Genomic Markers of Ancient Anaerobic Microbial Pathways: Sulfate Reduction, Methanogenesis, and Methane Oxidation

6 Pages·2003·2.6 MB·

by  Andreas Teske

Reference: Biol. Bull. 204: 186-191. (April 2003)
2003 Marine Biological Laboratory
Genomic Markers of Ancient Anaerobic Microbial
Pathways: Sulfate Reduction, Methanogenesis, and
Methane Oxidation
ANDREAS TESKE'-*'t ASHITA DHILLON2 AND MITCHELL L. SOGIN2
.
lBiology Department. Woods Hole Oceanographic Institution. Woods Hole. Massachusetts 02543: and
'Josephine Bay Paul Centerfor Comparative Molecular Biologv and Evolution. Marine Biological
Laboratory, Woods Hole. Massachusetts 02543
Genomic markers for anaerobic microhial processes in genie archaea become the dominant anaerobic microbial
marine sediments sulfate reduction, methanogenesis, and population. Autotrophic methanogens utilize hydrogen as
anaerobic methane oxidation reveal the structure ofsi'l- energy source for the reduction ofCO, to methane; special-
fate-reducing, methanogenic, andmethane-oxidizing micro- ized genera of methanogens are also capable of inter- and
hial communities (including uncultured members): thev al- intramoleculardisproportionate ofC and C-, carbon com-
t
low inferences about the evolution of these ancient pounds (methanol. methylamines, acetate) to methane and
microhial pathways: and the\ open genomic windows into CO2 (Boone et a/., 1993). Where methane and sulfate co-
extreme microbial habitats, such as deep subsurface sedi- exist (for example, at the interface of sulfate-reducing and
ments and hydrothermal vents, thai arc analogs for the methanogenic sediment layers, or at marine methane seeps
early Earth and for extraterrestrial microbiota. and vents), sulfate-dependent anaerobic methane oxidation
Sulfate reduction and methanogenesis are two terminal takes place; methane of biogenic origin is oxidized to CO-,
anaerobic bioremineralization pathways that convert low- with sulfate as the terminal electron acceptor (Valentine and
molecular-weight products of other bacterial processes Reeburgh. 2000). As proposed originally on the basis of
(degradation of polymers, fermentation) to CO2 and meth- biogeochemical field data and thermodynamic consider-
ane. Sulfate-reducing bacteria are physiologically and phy- ations (Hoehler et al.. 1994), anaerobic methane oxidation
logenetically highlydiverse (Castroeta!.. 2000; Widdel and is carried out by syntrophic consortia of methanotrophic
Bak. 1992): they oxidize a wide variety of low-molecular- archaea and sulfate-reducing bacteria, in which the sulfate-
weight compounds (short-chain fatty acids, alcohols, al- reducing partner catalyzes the electron transfer from meth-
kanes, aromatic compounds, acetate) to CO,. In marine ane to sulfate and assimilates a portion of the methane
sediments, the range of sulfate-reducing bacteria is limited oxidation products (Boetius ct al.. 2000; Orphan et al.,
by sulfate availability. When sulfate is depleted, methano- 20()lb). Methanotrophic archaea of different phylogenetic
affiliation can form dense, highly ordered clusters with
sulfate-reducing syntrophs. or may occur in less tight asso-
tCurrentAddress: DeparlmemofMarineSciences.CB#3300.Venable ciations (Orphan et al.. 2002).
Hall 12-1. University of North ( mlma at Chapel Hill, Chapel Hill, NC The sulfate-reducing, methanogenic, and methane-oxi-
27599.
