 16S rRNA
andclusteringalgorithmstoavoiderroneoustaxaandoveres- clones from 27 common marine taxa (from nine Bacterial
timationofspeciesrichness(109,110). and two Archaeal phyla) to show that small differences in
Next-generationsequencingapproacheshavealsoallowed PCR primers (and different clustering methods) can yield
worktobeginmetagenomicsstudiesofmicrobialeukaryotes largedifferencesinapparentrelativeabundancesofreported
(111).Thesestudiesarestillconstrainedbythemuchlarger taxa. However, one primer pair and informatics pipeline
genomes of eukaryotes, and therefore the difficulties of they tested, using a particular version of 515F-926R (V4–
obtainingsufficientsequencestoreconstructthegenomeof V5),providedaccurateestimatesofrelativecloneabundance
aparticularspecies.Alongwithadvancesinsequencingabil- (r2=0.95)whencomparingobservedversusexpectedclone
ity,studiesofeukaryoticmetagenomicshavebeenfacilitated abundance.
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15
4.2.2-10 ▪ AQUATICENVIRONMENTS
Culture-IndependentDiversityStudies crenarchaeolwasderivedlargelyfromCO ,suggestingauto-
2
trophic metabolism (137, 138). An experiment showing
Thefirstgroupstobeidentifiedusingcultivation-independ- uptakeof13C-labeledbicarbonateintothesearchaeallipids
ent molecular techniques from the marine plankton (121) (139)directlypointedtoautotrophyinthisgroup.Chemoli-
werethebacterialSAR11cluster(agroupofcloselyrelated thoautotrophywasfirsthintedatbyVenteretal.(105),whose
gene sequences, or phylotypes) and marine picoplanktonic metagenomicanalysisshowedanapparentarchaealscaffold
unicellularcyanobacteriaSynechococcusandProchlorococcus). thatcontainedgenessuggestiveofammoniaoxidation.Fur-
Of these, the SAR11 cluster was completely unknown, but therevidencecamefromSchleperetal.(140),whodetected
the cyanobacteria had previously been recognized by their severalammoniaoxidationgenesinorderdirectlyadjacentto
unique pigment fluorescence; Waterbury et al. (122) and aThaumarchaeota16SrRNAgeneinasoil-derivedmetage-
Johnsonetal.(123)usedepifluorescencetoobserveSynecho- nomicclone.TheissuewasdirectlyresolvedwhenKonneke
coccus,andChisholmetal.(124)discoveredProchlorococcus etal.(141)isolatedarelatedmarinearchaeonfromsediment
usingflowcytometry.Thesecyanobacteriawerelaterisolated ofamarineaquarium,andthisorganism,CandidatusNitroso-
andgrowninphytoplanktonculturemedia.Thesetwogroups pumilus maritimus, was found to have a chemoautotrophic
aregenerallycommonintheeuphoticzone,withSAR11typ- metabolism, quantitatively oxidize ammoniato nitrite, and
icallycomprisingonethirdoftheplanktonicbacteria(125), containanarchaealammoniamonooxygenasegene(amoA)
andthecyanobacteriacommoneverywherebutpolarwaters. andinterestinglydidnotgrowheterotrophically.Cultivation
Probablythebiggestsurprisetocomefromtheapplication experiments showed this organism has a high affinity for
ofmoleculartoolswasthediscoverybyFuhrmanetal.(126) ammonium,allowingittooutcompetebacterialnitrifiersat
ofabundantarchaeainthedeepsea.Thearchaeawerefound low (submicromolar) concentrations as are typical in the
tobeinauniquephylogeneticclusterthatwasonlydistantly sea(142).Thecompletegenomeoftheorganismhasshown
relatedtoanypreviouslyknownarchaea,butthe“closestrel- noveladaptationsfornitrificationandautotrophy(143).
atives”(notreallycloseatall)wereextremethermophiles.A Even if the Thaumarchaea are primarily chemolitho-
subsequent study also found archaea to be present in near- autotrophs, field datasuggest alevel of mixotrophy, in that
surface coastal waters, albeit relatively rare (<2% relative someorganicsubstratesarebeingincorporatedintobiomass.
abundance). This study used a PCR technique specifically A stable isotope study using cells collected at 670m depth
targetedarchaea,andfound“marineCrenarchaea”plusasec- off Hawaii estimated that about 80% of the carbon incor-
ondgroupbelongingtothephylumEuryarchaea(127). poratedintoarchaea-specificlipidscamefrominorganicsour-
Upuntilthistime,allknownArchaeawerethoughttobe ces and about 20% from organic compounds (144, 145).
