archaeal communities in a tropical estuarine ecosystem: guanabara bay, brazil

MicrobialEcology

Archaeal Communities in a Tropical Estuarine Ecosystem:Guanabara Bay, Brazil

Ricardo P. Vieira1,2, Maysa M. Clementino3, Alexander M. Cardoso2, Denise N. Oliveira4,Rodolpho M. Albano4, Alessandra M. Gonzalez1, Rodolfo Paranhos1 and Orlando B. Martins2

(1) Instituto de Biologia, Departamento de Biologia Marinha, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil(2) Instituto de Bioquımica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil(3) Instituto Nacional de Controle da Qualidade em Saude—INCQS/FIOCRUZ, Rio de Janeiro, 21045-900, Brazil(4) Departamento de Bioquımica, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 20551-013, Brazil

Received: 25 January 2007 / Accepted: 18 April 2007 / Online publication: 26 June 2007

Abstract

Guanabara Bay is an eutrophic estuarine system located ina humid tropical region surrounded by the second largestmetropolitan area of Brazil. This study explores thecontrasting environmental chemistry and microbiologicalparameters that influence the archaeaplankton diversity ina pollution gradient in Guanabara Bay ecosystem. Theenvironments sampled ranged from completely anoxicwaters in a polluted inner channel to the adjacent,relatively pristine, coastal Atlantic Ocean. Partial archaeal16S rDNA sequences in water samples were retrieved bypolymerase chain reaction (PCR) and analyzed using de-naturing gradient gel electrophoresis (DGGE), cloning,and sequencing. Sequences were subjected to phylogeneticand diversity analyses. Community structure of the free-living archaeal assemblages was different from that of theparticle-attached archaea according to DGGE. Genelibraries revealed that phylotype identification was consis-tent with environmental setting. Archaeal phylotypesfound in polluted anoxic waters and in more pristinewaters were closely related to organisms that havepreviously been found in these environments. However,inner bay archaea were related to organisms found in oil,industrial wastes, and sewage, implying that water pollu-tion controls archaea communities in this system. Thedetection of a substantial number of uncultured phylo-types suggests that Guanabara Bay harbors a pool of novelarchaeaplankton taxa.

Introduction

Estuaries present strong chemical and biological gra-dients established by the mixing of continental watersand coastal seawater. In urbanized areas, environmentalimpacts such as industrial and domestic pollution areadded by human interference. Estuarine habitats canhost a great amount of biological diversity because oftheir position at the interface between freshwater andmarine environments [22, 23]. Archaeal communitycomposition along estuaries has been shown to beaffected by distinct environmental conditions, includingphysical and chemical factors, as well as biotic conditions[12, 46]. Guanabara Bay, which is surrounded by a largecity, can be viewed as a model of tropical impactedecosystems representative of many coastal metropolitanareas of the world.

The application of genomic techniques to elucidateissues in environmental microbiology is significantlyexpanding our understanding about marine microbialdiversity, evolution, metabolism, and ecology [5, 6, 14].Microbial genomic plasticity determines the relativefitness of ecotypes in response to key abiotic variables,and hence regulates their distribution, activity, andabundance in nature. Correlation of prokaryotic genomicvariation with ecological parameters has been demon-strated in marine ecosystems [14]. The study of compo-sition and function of archaeal communities is crucial forunderstanding local and global biogeochemical processes.The 16S rDNA gene libraries combined with polymerasechain reaction-denaturing gradient gel electrophoresis(PCR-DGGE) analysis has provided valuable qualitativeand quantitative pictures of archaeal diversity that allowcomparison of communities in different habitats [8, 18,

Correspondence to: Alexander M. Cardoso; E-mail: [email protected]

DOI: 10.1007/s00248-007-9261-y & Volume 54, 460–468 (2007) & * Springer Science + Business Media, LLC 2007460

32, 35, 36]. Based on quantitative molecular surveys ofseveral coastal and deep ocean waters, evidence forplanktonic archaea has been reported [13, 17].

Guanabara Bay is an extremely dynamic ecosystemlocated at the interface between freshwater and adjacentcoastal ocean waters [33]. Spatial and temporal salinityand pollution gradients found in such estuarine environ-ments are caused by the variability of several factors suchas currents, winds, and tides, as well as anthropogenicinputs. Diversity of archaea communities along estuaries islikely to be affected by these variable environmentalconditions [12, 46]. In this study, we characterizedarchaeaplankton diversity in four representative habitatswithin and near Guanabara Bay. Samples were takenalong a gradient of pollution going from the innerchannel (CM), which receives heavily polluted waste waterat Fundao Island, characterized by oil and sewage-pollutedwaters [7, 34], to relatively pristine seawater, 5 km off thebay’s entrance, in the adjacent Atlantic Ocean. Using amolecular approach, we showed that archaeal communi-ties are composed of different taxa and also discuss howthe spatial distribution of these populations may belinked to nutrient and pollution levels.

