Nitrogen Fixation Among MarineBacterioplankton

Kjärstin H. Boström

Department of Biology & Environmental ScienceUniversity of Kalmar

Sweden2006

AKADEMISK AVHANDLING

som för avläggande av Filosofie Doktorsexamen vid Naturvetenskapliga Fakulteten vid högskolan i Kalmarkommer att offentligt försvaras fredagen den 20 januari 2006

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Doctoral thesis 2006University of KalmarFaculty of Natural SciencesDissertation series No. 26

Kjärstin H. BoströmDepartment of Biology and Environmental ScienceUniversity of Kalmar, SE 391 82 Kalmar, Sweden

Supervisor:Dr. Lasse Riemann, Assistant ProfessorDepartment of Biology and Environmental ScienceUniversity of Kalmar, SE 391 82 Kalmar, Sweden

Opponent:Dr. Grieg Steward, Assistant ProfessorDepartment of OceanographyUniversity of Hawaii, Honolulu, HI 96822, USA

2006 Kjärstin H. BoströmISBN: 91-89584-52-X, ISSN: 1650-2779, pp. 1-26Printed by: Högskolans tryckeri, Kalmar

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TABLE OF CONTENTS

TABLE OF CONTENTS.............................................................................................................4

ABSTRACT...................................................................................................................................5

SVENSK SAMMANFATTNING ...............................................................................................6

LIST OF PUBLICATIONS.........................................................................................................8

INTRODUCTION ........................................................................................................................9

BACTERIOPLANKTON IN THE MARINE FOOD WEB ......................................................................9CULTIVATION AND BACTERIOPLANKTON DIVERSITY................................................................9BIOGEOCHEMICAL SETTING .....................................................................................................11NITROGEN.................................................................................................................................12

The nitrogen cycle...............................................................................................................12N2-fixation ...........................................................................................................................13Regulation of N2-fixation....................................................................................................14Methods to measure N2-fixation.........................................................................................15

AIMS OF THESIS......................................................................................................................16

THESIS OUTLINE ....................................................................................................................17

CONCLUDING REMARKS.....................................................................................................21

REFERENCES ...........................................................................................................................22

ACKNOWLEDGEMENTS.......................................................................................................26

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ABSTRACTWhile bacterioplankton indisputably control vital biogeochemical paths in the cycling ofcarbon and nutrients in the world’s oceans, our knowledge about the functional and geneticdiversity of bacterioplankton communities is negligible. In this thesis, molecular and moretraditional microbiological methods were used to study the specific function of N2-fixationand in a general sense diversity of marine bacterioplankton species.

Most oceans are nitrogen limited and, therefore, adaptive to bacterioplankton capable ofN2-fixation. Recent studies have found nifH genes (coding for the nitrogenase enzyme)related to diverse heterotrophic bacteria in oceanic seawater samples indicating that, alongwith cyanobacteria, also heterotrophic bacteria benefit from N2-fixation. Here, molecular andcultivation methods were used to examine diazotrophic bacterioplankton in the Baltic Sea.We successfully isolated heterotrophic N2-fixing bacteria belonging to the γ-proteobacterialclass by means of low-nitrogen plates and semi-solid diazotrophic medium tubes. Theisolates required low-O2 conditions for N2-fixation. Using Real-time PCR it was found thatheterotrophic bacterioplankton carrying the nifH gene was abundant (3 x 104 nifH genecopies L seawater-1) at locations in the Southwest Baltic proper.

With the aim to identify the main N2-fixing organisms in Baltic Proper surface waters, aclone library of nifH gene transcripts (RNA) was generated. Clone inserts were exclusivelyrelated to Aphanizomenon sp. and Nodularia sp. Using quantitative real-time PCR it wasfound that the nifH gene expression from Nodularia sp. was highly variable between stationsin the Baltic Proper but was 10-fold higher during mid summer relative to early summer andfall. A diel study showed a 4-fold increase in Nodularia transcript concentrations at early tomid day relative to rest of the day. Real-time PCR was found to be a powerful and highlysensitive method for measuring gene expression.