“extremophiles”—adaptedforeitherveryhightemperatures Interestingly, it has also been claimed that the genomes
(thermophiles), extremely salty conditions (halophiles), or of deep-sea(>2,000m depth) members of the Thaumarch-
strictly anaerobic environments (methanogens). Yet these aea,aswellasthoselivinginequatorialwaters,rarelycontain
organismswerepresentincoldorcoolwateratordinarysalin- the amoA gene and thus may be primarily chemoorgano-
ity and high oxygen concentrations. Fluorescent in situ trophs(48).
hybridization (FISH) measurements from deep-sea samples Major bacterial groups that have been documented
have since showed that the archaea may make up appro- from seawater using 16S rRNA characterization include
ximately 40% of the total countable prokaryotes, with the some that are also known from culture (e.g., Alteromonas,
percentagereachingto60%at200mdepthintheMediterra- Roseobacter), and several that are phylogenetically distant
nean (35, 36). An extensive time series of FISH measure- from standard cultures. The most common groups are, in
ments near Hawaii confirmed that the archaea are indeed rough order of their relative abundance in clone libraries
very abundant throughout the year from below the photic frommosttoleastabundant:SAR11(relativesofPelagibac-
zonetoatleast4,000m,andtypicallyconstituting30–40% ter ubique), Roseobacter, SAR86, cyanobacteria, SAR116,
ofthetotalprokaryotespresentinwatersdeeperthanafew SAR202,SAR234,andMarineGroupA.TheSARdesigna-
hundred meters (128), with similar results found elsewhere tion followed by a number is an arbitrary sequential clone
(129,130)(Fig.4).Theyhavebeenreportedfrommanypla- identifierfromSargassoSeacloningstudiesdonebytheGio-
ces, including the Atlantic, Pacific, and Southern Oceans, vannonilabthatdidmostoftheearlysystematiccatalogingof
andaredynamiccomponentsoftheplankton,themostcom- clones.Summariesofthedataandphylogeneticrelationships
montypebyfarbeingthe“marineCrenarcheaea”(128,129, ofthesegroupscanbefoundinFuhrmanandHagstrom(102)
131). While the marine archaea have been reported to be andGiovannonietal.(101).
dominated by a few major “phylotypes” (132), they also
havebeenshowntohaveagreatdealofmicrodiversitywithin
thesephylotypes,suggestingtherearemanykindsofcloserel- MolecularGeneticDiscoveriesinBacterialand
atives coexisting (133). Interestingly, this group of archaea ArchaealMarineBiology
maylikelybethemostabundantkindoforganismonEarth, As described earlier, metagenomics is an extension of the
giventhehugevolumeofthedeepseaandtheirhighabun- ideasusedinthe16SrRNAcloningstudies,inthatallgenes
dancethere(134).Ithasrecentlybeenproposedthat“marine from the native microorganisms are separated and cloned
Crenarchaea”beelevatedtotheirownmajorphylumoutside without having cultivated the organisms. These methods
theCrenarchaeota,calledtheThaumarchaeota,onthebasis early on started to find unexpected and very interesting
ofdeepphylogeneticbranchingandfundamentaldifferences results.Thebestexamplesinvolveunexpectedmarinephoto-
between them and the Crenarchaeota (135). The Thau- trophy, the first of which is discovery of a nonchlorophyll
marchaeota possess the uniquely archaeal membrane lipid photosynthetic bacterial pigment, called proteorhodopsin
crenarchaeol,andarenowalsoknowntobeabundantinsoils. (146). The gene was found on a large environmentally
The physiology of the Thaumarchaeota has been an derived fragment of DNA that also had a gene coding for
intriguing area of study. Initially, an autoradiography-FISH 16S rRNA from the so-called SAR86 group (one of
combination approach demonstrated that they take up thegroupscommoninseawater).Thispigmentcanactasa
amino acids (130, 136). But subsequent data showed that light-drivenprotonpump,thoughttopermitcellstogenerate
Downloaded from www.asmscience.org by
IP: 66.208.62.130
On: Thu, 03 Mar 2016 18:52:15