Methods

Sampling and Site Characterization. Surface watersamples (1-m depth) were collected on board the AstroGaroupa oceanographic vessel using a Niskin bottle (10 L),in four different sites, during high tide on July 10, 2003.The chosen sites represented of moderately pollutedwaters inside Guanabara Bay (B1), waters at the bay’sentrance (IL), and pristine seawaters 5 km offshore in thecoastal Atlantic Ocean (5K). Another sample was takenfrom an enclosed inner channel, Mare channel (CM),alongside Fundao Island, which receives industrial wasteand raw domestic sewage from Cunha River (Fig. 1A). Tocharacterize sampling sites, microbiological and chemicalparameters were determined according to standardoceanographic methods [19, 38].

Microbiological Parameters. Microbial abundance(MA) was determined by flow cytometry (CyAn ADPflow cytometer [Dako]) after nucleic acid staining with2.5 mM syto13 fluorochrome in samples fixed with 2%paraformaldehyde [3]. Microbial production (MP) wasanalyzed by 3H-leucine incorporation technique [28].Specific production (SP) was calculated as the ratio MP/MA.

DNA Extraction. We sampled superficial water (1-mdepth) in the same four sites described above (CM, B1, IL,and 5K). Water was filtered with a 3-mm filter, whichcaptures colonial microbes, particle-attached archaea, andalso archaeal symbionts from phytoplankton andzooplankton organisms that are abundant in tropical

coastal waters. The free-living planktonic microbes wereconcentrated on a Sterivex-filter (0.22 mm). DNA wasprepared by standard methods [43]. Briefly, 50 mL offreshly prepared lysozyme (1 mg/mL) was added to filterunits containing lysis buffer (0.75 M sucrose, 20 mMethylenediamine tetraacetic acid (EDTA), 50 mM Tris–HCl[pH 8.0]), and the units were incubated at 37-C for 45 min.Then, 50 mL of freshly prepared proteinase K (0.2 mg/mL)and 200 mL of 10% sodium dodecyl sulfate (SDS) wereadded, and incubated at 55-C for 1 h. Lysates wereremoved with sterile 3-mL syringes, and the filter unitswere each rinsed with 1 mL of lysis buffer and incubatedfor 15 min. The rinse buffer and lysates were pooled. Crudelysates were extracted once with phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0) and once withchloroform-isoamyl alcohol (24:1). The nucleic acids inthe aqueous phase were precipitated overnight with twovolumes of ethanol at _20-C, centrifuged, washed with70% ethanol, dried, and then dissolved in 100 mL TE (10 mMTris–HCl, 0.1 mM EDTA, pH 8.0). DNAs were furtherpurified by a low-melting agarose gel procedure andquantified in a 1% agarose gel stained with SYBR green(FMC Bioproducts, Rockland, ME, USA). Afterelectrophoresis the gel was digitalized in a Storm ImageScanner (GE Healthcare, Little Chalfont, UK).

Denatu r ing Grad ien t Ge l E lec t rophores is

(DGGE). Polymerase chain reaction products ofpartial 16S rDNA regions from environmental sampleswere analyzed by DGGE as described previously [35]. AGC clamp was added to archaea universal primer 1100Afc (5¶-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGC C C C C G C C A A C C G T C G A C A G T C A G G Y A A CGAGCGAG-3¶) and PCR was performed with 1400 Ar(5¶-CGGCGAAT TCGTGCAAGGAGCAG GGAC-3¶)[30]. PCR was run in 50-mL reaction mixtures (2.5 mMMgCl, 0.2 mM diethylnitrophenyl thiophosphates(dNTPs), 50 pmol of each primer, 2.5 U of Platinum TaqDNA polymerase, and PCR buffer (Invitrogen, Carlsbad,CA, USA) and 1 mL of each DNA sample. DGGE was runin a D-Code system (BioRad, Richmond, CA, USA). PCRsamples were applied directly onto 6% (w/v) poly-acrylamide gels in 1�TAE, with denaturing gradientfrom 20 to 60% (where 100% of denaturant buffercontains 7 M urea and 40% formamide). Electrophoresiswas performed at constant voltage (200 V) at 60-C for 18 h.After electrophoresis, the gel was incubated for 15 min inSYBR green (FMC Bioproducts, Rockland, ME, USA), andthe image was digitalized in a Storm Image Scanner (GEHealthcare, Little Chalfont, UK).