Since nucleic acids are a prerequisite for molecular analyses of bacterioplanktondynamics a protocol to extract DNA from seawater samples was developed with the aim tomaximize the yield of high-quality DNA. Each step in the protocol was important for theefficiency of extraction. The obtained extraction efficiencies were up to 92% for seawatersamples and up to 96% for isolates. The protocol provides a guideline for DNA extractionfrom seawater samples for other studies.

In a global sampling campaign (9 locations from polar, tropical and temperate regions)we sampled DNA from surface water and constructed 16S rRNA gene libraries to investigatediversity and biogeography of bacterioplankton. Approx. 80% of the sequences found weresimilar to sequences already deposited in GenBank, indicating that a large fraction of themarine bacterioplankton already has been sampled, which in turn suggests a limited globalbacterioplankton diversity.

This thesis have improved our knowledge about the composition and nifH geneexpression of the diazotrophic bacterioplankton community in the Baltic Sea and contributesignificantly to the discussion on global marine bacterioplankton diversity and biogeography.

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SVENSK SAMMANFATTNING

Östersjön är ett av världens största brackvattensystem. Den ekologiska balansen i detta hav ärhotad på grund av övergödning. Mycket arbete har därför fokuserats på att reducerautsläppen av näringsämnen, speciellt kväve. Dessa ansträngningar kan dock motverkas avbakterier som har förmåga att omvandla luftens kväve till metaboliskt användbart ammonium(kvävefixering).

På sommaren är Östersjöns primärproduktion begränsad av kväve, med följden att detårligen uppstår massiva blomningar av kvävefixerande bakterier, framför allt cyanobakterier.Dessa är främst Aphanizomenon och Nodularia, men inte endast de fototrofa cyano-bakterierna har förutsättningar att fixera N2. NifH gener (genen som kodar för nitrogenas)bärs också av heterotrofa bakterioplankton, vilket har visats i studier i främst Atlanten ochStilla havet. Med hjälp av två olika odlingsmetoder lyckades vi isolera heterotrofakvävefixerande bakterier tillhörande klassen γ-proteobakteria från Östersjön. Svårighetenmed att finna dessa bakterier ligger i att de kräver en miljö med mycket låg syrehalt för attkunna fixera kväve.

Resultaten från denna studie ledde oss vidare till att undersöka vilka organismer somuttrycker nifH genen (och då troligen även fixerar kväve) i Östersjön. En av de bakterier somisolerats kunde påvisas med Realtids PCR i ett relativt stort antal (3 x 104 nifH genkopior perliter) vid en av de ursprungliga provtagningsstationerna. För att söka rätt på de olikaorganismtyper som uttrycker nifH skapades ett klonbibliotek baserat på mRNA extraheratfrån havsvatten. Det visade sig då att alla de närmare 100 kloner som sekvenserades tillhördeantingen Aphanizominon eller Nodularia. De heterotrofa bakteriernas nifH genuttryck vartroligen i jämförelse med dessa cyanobakterier alltför lågt för att kunna detekteras. RealtidsPCR mätningar av Nodularias nifH genuttryck visade på en stor variation mellan de olikaprovtagningsstationerna samt mellan de olika provtagningstillfällena. Vi fann dock en kraftigökning under juli med en nedgång igen i augusti. En dygnscykelstudie visade att NodularianifH genuttrycket ökade under förmiddagen med en topp mitt på dagen för att sedan minskaigen. Detta troligen med anledning av att den energikrävande kvävefixeringsprocessen skerunder de ljusa timmarna då cellen får energi från fotosyntesen.

I de molekylärbiologiska metoderna som används för att få information om identitet ochaktivitet hos skilda organismer krävs att DNA och RNA kan extraheras från prover tagna inaturliga vattenmiljöer. Även om antalet bakterier tillsynes är högt, så är mängden DNA ochRNA per liter havsvatten relativt låg, därför krävs ett väl fungerande protokoll för dennaextraktion. I en inledande studie i denna avhandling optimerades en metod för att utvinnaDNA. Ett antal sådana protokoll finns publicerade men dessa har ofta lågt utbyte. Det nyaprotokollet har hög effektivitet, vilket gör att små provvolymer kan användas (2 ml jämförtmed tidigare flera liter) och därmed ökar hanterbarheten. Vi visar i denna studie att varje steg

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i DNA-extraktionsprotokollet är viktigt för att ge en hög effektivitet. Detta protokoll kan medfördel användas som vägledning för många olika typer av studier.