*To whom correspondence shouki be addressed. E-mail: teske@email. dizing microbial populations that are found in anaerobic
unc.edu marine microbial ecosystems today are the modern descen-
The paper was originally presented at a workshop titled Outcome* of dants of ancient microbiota whose isotopic imprints are
Genome-Genome Inicrafiinnx. The workshop, winch was held at the J. pervasive in the carbon- and sulfur-isotopic record, from the
Erik Jonsson Center of the National Academy ofSciences. Woods Hole. present back to the Archaean-Proterozoic transition (Knoll
AMadsvsaanccheusdetSttsu,difesromin 1t-he3 SMpaayce2L0i0f2e.Swcaisencsepsonastortehde Mbyaritnhee CBeinotleogricfaolr and Canfield, 1998). The only oxidants that these pathways
CO
Laboratory, and funded by the National Aeronautics and Space Adminis- require are : or carbonate, and sulfate, which existed
tration underCooperative Agreement NCC 2-1266. before the photosynthetic oxygenation of the Earth's bio-
186
GENOMIC MARKERS OF ANAEROBIC MICROBIAL PATHWAYS 187
sphere and the appearance offree oxygen in the atmosphere would be the most difficult and energy-demanding step to
and the marine water column. Isotopic evidence for widely reverse (Hoehler and Alperin. 1996). Current full-genome
expressed microbial sulfate reduction, in the form of 34S- sequencing efforts using purified ANME-1 and ANME-2
depleted sedimentary sulfides. goes back to the middle and archaeafrom environmental samples are testing whetherthe
early Proterozoic. 2.2 to 2.3 billion years ago (Canfield et genomes of these methanotrophic archaea carry coenzyme
M
til.. 2000). The mineralization of organic matter by metha- methyl reductase genes (Orphan el ul.. 2002).
nogenesis, followed by methane oxidation, may even pre-
date the onset of sulfate reduction. The carbon isotopic The Guaymas Basin Hydrothcrmal Vent Sites as
imprint of this process, in the form of highly '^C-depleted Model System
kerogen (5UC > -60%c), is found in late Archaean and To search for deeply branching and (possibly) ancestral
eaanrdlyMoPorrotee,ro1z9o9i2c).keTrhoigseniss,oto2p.i8cbriellcioorndyweaasrsoroilgdin(aSltlryauisns- rmeeptrheasneen-toatxiivdeiszinogf misuclrfoaotreg-arendiuscmisng,andmetthehiarnokgeeynigce,nesanidn
terpreted as evidence for widespread aerobic methane oxi- modern environments, we focus on hydrothermal vent hab-
dation (Hayes, 1994). Anaerobic methane oxidation is more itats. Hydrothermal vents represent some ofthe earliest and
likely, since evidence for the stepwise and pervasive oxy- best protected microbial habitats that may have survived
genation ofthe proterozoic biosphere begins to appear only repeated environmental disturbances in the surface bio-
at alatertime, about 2.2 billion years ago (Des Maraisetul.. sphere; vents can in principle occur on every planet with
1992). oceans and active plate tectonics or volcanism. On Earth,
hydrothermal vents sustain complex microbial ecosystems
Key Genes for Sulfate Reduction and Anaerobic that utilize inorganic energy sources (such as sulfide, hy-
Methane Cycling drogen, and reduced metals) and geothermal sources of
carbon (such as methane. CO^, and geothermally synthe-
The antiquity and evolutionary significance of these mi-
sized low-molecular-weight organic compounds) (Kelley et
crobial pathways is shown in the high degree of phyloge- ul.. 2002). The hydrothermally active sediments of the
netic conservation of their key genes and key enzymes. In Guaymas Basin (Gulf of California. Mexico) provide a
esunlzfyamtee-raeddeuncoisnignper-o5k'a-rpyhootsepsh,otshuelfaaptsegreendeucctoadsee,s fwohritchheckaety- rtehleatmiivcerloybiwaelll-csotmumduineditmioedselthastysatreeminfovroltvheedcionmpsluelfxaitteyroe-f
assuulllylfziaettsee(trFherediueacdcrttiiicovhna,,ti2do0sn0r2aA)nB.d,Ascuosbdesecesoqnufdeornktetyhreegdeaulncpethioaofnadnoidfsssbiuemltifalaatsteuobrty-o dtiuocntsi,onl,ipimdetbhiaonmoagrekneersiasn,alaynseds,me1t6hSanreRNoAxi.daatniodn.fuCnuclttiiovnaa-l
gene sequencing are beginning to reveal unusually complex
units of the enzyme dissimilatory sulfite reductase, which microbial communities that include sulfate-reducing pro-
catalyzes the reduction of sulfite to sulfide (Wagner el ul., karyotes, methanogenic archaea, and anaerobic methanotro-
sa1re9cr9hv8a)ee.dalTinhessueldvfsearrtaeAlBrdaeendeudpcleayrpss.brgaeWnnhcehesinnagresppphehycylilfaoicgoefngebetanicectaelrtilraylancsaofnned-r puSlph.ei.cci2f0ai0rc2ca;hllayeD,ahirlaelnsoudlnttsehteoifurl.t.shueulnGfpauutabely-.rm)eadsuwicliBlnagsailsnsyonsthurerolvppehysto((TiFedisegnk.tei1fe)yt.