16S rDNA Gene Library Construction. Four 16SrDNA gene libraries were constructed from free-livingplanktonic archaea collected at distinct environments(CM, B1, IL, and 5K) to compare archaeaplankton

R.P. VIEIRA ET AL.: ARCHAEAPLANKTON COMMUNITY STRUCTURE 461

diversity. PCR was performed in 50-mL reaction mixtures(2.5 mM MgCl, 0.2 mM dNTPs, 50 pmol of each primer,2.5 U of Platinum Taq DNA polymerase, and PCR buffer(Invitrogen, Carlsbad, CA, USA) for 1 mL of each DNAsample, using the universal archaeal primer 21 F (5¶-TTCCGGTTGATCCYGCCGGA-3¶) [13] and universalprimer 907 R (5¶-CCGTCAATTCCTTTGAGTTT-3¶) [2].PCR amplification began with a 5-min denaturing stepat 94-C; followed by 30 cycles of 94-C for 1.5 min,50-C for 1.5 min, and 72-C for 2 min, and a finalextension cycle at 72-C for 10 min. PCR productswere purified with GFX PCR DNA and gel bandpurification kit in accordance with the manu-facturer’s instructions (GE Healthcare, Little Chalfont,UK). The PCR fragments (880 bp) were cloned intothe pGEM-T cloning vector (Promega) and used totransform E. coli DH10B competent cells. Ninety-sixpositive colonies from each transformation were pickedand stored at _70-C.

Sequence Analyses. Forty-eight clones from each16S library were randomly picked and submitted tosequence analysis. DNA from each clone was preparedby alkaline lysis and reads were obtained after sequencingreactions with vector primer (T7) by capillary elec-trophoresis on a MegaBace1000 using DYENamic dyeterminator cycle sequencing kit (GE Healthcare, LittleChalfont, UK). Chromatograms were transformed intoFasta format sequences with Phred software [15] andreads with less than 300 pb were removed. A total of 140valid reads with Phred score Q20 were compared withsequences in the Ribosomal Database Project II (RDP II).Chimeric sequences were identified and removed usingCHECK-CHIMERA [10]. Alignments with representativearchaeal sequences obtained at GenBank databases werecarried out using ClustalX program [44] to comparesequences. Partial 16S rDNA sequences generated in thisstudy have been deposited in the GenBank databaseunder accession numbers DQ913105 to DQ913239.

0

20

40

60

80

100

CM BI IL 5K

Am

monia

and

Tota

l Phosp

horo

us

(µM

)

0.0

0.5

1.0

1.5

2.0

Nitra

te (µ

M)

Tid

eA

N

S

EW

N

S

EW

0

2

4

6

8

CM BI IL 5K

Dis

solv

ed

Oxy

ge

n (m

L.L-1

)

Ch

loro

ph

yll a

(µg.

L-1)

0

10

20

30

40

50CB

SP

M (m

g.L-1)

Figure 1. (A) Guanabara Bay, its drainage basin ( ), metropolitan area of Rio de Janeiro ( ; enlarged map on the right) and samplingsites: CM, B1, IL, and 5K. (B) Dissolved oxygen (0), chlorophyll a (Ì), and suspended particulate matter—SPM (&). (C) Ammonia (Ì),nitrate (Í), and total phosphorous (&).

462 R.P. VIEIRA ET AL.: ARCHAEAPLANKTON COMMUNITY STRUCTURE

Diversity and Phylogenetic Analyses. Sequenceswere clustered as operational taxonomic units (OTUs) atan overlap percent identity cut-off of 75% and 97% usingCAP3 program [24]. The diversity of archaea phylotypeswas further examined using rarefaction analysis [21, 25].Phylogenetic trees were constructed by the neighbor-joining method [41] based on distance estimatescalculated by Kimura-2 algorithm [27]. Treeconstruction was performed with MEGA2 programversion 2.1 [31], and bootstrap analysis was performedwith 1000 replications.

Results

Environmental Chemistry. To address the question ofhow archaeaplankton diversity is linked to environmentaldata, superficial water was collected at four sites (Fig. 1A).Abiotic parameters were determined to monitor nutrientconcentration and chemical characteristics of each site inGuanabara Bay (Fig. 1B and C). Temperatures werenearly constant at all sites, varying from 21-C to 22-C,typical of winter estuarine conditions in Rio de Janeiro,latitude 23-S. Low salinity at CM reflects input ofdomestic sewage and waste-polluted freshwater fromCunha River. Moderate amounts of dissolved oxygenwere measured at B1 station, whereas high levels ofdissolved oxygen were detected in the pristine seawater(5K) off the bay entrance (Fig. 1B).

Analyses of nitrogenated compounds showed thehighest concentration of ammonia in CM, whereas inmore pristine seawater (IL and 5K) the main nitrogenouscompound was nitrate (Fig. 1C). These data support theoccurrence of a nitrification process along Guanabaraestuarine waters [37]. Ammonia-oxidizing processes arelargely mediated by bacteria, whereas archaea are nowrecognized as important agents of the marine nitrogenbiogeochemical cycle [29]. Phosphates were present athigh concentrations at CM and decreased progressivelyto much lower levels in more pristine seawater (Fig. 1C).Suspended particulate matter (SPM) was present atelevated values in the heavily polluted CM and inmoderately polluted B1 stations (Fig. 1B). SPM is knownas a hotspot because particles function as sites of intenseheterotrophic metabolism as a result of the presence ofdegradative exoenzymes secreted by numerous attachedmicroorganisms [4]. Measured chlorophyll was mainlyfrom phytoplankton origin, and also decreased frominner to outer bay waters (Fig. 1B).