På grund av att många havsbakterier inte kan bilda kolonier och alltså inte växa påtraditionella medier har det varit svårt att få en klar bild av artrikedomen. Molekylärbiologinhar dock gjort det möjligt att identifiera bakterier med hjälp av 16S rRNA genen, en enormmängd gensekvenser från världens alla hav har inkommit till den gemensamma databanken(GenBank). År 2002 gjordes en studie där man sammanställde informationen i dennadatabank, för att få en bild av artrikedomen i världshaven. Resultatet av denna studie var attdet i världshaven fanns färre bakterietyper än vad många forskare har spekulerat i. I dennaavhandlig har vi utfört en studie där vi gjorde en stor global provtagning för att se om dennaundersökning överensstämde med den datainformativa. Provtagning från nio lokalitetergjordes i de tempererade, tropiska och polarhaven. Ett genbibliotek från varje lokal gjordesoch kloner sekvenserades. Resultatet visar i likhet med den datainformativa undersökningenpå en begränsad artrikedom. 80% av gensekvenserna fanns redan i databanken, vilket tyderpå att de flesta arter redan har blivit funna. Dessutom visade det sig att få av bakteriernaåterfanns på alla ställen och många återfanns endast på ett ställe. Utöver detta visade det sigatt det fanns en ökad artrikedom ju närmare ekvatorn man kom, vilket tidigare har visats förstörre organismer.

Studierna i denna avhandling har ökat förståelsen för hur sammansättningen av detkvävefixerande bakteriesamhället i Östersjön ser ut samt bidragit till diskussionen om denglobala artrikedomen bland bakterioplakton och dess utbredning.

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LIST OF PUBLICATIONS

This thesis is based on the following papers that will be referred to in the text by romannumerals I-IV.

I Boström, K.H., Simu, K., Hagström, Å., Riemann, L. (2004) Optimization of DNAextraction for quantitative marine bacterioplankton community analysis. Limnologyand Oceanography: Methods 2: 365-373.

I I Boström, K.H., Riemann, L., Kühl, M., Hagström, Å. Isolation and genequantification of heterotrophic N2-fixing bacterioplankton in the Baltic Sea.Submitted.

III Boström, K.H., Riemann, L., Zweifel, U.L., Hagström, Å. Distribution of N2-fixation in the Baltic Sea. Submitted.

IV Pommier, T., Canbäck, B., Riemann, L., Boström, K.H., Simu, K., Lundberg, P.,Tunlid, A., Hagström, Å. Global sampling of marine bacterioplankton revealslatitudinal gradients of diversity and limited cosmopolitan distribution. Submitted.

Paper I is reproduced with the permission from the American Society of Limnology andOceanography, Inc.

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INTRODUCTION

Bacterioplankton in the marine food webThe oceans cover about 71% of the

planet surface and contain both the largestand the smallest organisms on Earth. Thirty-five years ago, free-living minusculeheterotrophic bacteria in the ocean werelargely ignored and thought to play a minorecological role (Khailov & Finenko 1970).However, with the development of newmethods for counting bacteria, based onfilters allowing efficient size fractionationand fluorescent staining (Zimmermann &Meyer-Reil 1974), it was realized thatmarine bacterioplankton are highlyabundant; on average 1 x 106 ml-1 (Hobbie etal. 1977, Porter & Feig 1980). Thus, thebiogeochemical importance of heterotrophicbacteria in the sea was established. Yet, theiridentity was almost completely unknownand has gradually been revealed over the lasttwo decades primarily through molecularbased phylogeny (Rappé & Giovannoni2003).