events are taken into account, the dsrAB and aps genes
novel sulfate-reducing. methanogenic. and methane-oxidiz-
allow a simultaneous phylogenetic and metabolic identifi- ing microorganisms in deep subsurface sediments, where
cation of sulfate-reducing prokaryotes (Klein et ul.. 2001; these processes are predominant (D'Hondt et ul., 2002).
Friedrich, 2002).
M These anoxic environments represent analogs to subsurface
Coenzyme methyl reductase is the key enzyme of life under extraterrestrial conditions in which an inhospita-
methanogenesis; it catalyzes the terminal and highly exer- ble surface environment might have driven microbial life
gonic step of the methanogenesis pathway, the reduction underground or never allowed its evolution within a pho-
and release ofthe coenzyme-M-bound methyl group as free
methane. The Coenzyme M methyl reductase gene (mrcA) totrophic. oxygenated biosphere.
is found in methanogenic archaea; it is sufficiently con- Guaymas Basin Microbial Communities
served and consistent with 16S rRNA phylogenies to allow
the identification of methanogenic archaeal lineages in en- Sulfate-reducing bacteria and archaea are dominant ter-
vironmental samples (Springer et ul.. 1995; Lueders et ul.. minal oxidizers oforganic matter in the Guaymas Basin, as
2001; Ramakrishnan etal.. 2001 ). At present it is not known shown by high rates of sulfate reduction measured over
whether anaerobic methane-oxidizing archaea are using a wide temperature ranges up to about 100C (Jorgensen el
version ofthis enzyme for the activation and reoxidation ot ul.. 1990. 1992; Elsgaard ct ul.. 1994; WeberandJorgensen,
methane. If anaerobic methane oxidation by archaea could 2002). Hyperthermophilic, autotrophic, or mixotrophic ar-
proceed through a reversal of classical methanogenesis chaea of the genus Archueoi>lobus were found by cultiva-
pathways, the Coenzyme M methyl reductase reaction tion (Burggraf et ul.. 1990) and 16S rRNA sequencing
188 A. TESKE ET AL
Beggiatoa spp., filamentous sulfide-
oxidizing bacteria at the oxic/anoxic
water/sediment interface
sediment - water
H
Sulfate reducers, here the
anaerobic hyperthermophilic archaeon
methane-oxidizing consortia Archaeoglobus profundus
archaea (red), bacteria (green)
H S + NO > H O -> S0 2
H2S + 2O3 -->2SO 2 +2H4 +
2 2 4
Prokaryotes involved
in sulfur and methane
cycling in the
Guaymas Basin
CH4 + SO42 "> SSOO422 ++ C4HH2C-O->OHH2S++H2+-O>H" 2 H2O
HCO3'+ HS'+ H2O HS>4 2 CO3+ 2 H O
2 2
t
Methanogenic archaea, here the CfrOo2mHv2einatceftlautied,and
ClyHsi4sforfoomrgvaennitcfsluubids,traantdespyro- Chy0p2er+th4eHrm2o-p-h>iCleHM4e+th2aHno2Opyrus kandleri cpyormoplyosuinsdosf organic
Figure 1. Simplified scheme of microorganisms and their reactions (sulfate reduction, methanogenesis,
methaneoxidation,sulfideoxidation)inthemethaneandsulfurcyclesintheGuaymasBasinhydrothermalvents.