Microbiological Parameters. Microbial abundance(MA), production (MP), and specific production (SP)were evaluated to characterize the four representativesites: CM, B1, IL, and 5K (Table 1). Prokaryotic countsvaried from 1.05�105 cells mL-1 in pristine coastalseawater (5K) to 3.36�107 cells mL-1 in heavily polluted

CM. Prokaryotic productivity varied from 80 ng C L-1 h-1

in pristine water to 8759 ng C L-1 h-1 in more pollutedwaters. We observed higher specific productivity (SP) inB1 station, probably caused by abundant availableorganic matter, nutrients, and dissolved oxygen. HigherSPM and chlorophyll values observed in B1 station alsosupport the very high SP in the moderately oxygenatedand nutrient-rich inner bay waters [23]. Stations IL and5K presented moderate SP and a better water quality.Although a high level of SPM suggests intensemetabolism, the lower SP observed in CM indicates lowmicrobial metabolic activity, probably caused byfermentative metabolism imposed to active microbes inthis anoxic environment.

Denaturing Gradient Gel Electrophoresis (DGGE)

Analysis of Free-living vs Particle-attached Archaea. Themain purposes of this experiment were to comparearchaeaplankton populations among the four representativesites (CM, B1, IL, and 5K), and to compare free-livingarchaea separated by filtration with those attached toparticulate material, symbionts, or aggregated colonialphylotypes [12]. The free-living picoplanktonic communitywas concentrated in a Sterivex unity, and environmentalgenomic DNA was prepared. After PCR amplification witharchaeal-specific clamped primers, the fragments of about300 bp were submitted to DGGE analysis to comparephylotype richness in the four distinct sites (Fig. 2). First,we observed different band patterns between communitieswhen the four sites were compared. Second, there was ahigher number of distinct bands for particle-attachedarchaea in each station. These results suggest a lowerdiversity of free-living archaea and a higher diversity ofdifferent phylotypes within particle-attached communities.Despite repeated efforts to produce a clear DGGE bandpattern for the particle-attached CM sample, we could notget resolved bands. Perhaps, this could be because of somecarried-over inhibitors coming from degraded organicmatter that abounds in this environment.

C o v e r a g e a n d D i v e r s i t y o f F r e e - l i v i n g

Archaeaplankton. To shed further light on free-livingmicrobial community structure, we constructed 16SrDNA libraries to map and compare archaea diversity inGuanabara Bay planktonic populations collected at CM,B1, IL, and 5K. Forty-eight clones from each library were

Table 1. Microbiological parameters of Guanabara Bay sites

Site CM B1 IL 5KaMA 3.36�107 2.34�106 1.16�106 1.05�105

bMP 8758.72 2804.56 727.77 79.89cSP 260 1198 627 760

aMicrobiol abundance (cells, mL_1)

bMicrobiol production (ng C L_1 h

_1)cSpecific production (ag C cell

_1 h_1), calculated as the ratio MP/MA


sequenced. Chimeric sequences were identified andremoved using the CHECK-CHIMERA software at RDPII http://rdp.cme.msu.edu) [10]. The valid reads withphred score Q20 were used for database query with onlineBLAST search in GenBank http://www.ncbi.nim. nih.gov).

To determine the relationships between archaealdiversity and taxons (OTUs and phylotypes), we per-formed clusterization experiments at two different strin-gency levels using Cap3 and rarefaction analysis software(Fig. 3). The purpose of these in silico experiments was tocompare archaea diversity at groups and species levelsand to determine the coverage of each library in distinctestuarine environments. Clusterization at low stringency(75%) created larger OTUs with many archaeal phylo-types and few singlets. Rarefaction analysis at lowstringency indicated that the four libraries were largeenough to give reliable coverage of OTUs. All rarefactioncurves reached a plateau, indicating that sequencedclones represented the main archaeal taxons found inGuanabara Bay ecosystem (Fig. 3A). At 97% identity,rarefaction curves suggest higher diversity of archaealspecies in more polluted sites (Fig. 3B). We observedhigh microdiversity [1, 16] at species level in thehypereutrophic environments (CM and B1), whereas inrelatively pristine seawater (IL and 5K) rarefaction curvessuggested lower species diversity. This probably occurs

because CM and B1 stations are located in regions thatduring high tide present great water mixture. Thus, theinterface between waste-polluted freshwater and coastalmarine waters in Guanabara Bay estuary has revealedremarkably high archaeaplankton species diversity.