Cultivation and bacterioplanktondiversity

Application of molecular methods inmicrobial ecology throughout the lastdecades has produced a tremendous amountof DNA sequences from marine bacteria.The ribosomal 16S RNA gene is the mostwidely used gene for phylogenetic studiesdue to its high degree of functionalconsistency and occurrence in all organisms(Woese 1987). Currently, the known marinebacterial diversity, based on the 16S rDNAsequences available in GenBank, is

comprised of 52 phyla of which 26 still haveno representatives in culture (Rappé &Giovannoni 2003; Fig. 1). Among marinebacterioplankton available in culture theclass γ-proteobacteria are the most common;including genera such as Vibrionaceae,Pseudomonas, Pseudoalteromonas, andShewanella. The second most common classis the α-proteobacteria with genera such asRoseobacter and Sphingomonas. In contrastto the cultured isolates, clone libraries ofPCR amplified community DNA frommarine waters are dominated by α- ratherthan γ-proteobacteria (Hagström et al. 2000,Moeseneder et al. 2005, Giovannoni &Rappé 2000).

Currently there are contrasting views onthe magnitude of marine bacterioplanktondiversity. In a recent shotgun sequencing-based effort to characterize diversity ofSargasso Sea bacterioplankton, Venter et al.(2004) identified 1164 distinct 16S rRNAgenes including 148 previously unknownbacterial species. When estimating diversityusing models based on depth of coverage asubstantial oceanic microbial diversity (upto 47,733 ribotypes) was suggested. Incontrast, by evaluating the number ofdifferent ribotypes and rate of new entriesinto GenBank, Hagström et al. (2002)estimated the total number of bacterialribotypes in the marine plankton to ~1100.In a recent update, this was increased to~2300 ribotypes (Pommier et al. 2005),which is still fairly low when consideringthe large depositions in GenBank in theintervening period (e.g. Venter et al. 2004).Since barriers to migration and dispersal areineffective in the continuous global ocean,

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Fig. 1. Schematic illustration of the phylogeny of the major plankton clades. Black letters indicate microbialgroups that seem ubiquitous in seawater. Red indicates groups found in the photic zone. Blue indicates groupsconfined to the mesopelagic and surface waters during polar winters. Green indicates microbial groups associatedwith coastal ocean ecosystems (redrawn from Giovannoni & Stingl 2005).

there is a strong tendency towards cosmo-politanism of microorganisms supportingthe hypothesis of a rather low diversity(Finlay 1998). Thus there is still controversyaround the magnitude of the diversity in thebacterial communities in the world’s oceans.

While some bacterial species/groups arewidespread, only few are known to have aworldwide distribution. Two of these belongto the cyanobacteria (Synechococcus andProchlorococcus; Carr & Mann 1994),while the most abundant bacteria in theworld’s oceans belong to the ubiquitous, andrecently cultivated, SAR11 cluster(Giovannoni et al. 1990, Rappé et al. 2002).To cultivate SAR 11, Rappé et al (2002)

used a dilution to extinction approach firstintroduced by Button et al. (1993), wherecommunity bacterioplankton samples arediluted in sterile seawater medium down to asingle bacterium and thereafter incubated forseveral weeks. It turned out that growth bythese previously uncultured bacteria issomehow hampered by nutrient-richconditions and that they propagateindependently rather than associated ingroups. Simu & Hagström (2004) suggestedthat the strategy of these bacteria might be toactively prevent colony formation since in alow-nutrient environment, such as the ocean,it might be advantageous to avoid thecompetition for nutrients implicit in colony

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Fig. 2. Conceptual drawing of the marine food web (Nielsen & Hansen 1999). Heterotrophic organisms areshown to the left, photosynthesizing organisms to the right, and mixotrophs in the middle. Most organisms andprocesses generate dissolved organic carbon (DOC) that are taken up by bacteria.

growth. Several recent studies, applyingmodified versions of the original dilution-to-extinction technique, have reported thecultivation of many previously unculturedmarine bacteria (Connon & Giovannoni2002, Page et al. 2004, Simu et al. 2005)emphasizing a large future potential for this

cultivation method.