Clockwise, sulfate-reducing bacteria and archaea (Archaeoglobus profimdiis), methanogenic archaea (Melh-
niKipyniskandleri), methane-oxidizingconsortia, and sultide-oxidizingbacteria(Beggiatoa spp.I. Fluorescence
insituhybridizationimageofanaerobicmethane-oxidizingconsortium,courtesyK. KnittelandA. Boetius(Max
PlanckInstituteforMarineMicrobiology.Bremen,Germany).Thebacterialandarchaealspeciesshownhereare
representatives ofmetabolically and phylogenetically diversified functional classes ofprokaryotes. It has tobe
noted that sulfate reduction and sulfate-dependent methane oxidation are almost certainly uncoupled in the
Guaymassediments.Thehighlydiversifiedsulfate-reducingprokaryoticcommunitycanoxidizeawiderangeof
substrates(H:andacetatearejustthe simplestexamples), whereasmethane-oxidizingsyntrophsaremostlikely
restricted to methane oxidation intermediates provided by their archaeal partners.
(Teske et al., 2002). Moderately thermophilic or mesophilic Methanococcus (Jones et al.. 1989; Canganella and Jones,
fatty acid oxidizing sulfate reducers have been cultured 1994) and Methanopyrus (Kurr et al.. 1991). and members
from Guaymas (Rueter et al.. 1994). Surveys with 16S of the formate-utilizing, mesophilic or moderately thermo-
rRNA detected predominantly members of the propionate- philic family Methanomicrobiales (Teske et al., 2002).
oxidizing, acetate-producing family Desulfobulbaceae Methane produced by these diverse methanogenic commu-
(Teske et al., 2002) and members of the acetate-oxidizing nities combines with the methane pool originating from
family Desulfobacteriaceae (Dhillon et <//., 2002). A molec- pyrolysis of organic matter buried in the Guaymas sedi-
ular survey based on dsrAB genes revealed the existence of ments; the resulting methane concentrations in the Guaymas
novel sulfate reducers in the Guaymas Basin that are not vent fluids are orders of magnitude higher than at non-
related to any cultured group of sulfate-reducing pro- sedimented, bare lava vent sites (Welhan, 1988).
karyotes (Dhillon et al.. 2003). Anaerobic methanotrophic communities in the Guaymas
Methanogenic archaea in the Guaymas Basin include Basin include ANME-1 and ANME-2 archaea, as shown by
hyperthermophilic, autotrophic methanogens of the genera 16S rRNA gene sequence analysis and '^C-isotopic analysis
GENOMIC MARKERS OF ANAEROBIC MICROBIAL PATHWAYS 189
of diagnostic archaeal lipids (Teske el ai, 2002). The taxonomy ormicrobial ecology. At the same time, itreveals
sulfate-reducing syntrophs in the Guaymas Basin sediments the evolutionary divergence that has accumulated in these
could not be identified unambiguously by 16S rRNA se- genes since the early Proterozoic or the Archaean, and the
quencing; their l3C-depleted membrane lipids (mono- and phylogenetic depth ofthese metabolisms inthebacterial and
dialkylglycerol ethers) indicate deeply branching bacteriaor archaeal tree oflife. With a growing database, homologous
sulfate-reducing bacteria of the family Desulfosarcinales and ancestral traits of these genes, including secondary
(Teske et ai. 2002). In classical ANME-2 consortia at structure motifs and conserved sites, can be found that
Hydrate Ridge and Eel River Basin, the archaeal core was significantly increase our understanding of environmental
surrounded by an outer layerofsulfate-reducing bacteria of and functional constraints that have shaped the evolution of
the family Desulfosarcinales (Boetius et ai, 2000; Orphan these ancient microbial pathways and enzymes.