Phylogenetic Analysis of Guanabara Bay 16SrDNA Gene Libraries. Estuarine microbial com-munities are composed of local organisms and thoseoriginating from other ecosystems such as fluvial, oceanic,and even terrestrial habitats [12, 46]. Overall, among 134valid reads, most of our sequences were affiliated toenvironmental uncultured archaeal species and only fewsequences were closely related to cultivated phylotypes.Many OTUs from Guanabara Bay were characteristic ofparticular environments. Members of thermoplasmatalesand methanogenic archaeon groups, characteristic of anaer-obic habitats, were dominant in CM. Typical estuarineeuryarchaeon were predominated in B1 and in outer baylibraries (IL and 5K) cosmopolitan marine euryarchaeota

0 10 20 30 40 500.0

2.5

5.0

7.5

10.0

B1

5k

IL

CM

Clones

OTU

s

0 10 20 30 40 500

5

10

15

20

25

CM B1

IL

5K

Clones

Phy

loty

pes

A

B

Figure 3. Rarefaction curves from different environments (CM,B1, IL, and 5K). Clusterization at (A) 75% or (B) 97% identity.

Figure 2. DGGE analysis from archaeaplankton communitiesobtained at CM, B1, IL, and 5K stations. Arrows indicates free-living phylotypes at the left side and particle-attached at the right side.


http://rdp.cme.msu.edu

http://www.ncbi.nim.nih.gov

E N V I R O N M E N T A L

Uncultured crenarchaeote AF223114 Crenarchaeon from coast Africa ocean AY225426

5K OTU 3 (7 clones) DQ913221

Archaeon from symbiotic communities in marine sponges AY192627

B1 OTU 6 (1 clone) DQ913131 B1 OTU 7 (1 clone) DQ913136

Crenarchaeote from marine sediment AJ870955

IL OTU 7 (1 clone) DQ913179 Picrophilus torridus AE017261

Thermoplasma volcanium AJ299215 Thermoplasma acidophilum M38637 Acidianus brierleyi X90477

Sulfolobus solfataricus X20478 Sulfolobus tokodaii AB022438

Aeropyrum pernix D83259

Hyperthermus butylicus X99553 Pyrolobus fumarii X99555

100

100

93 97

91

59

100

55

100

66

96

67

0.05

A

C U L T U R E D

Marine archaeon AF121990


IL OTU 2 (10 clones) DQ913173

Archaeon from coast Africa ocean AY225435

B1 OTU 1 (3 clones) DQ913130 Archaeon from deep-sea hydrothermal vent water AB193997




Marine coastal archaeon M88078

Marine coastal archaeon M88077


Euryarchaeote from surface microlayer of corals AY380725

B1 OTU 2 (12 clones) DQ913132

IL OTU 4 (4 clones) DQ913177 Euryarchaeote from surface microlayer of corals AY380646

Marine euryarchaeote AF257277 IL OTU 6 (1 clone) DQ913182



Euryarchaeota from Virgin Islands corals AY380717

CM OTU 5 (2 clones) DQ913127

Thermoplasmatales archaeon AY323220

Marine archaeon from southern Aegean Sea AF290535 5K OTU 6 (1 clone) DQ913217

5K OTU 7 (1 clone) DQ913234

Methanosarcina barkeri M59144

Methanosarcina mazei AB065296

Methanosarcina acetivorans M59137

Methanococcoides burtonii X65537 CM OUT 1 (3 clones) DQ913105

Methanocorpusculum parvum M59147

CM OUT 4 (7 clones) DQ913111

Toluene-degrading methanogenic archaeon AF423187


Euryarchaeote from plankton of a boreal forest lake AJ131268 Methanospirillum hungatei M60880