Biogeochemical settingThe detection of large numbers of

bacterioplankton and estimates of highbacterial production rates (Hagström et al.1979, Fuhrman & Azam 1982) appearing in

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the beginning of the 1980s, implied theimportance of bacterioplankton in themarine carbon and nutrient cycling. Theformulation of a microbial loop wastherefore an important conceptual step in therestructuring of the marine food webcompared to the views of the 1970´s (Azamet al. 1983; Fig. 2). In the resultingbiogeochemical models up to 50% of thecarbon produced by planktonicphotosynthesis passes through thebacterioplankton community (Cole et al.1988). However, it should be noted thatwhile nutrient cycling comprise bothmineral salts and organic components thecurrent understanding of the circulation ofmatter in the marine environment isprimarily based on the flow of carbon. Theobvious reason for the choice of carbon liesin the advent of the radioisotopemeasurement of primary production(Steemann-Nielsen 1952). With carbon usedas a tracking element the flow of forinstance nitrogen and phosphorous in themarine environment has largely beencalculated as ratios in relation to the carbonflow. However, considering that the primaryproductivity in most oceans is nitrogenlimited (Howarth 1988), it is essential toobtain a detailed understanding of thefactors controlling the nitrogen cycle as wellidentifying the participating organisms.

Nitrogen

The nitrogen cycleThe fundamental steps of the nitrogen

cycle involve primarily microbiologicaltransformations between elemental nitrogen(gas), inorganic and organic nitrogen

(Arrigo 2005; Fig. 3). As bacteria and fungimineralize organic compounds in the questfor reduced carbon, ammonium (NH4

+) isreleased. This ion can be oxidized to nitrite(NO2

-, by ammonium oxidizers) and furtherto nitrate (NO3

-, by nitrite oxidizers) in thenitrification process. Phytoplankton andheterotrophic bacteria can directly assimilateNO3

- and NH4+, with ammonium as the

preferred source of nitrogen due to itsreduced state. In the absence of oxygen asthe terminal electron acceptor, NO3

- can bereduced in three steps to N2 gas bydenitrification. The anaerobic denitrificationwas long thought to be the only processremoving fixed nitrogen from theenvironment; however, recently it wasdiscovered that ammonium may be oxidizedanaerobically in sediments in the presence ofNO2

- in a process called anammox. Thispathway is thought to contributesignificantly to benthic N2 production(Thamdrup & Dalsgaard 2002). The loss ofnitrogen in the form of N2 gas and to someextent nitrous oxide (N2O) into theatmosphere is compensated for by N2-fixation, the enzyme-mediated processreducing N2 gas to ammonium:

NitrogenaseN2 + 8H+ + 8− + 16ATP → 2NH3 + H2 + 16ADP + 16Pi

Recent estimates of global ocean N2-fixation are in the range of 100-200 Tg Nyr-1, similar to terrestrial N2-fixation, butcarry large uncertainties (Karl et al. 2002).Since nitrogen often limits marine primaryproductivity and dissolved nitrogen gas isfreely available, N2-fixation appears to be

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Fig. 3. The marine nitrogen cycle. PON, particulate organic nitrogen, including phytoplankton; DON, dissolvedorganic nitrogen; DNRA, dissimilatory nitrate reductase to ammonium (redrawn from Arrigo 2005).

a key process for marine productivity(Capone 2000).

N2-fixationProkaryotes are the only organisms

capable of N2-fixation and, therefore,favored in the nitrogen limited oceans (Karlet al. 2002). The key enzyme involved inN2-fixation is nitrogenase. The high degreeof similarity of protein sequences ofnitrogenase among microorganisms suggestsan early origin or lateral gene transfer of thisgene complex among prokaryotic lineages(Postgate & Eady 1988 cited by Zehr et al.2003a). This may also explain whydiazotrophs (N2-fixers) are found amongsuch diverse groups of bacteria. Theseinclude representatives from:

• Heterotrophic anaerobic (e.g., Clostridium),

microaerophilic (e.g., Klebsiella), and aerobic

(e.g, Azotobacter) bacteria.

• Phototrophic bacteria (e.g., Rhodospirillum)

including cyanobacteria (filamentous, uni-

cellular, heterocystous, and non-hetero-

cystous).

• Chemolithoautotrophic bacteria (e.g., Thio-

bacillus).

• Archaea (e.g., Methanococcus).