et ul., 2001a, b). In Guaymas Basin samples that yielded However, in spite of continuing primer development
ANME-2 sequences, fluorescence in situ hybridization re- (Klein et ai, 2001). it is not certain whether PCR-based
vealed a different structure; archaeal cells were intertwined approaches can reliably detect all environmental genes of
with irregular lobes of syntrophic bacteria that did not interest, in particular the most ancestral and deeply branch-
hybridize with the probe for members of the Desulfosarci- ing lineages or key genes with nonconserved primer sites.
nales (Knittel et ai. 2002). A similar structure ofANME-2 Primersite conservation can neverbe taken forgranted. For
archaea intertwined with unidentified bacteria has been ob- example, 16S rRNA sequence motifs that were regarded as
served in anaerobic methane-oxidizing consortia from Eel universally conserved show substantial variation between
River Basin (Orphan et ai, 2002), and in the Haakon- different bacterial lineages (Daims et ai, 1999), and can
Mosby Mud Volcano in the Norwegian Arctic Ocean (Knit- render 16S rRNA gene amplification with standard primers
tel et ai, 2002). impossible (Huber et ai, 2002). To circumvent primer lim-
Autotrophic sulfide-oxidizing bacteria of the genus Beg- itations, surveys of PCR-accessible genes (such as dsrAB,
giatoa (Nelson et ai, 1989) that grow in dense mats on the apsA, and mrcA) could be extended by shotgun cloning and
sediment surface assimilate CO2 from seawater and sedi- fosmid library construction followed by sequence analysis.
ments; the latter CO2 pool includes contributions from sul- This approach also addresses the problem of phylogenetic
fate reduction and methane oxidation. The assimilation of congruence ofdifferent marker genes with partially discor-
methane oxidation products by sulfur-oxidizing bacteria dant phylogenies. Proving the affiliation ofdifferent marker
U
appears to be highly variable. The C isotopic signals of genes to each other and to their host organism requires an
Beggiatoci biomass from hydrocarbon and methane seeps extensive database ofpurecultures and strains, as shown for
range from typical valuesofabout -20%oto -30%c(Sassen the 16S rRNA, dsrAB and apsA genes in sulfate-reducing
et ai, 1993) to strong I3C depletion indicative of assimila- prokaryotes, and their partially discordant gene trees
tion ofmethane oxidation products (Paull etai, 1992). The (Friedrich, 2002). A genomic solution has to be found if
oxidation of sulfide and other sulfur intermediates (pro- marker genes belong to uncultured lineages in which this
duced by sulfate-reducing bacteria) by Beggiatoa spp. de- ground-truthing approach is not possible. Inferences based
pends on the availability ofoxygen ornitrate as the terminal on co-occurrence at particular sampling locations with spe-
electron acceptor (McHatton et ai, 1996). Therefore, Beg- cific geochemical regimes cannot prove that novel phylo-
giatoa spp. and other free-living and symbiotic sulfide- types (for example 16S rRNA and dsrAB) belong to the
oxidizing bacteria that represent the basis ofthe food chain same source organism (Thomson et ai, 2001). Tying to-
at hydrothermal vents could not survive in a strictly anoxic gether such phylotypes in the absence of cultures may
microbial habitat that does not receive molecular oxygen require an extensive database oflong genomic fragments, in
from the photosynthetic biosphere. In this way, the Guay- which multiple key genes serve as "phylogenetic anchors"
mas Basin shows the caveats and limits of early-earth or that identify the source organism of a larger genomic frag-
astrobiological analogs. Also, sulfate reduction and metha- ment and its phylogenetic position in addition to its function
nogenesis in the Guaymas sediments are ultimately fuelled (Bejaet ai, 2000).
by high sedimentation of terrestrial organic matter and
upper water column primary production; in other words, Acknowledgments
they depend on products of the oxygenated, photosynthetic This study was supported by NASA Astrobiology Insti-
biosphere. tute "Environmental Genomes." Sampling at the Guaymas
hydrothermal vents was supported by NSF (LExEN pro-
Potential of Conserved Functional Genes for Genomics gram, Grant OCR 9714195).
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