Methanogenium sp. AY177815

Methanoplanus petrolearius U76631

CM OTU 6 (1 clone) DQ913129

Archaeon from anoxic sediment AF142979

CM OTU 3 (10 clones) DQ913125


Halobacterium salinarum AJ496185

Halorubrum lacusprofundi U17365

Haloferax volcanii AB074566

Haloarcula marismortui AF034619

Halobaculum gomorrense L37444

Methanothermobacter thermautotrophicus Z3715655 Methanosphaera stadtmanae M59139

B1 OUT 5 (1 clone) DQ913137

Uncultured euryarchaeote AJ937874

CM OUT 2 (2 clones) DQ913112

Archaeon from deep-sea hydrothermal vent water AB019752 100

83 100

100

100

100

68

100

61 100

99

81

78

96

100

95

100

73

98

68

94

68

82

52

72

91 100

89 96

100

75 100

82

63

100

99

100

100

99

96

0.05

B

ME T H A N O G E N I C

M A R I N E

A R C H A E O N


and crenarchaeota species were found (Fig. 4). CM is con-nected with inner polluted bay waters and receives heavilypolluted freshwater from Cunha River [20]. This channelpresented the highest levels of environmental degradation,exemplified by anoxic conditions and high amounts ofammonia. Composition of archaea community in thishypereutrophic site is quite distinct from the others (B1,IL, and 5K), with several phylotypes related to organismsfrom methanogenic environments. At other areas of the bay,archaeal communities are also different: inner bay waters(B1) presented typical estuarine archaea groups and aeuryarchaeon affiliated to Methanoplanus petrolearius, isolat-ed from petroleum wells. In relatively oligotrophic waters(IL and 5K), uncultured ubiquitous marine euryarchaeotaand crenarchaeota phylotypes were identified. The majorestuarine resident group was the marine euryarchaeota frommoderately polluted inner bay water (B1), whereas themarine crenarchaeota was only present in pristine seawater(IL and 5K). Crenarchaeota identified in outer bay sitesare closely related to other environmental sequencesand distantly affiliated to cultivable hyperthermophiliccrenarchaeon (Fig. 4A). Euryarchaeota from IL and 5Kgrouped largely with an ubiquitous marine euryarch-aeon group, whereas inner bay sites presented manyspecies related to the methanosarcina group (Fig. 4B).

Discussion

Our results describe Guanabara Bay archaeaplanktoncommunity structure. The comparison between four 16SrDNA gene libraries from the distinct planktonic habitatsreported in this article reveals some archaea singularitiesin an urban estuarine ecosystem, such as the presence ofphylotypes associated to products of anthropogenicactivities, particularly domestic waste and oil pollution.This imposes an anaerobic metabolism to activemicrobes in these waters, creating a selective pressurethat could favor methanogenic archaea metabolic activ-ity. Abundance of species with hydrocarbon degradationphenotypes probably reflects microbial community adap-tations to periodic incidents with oil, fuels, and,sporadically, crude petroleum spillage in bay waters [7].Many archaeal phylotypes seem to be confined to specificgeographical regions or to environments that havesimilar geochemistry, whereas other marine groups seemto be widely distributed.

Many studies point to a high proportion of cren-archaeota in mesopelagic microbial communities [18, 26].It is noteworthy that a 16S gene library constructed in aprevious study from superficial Guanabara Bay watersrevealed 40% of crenarchaeotes [9] contrasting to 8%found in this investigation (Fig. 4A). Using BLASTsearches, we observed that the winter archaeal commu-nities described in this article are composed mainly ofcoastal planktonic phylotypes, whereas species related toammonia-oxidizing Nitrosopumilus maritimus [29] werefound in the summer [9]. The occurrence of a higherpercentage of pelagic phylotypes in superficial water inthe study of Clementino et al. [9] may be explained bythe active upwelling phenomenon that occurs in thisregion exclusively during the summer [45], when theirsamples were collected. This also points to a dynamicseasonal variation in Guanabara Bay estuarine microbialcommunities.

The phylogenetic tree reconstruction studies showedlow taxonomic consistency and polytomy within archaea.It is impossible to construct unique taxonomically consis-tent phylogenetic trees by grouping environmental eur-yarchaeota and crenarchaeota with cultured archaeasequences. Therefore, we constructed two separate phylo-genetic trees (Fig. 4A,B). We agree with previous sugges-tions that a phylogenetic review is needed in the Archaeadomain [40].

Furthermore, the approaches utilized in this workare important to obtain insights into the nature ofuncultivated phylotypes [42]. The discovery of newspecies may be helped by improved molecular ecologicalsearches, more sophisticated cultivation techniques, andby metagenomic approaches [11, 29, 39]. It is alsoimportant to address in this article the necessity of per-forming more studies on archaeal diversity in aquaticenvironments, specifically of tropical coasts, rivers, andlakes, where little is known about biogeography andecological characteristics of archaea.

Acknowledgments

We acknowledge Genome Sequencing facilities coreJohanna Dobereiner, IBqM/UFRJ, Limnology Laboratoryof UFRJ for the access to liquid scintillator and officersand crew of OV Astro Garoupa. We are grateful toPETROBRAS and Astromarıtima for kindly providingship time and fuel. We also thank Leonardo Cesar A. daSilva for his valuable help with Rarefaction analysisprogram. We are grateful to Fernando N. Pinto forsample collection and Guilherme R. S. Muricy formanuscript review. We also thank Laboratory of Ecologyand Taxonomy of Microorganisms IMPPG/UFRJ forDGGE facilities. This work was supported by a FAPERJgrant (150.121/04); RHAE—PUC; Pronex 171.177/03(CNPq/FAPERJ); Finep/CTPetro No. 21.01.0278.00.

R Figure 4. Neighbor-joining phylogenetic tree construction frompartial 16S rDNA sequences. One access number of each OTUis shown on the tree. Bootstrap values (1000 replicates) higherthan 50% are shown. Scale bar represents the 5% substitu-tion percentage. (A) Crenarchaeota and (B) Euryarchaeota.