The filamentous non-heterocystouscyanobacterium Trichodesmium has beenregarded as a main N2-fixer in the openocean (Carpenter & Romans 1991, Caponeet al. 1997) together with the diatom-associated endosymbiotic R i c h e l i a(Carpenter et al. 1999). Recently however,several studies have shown that unicellular

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cyanobacteria contribute significantly tooceanic N2-fixation (Zehr et al. 2001, Falcónet al. 2004, Montoya et al. 2004).Heterotrophic bacteria presumably able tofix nitrogen (carrying the nitrogenase gene)have also been found in various marinewaters (Zehr et al. 1995, Zehr et al. 1998,Short et al. 2004, Jenkins et al. 2004,Church et al. 2005a, Bird et al. 2005).Further, recent studies by Church et al.(2005b) and Zani et al. (2000) documentedthe expression of nitrogenase by hetero-trophic bacterioplankton. However, whetherunicellular cyanobacteria and heterotrophicbacterioplankton significantly contributewith fixed nitrogen to the nitrogen pool isstill unclear.

Regulation of N2-fixationThe cellular process of breaking the

triple bound in N2 is an energy demandingprocess and, therefore, highly regulated ontranscriptional, translational, and proteinactivity levels (Merrick & Edwards 1995).Nitrogenase, the enzyme responsible for N2-fixation, consists of the proteinsdinitrogenase and dinitrogenase reductase.The most extensively examined nitrogenasegene is the n i f H gene coding fordinitrogenase reductase, which is an iron(Fe) containing protein made up of twoequal subunits. The nifH gene is relativelyconserved and can therefore be used as aphylogenetic marker, although it is not asconserved as the 16S rRNA gene (Zehr et al.2003a). The current view of the diversity ofdiazotrophic marine bacterioplankton ismainly based on nifH gene sequences. Theother protein, dinitrogenase, harbors the

substrate-binding site where N2 is reduced toNH4

+ in the presence of a Fe-Molybdenum(Mo) cofactor. Dinitrogenase is composed oftwo hetero-dimers each consisting of twosubunits alpha and beta, encoded by nifKand nifD (Paerl & Zehr 2000). In addition tothese structural genes there are severalregulatory nif genes in the n i f operon(Collins & Brill 1985, Merrick & Edwards1995). At least 17 nif genes have beencharacterized in Klebsiella pneumoniae(Merrick 1983). Some diazotrophs have anadditional alternative nitrogenase usingvanadium (V) or Fe as cofactor instead ofMo. These different nitrogenases areencoded by separate sets of genes(vnfH/vnfDGK and anfHDGK, respectively;Bageshwar et al. 1998, Joerger et al. 1989).Nitrogenase nifH genes cluster into fourbasic groups (Zehr et al. 2003a):

Cluster I: The conventional Mo-containingnifH and some vnfH.

Cluster II: The anfH and nifH from someArchaea.

Cluster III: A diverse group, many that arestrictly anaerobes.

Cluster IV: A divergent, loosely coherentgroup with nifH-like sequences.

Nitrogenase is sensitive to oxygen andmay be inhibited reversibly (at low oxygenconcentrations) or irreversibly (at highoxygen concentrations) (Guerinot &Patriquin 1981, Urdaci et al. 1988). ManyN2-fixing bacteria are anaerobic orfacultative anaerobic to protect the oxygen-sensitive nitrogenase; however, some marinecyanobacteria have managed to protect the

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nitrogenase in oxygen-free, non-photo-synthesizing cells i.e. heterocysts (Paerl &Zehr 2000). These cells spatially separatethe oxygen-producing photosynthesis fromthe oxygen sensi t ive N2-fixation.Cyanobacteria with no heterocysts havefound other ways to protect the nitrogenasesuch as a temporal separation ofphotosynthesis (day) and N2-fixation (night)(Bergman et al. 1997, Church et al. 2005b).Trichodesmium , a multicellular cyano-bacterium, has an alternative strategy with adiel cycle with highest N2-fixation rateduring the day when also oxygenconcentration is highest due to photo-synthesis (Chen et al. 1998, Church et al.2005b, Bergman et al. 1997, Lin et al.1998). For protection of the nitrogenaseTrichodesmium has the N2-fixation allocatedto a few cells that do not photosynthesize(Berman-Frank et al. 2001). Fixing nitrogenduring the day is advantageous from anenergetic point of view as energy for theenergy demanding N2-fixation process isprovided from photosynthesis. Analternative way to protect the nitrogenase isto maintain a low intracellular oxygenconcentration through a high respiration rate(Bergman et al. 1997), which has also beendocumented in e.g. the well-studied aerobicsoil bacterium Azotobacter vinelandii (Postet al. 1983).