References

1. Acinas, SG, Klepac-Ceraj, V, Hunt, DE, Pharino, C, Ceraj, I,Distel, DL, Polz, MF (2004) Fine-scale phylogenetic architecture ofa complex bacterial community. Nature 430: 551–554

2. Amann, RI, Stromley, J, Devereux, R, Key, R, Stahl, DA (1992)Molecular and microscopic identification of sulfate-reducingbacteria in multispecies biofilms. Appl Environ Microbiol 58:614–623

3. Andrade, L, Gonzalez, AM, Araujo, FV, Paranhos, R (2003) Flowcytometry assessment of bacterioplankton in tropical marineenvironments. J Microbiol Methods 55: 841–850

4. Azam, F, Long, RA (2001) Sea snow microcosms. Nature 29:497–498

5. Bano, N, Ruffin, S, Ransom, B, Hollibaugh, JT (2004) Phylogeneticcomposition of Arctic Ocean archaeal assemblages and comparisonwith Antarctic assemblages. Appl Environ Microbiol 70: 781–789

6. Beja, O, Koonin, EV, Aravind, L, Taylor, LT, Seitz, H, Stein, JLet al (2002) Comparative genomic analysis of archaeal genotypicvariants in a single population and in two different oceanicprovinces. Appl Environ Microbiol 68: 335–345

7. Brito, EM, Guyoneaud, R, Goni-Urriza, M, Ranchou-Peyruse, A,Verbaere, A, Crapez, MA, Wasserman, JC, Duran, R (2006)Characterization of hydrocarbonoclastic bacterial communitiesfrom mangrove sediments in Guanabara Bay Brazil. Res Microbiol57: 752–762

8. Casamayor, EO, Schafer, H, Baneras, L, Pedros-Alio, C, Muyzer, G(2000) Identification of and spatio-temporal differences betweenmicrobial assemblages from two neighboring sulfurous lakes:comparison by microscopy and denaturing gradient gel electro-phoresis. Appl Environ Microbiol 66: 499–508

9. Clementino, MM, Fernandes, CC, Vieira, RP, Cardoso, AM,Polycarpo, CR, Martins, OB (2007) Archaeal diversity in naturallyoccurring and impacted environments from a tropical region. JAppl Microbiol. doi: 10.1111/j.1365-2672.2006.03230.x (in press)

10. Cole, JR, Chai, B, Marsh, TL, Farris, RJ, Wang, Q, Kulam, SA et al(2003) The Ribosomal Database Project (RDP-II): previewing anew autoaligner that allows regular updates and the newprokaryotic taxonomy. Nucleic Acids Res 31: 442–443

11. Cottrell, MT, Waidner, LA, Yu, L, Kirchman, DL (2005) Bacterialdiversity of metagenomic and PCR libraries from the DelawareRiver. Environ Microbiol 7: 1883–1895

12. Crump, BC, Baross, JA (2000) Archaeaplankton in the ColumbiaRiver, its estuary and the adjacent coastal ocean USA. FEMSMicrobiol Ecol 1: 231–239

13. DeLong, EF (1992) Archaea in coastal marine environments. ProcNatl Acad Sci USA 12: 5685–5689

14. DeLong, EF, Karl, DM (2005) Genomic perspectives in microbialoceanography. Nature 15: 336–342

15. Ewing, B, Hillier, L, Wendl, M, Green P (1998) Base-calling ofautomated sequencer traces using phred. I. Accuracy assessment.Genome Res 8: 175–185

16. Fuhrman, JA, Campbell, L (1998) Marine ecology: microbialmicrodiversity. Nature 393: 410–411

17. Fuhrman, JA, McCallum, K, Davis, AA (1992) Novel majorarchaebacterial group from marine plankton. Nature 356: 148–149

18. Giovannoni, SJ, Stingl, U (2005) Molecular diversity and ecologyof microbial plankton. Nature 15: 343–348

19. Grasshoff, K, Kremling, K, Erhardt, M (1999) Methods of seawateranalysis, 3rd edn. Wiley-VCH Verlag, Germany, 600 pp

20. Hagler, AN, Mendonca-Hagler, LC (1981) Yeast from marine andestuarine waters with different levels of pollution in the state ofRio de Janeiro Brazil. Appl Environ Microbiol 41: 173–178

21. Heck, Jr KL, van Belle, G, Simberloff, D (1975) Explicit calculationof the rarefaction diversity measurement and the determination ofsufficient sample size. Ecology 56: 1459–1461

22. Hewson, I, Fuhrman, JA (2004) Richness and diversity ofbacterioplankton species along an estuarine gradient in MoretonBay Australia. Appl Environ Microbiol 70: 3425–3433

23. Hoch, MP, Kirchman, DL (1993) Seasonal and inter-annualvariability in bacterial production and biomass in a temperateestuary. Mar Ecol Prog Ser 98: 283–295

24. Huang, X, Madan, A (1999) CAP3: a DNA sequence assemblyprogram. Genome Res 9: 868–877

25. Hurlbert, SH (1971) The nonconcept of species diversity: acritique and alternative parameters. Ecology 52: 577–586

26. Karner, MB, DeLong, EF, Karl, DM (2001) Archaeal dominance inthe mesopelagic zone of the Pacific Ocean. Nature 409: 507–510

27. Kimura, M (1980) A simple method for estimating evolutionaryrates of base substitutions through comparative studies ofnucleotide sequences. J Mol Evol 16: 111–120

28. Kirchman, DL, K’nees, E, Hodson, R (1985) Leucine incorpora-tion and its potential as a measure of protein synthesis bybacteria in natural aquatic systems. Appl Environ Microbiol 49:599–607

29. Konneke, M, Bernhard, AE, de la Torre, JR, Walker, CB,Waterbury, JB, Stah, DA (2005) Isolation of an autotrophicammonia-oxidizing marine archaeon. Nature 437: 543–546

30. Kudo, Y, Nakajima, T, Miyaki, T, Oyaizu, H (1997) Methanogenflora of paddy soils in Japan. FEMS Microbiol Ecol 22: 39–48

31. Kumar, S, Tamura, K, Jakobsen, IB, Nei, M (2001) MEGA2:molecular evolutionary genetics analysis software. Bioinformatics17: 1244–1245

32. Massana, R, DeLong, EF, Pedros-Alio, C (2000) A few cosmopol-itan phylotypes dominate planktonic archaeal assemblages inwidely different oceanic provinces. Appl Environ Microbiol 66:1777–1787

33. Mayr, LM, Tenenbaun, DR, Villac, MC, Paranhos, R, Nogueira, CR,Bonecker, SLC, Bonecker, AC (1989) Hydrobiological characteriza-tion of Guanabara Bay Coastlines of Brazil. American Society ofCivil Engineers 1: 124–139

34. Meniconi, MDG, Gabardo, IT, Carneiro, MER, Barbanti, SM, Cruzda Silva, G, Massone, CG (2002) Brazilian oil spills chemicalcharacterization. International Society of Environ Forensics 3:303–321

35. Muyzer, G, DeWaal, EC, Uitterlinden, AG (1993) Profiling ofcomplex microbial populations by denaturing chain reaction-amplified genes-coding for 16S Ribosomal-RNA. Appl EnvironMicrobiol 59: 695–700

36. Murray, AE, Preston, CM, Massana, R, Taylor, LT, Blakis, A, Wu, K,DeLong, EF (1998) Seasonal and spatial variability of bacterial andarchaeal assemblages in the coastal waters near Anvers IslandAntarctica. Appl Environ Microbiol 64: 2585–2595

37. Paranhos, R, Pereira, AP, Mayr, LM (1998) Diel variability ofwater quality in a tropical polluted bay. Environ Monit Assess 50:131–141

38. Parsons, TR, Maita, Y, Lalli, CM (1984) A manual of chemical andbiological methods for seawater analysis. Oxford Pergamon Press,173 pp

39. Rappe, MS, Giovannoni, SJ (2003) The uncultured microbialmajority. Annu Rev Microbiol 57: 369–394

40. Robertson, CE, Harris, JK, Spear, JR, Pace, NR (2005) Phyloge-netic diversity and ecology of environmental Archaea. Curr OpinMicrobiol 8: 638–642

41. Saitou, N, Nei, M (1987) The neighbour-joining method: a newmethod for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425


http://dx.doi.org/10.1111/j.1365-2672.2006.03230.x

42. Schleper, C, Jurgens, G, Jonuscheit, M (2005) Genomic studies ofuncultivated Archaea. Nat Rev Microbiol 3: 479–488

43. Somerville, CC, Knight, IT, Straube, WL, Colwell, RR (1989)Simple rapid method for direct isolation of nucleic acids fromaquatic environments. Appl Environ Microbiol 55: 548–554

44. Thompson, JD, Gibson, TJ, Plewniak, F, Jeanmougin, F, Higgins, DG(1997) The ClustalX windows interface: flexible strategies for multiple

sequence alignment aided by quality analysis tools. Nucleic Acids Res24: 4876–4882

45. Valentin, JL (1984) Analysis of hidrobiological parameters in theCabo Frio (Brazil) upwelling. Mar Biol 82: 259–276

46. Zeng, Y, Li, H, Jiao, N (2007) Phylogenetic diversity of planktonicarchaea in the estuarine region of East China Sea. Microbiol Res162: 26–36


archaeal communities in a tropical estuarine ecosystem: guanabara bay, brazil

Documents