Methods to measure N2-fixationN2-fixation may be measured using

either direct or indirect methods. The stableisotope assay is an example of a directmethod whereby the incorporation of N2,artificially enriched with 15N2, into biomass

is measured (Paerl 1998). While this methodis sensitive and nonselective, it is rather timeconsuming, and requires elaborateequipment such as a mass spectrometer. Theacetylene reduction assay technique is amore widely used indirect method, whichmakes use of the capability of nitrogenasenot only to break the triple bond in N2 gasbut also the triple bond in acetylene. Addingacetylene to a sample to measure theresulting ethylene production with a gaschromatograph is an old and robust method,though with a limited sensitivity (Capone1993, Moisander et al. 2003). Due to thelimited sensitivity measurements are oftenperformed on concentrated plankton tows,which exclude unicellular organisms.Though, the sensitivity of the acetylenereduction method has lately beensignificantly improved (Staal et al. 2001),the use of concentrated samples seems still arequirement. In light of the recent findingsof genetic potential among unicellularcyanobacteria as well as heterotrophicbacteria, this size exclusion may havegenerated serious underestimates of N2-fixation.

While these methods have yieldedimportant information about N-flux andprovided our existing estimates of N2-fixation, they provide no insights into theidentity of the organisms behind the processsince they are applied on whole planktoncommunities. Hence, our current knowledgeabout species specific contribution to globalmarine N2-fixation is limited.

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AIMS OF THESIS

Following the introduction of molecular biology techniques into microbial ecology nifHsoon emerged as an exciting possibility to assay at the same time expression and identity of afunctional gene. To determine the spatial and temporal scales at which cyanobacteria andheterotrophic bacteria actively fix nitrogen and accurately quantify their importance relativeto anthropogenic inputs of nitrogen, stood out as a major challenge for my thesis. A mainpriority was to gain insight into the diversity of organisms fixing nitrogen, but also tocultivate them, and establish a coupling between laboratory-based analyses and in situfunction.

Though, still, knowledge on bacterioplankton diversity remained a prerequisite for athorough understanding of marine biogeochemical cycling of nitrogen and other elementsessential to life in the ocean. Subjects such as diversity and biogeography of marinebacterioplankton in general could not be excluded from discussions on the specific organismsresponsible for N2-fixation.

The specific aims of the present thesis were:

To develop and fine-tune methods to identify marinebacteria at the species level with particular emphasis onN2-fixers

To identify the main N2-fixing organisms in the BalticSea and in particular to determine the importance ofheterotrophic bacteria for planktonic N2-fixation

To use molecular expression to examine spatial andtemporal variability of nitrogenase activity for singlebacterial species

To increase the understanding of bacterioplanktondiversity and distribution in the global ocean.

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THESIS OUTLINE

Molecular-based methods have becomean integral part of microbial ecology. Mostof these methods rely on nucleic acidsextracted from environmental samples. Therelatively low number of organisms alongwith their limited DNA/RNA contents posesa serious problem for marine microbialecologist when extracting nucleic acids fromseawater. Today a variety of relativelyinefficient protocols for extraction ofbacterioplankton DNA/RNA are in use. InPaper I, we optimized and developed aprotocol to extract DNA from seawatersamples and obtained extraction efficienciesof up to 92% for seawater samples and up to96% for isolates. In most previous protocols,several liters of seawater had to be filtered torecover satisfactory DNA amounts;however, the high efficiency of this protocolallows for 2 ml samples to be centrifugedand extracted. Each step in the protocol wasimportant for the efficiency of extraction:e.g., a prolonged proteinase K incubationincreased the extraction efficiency two fold,ethanol-precipitation may cause a DNA lossof ≈30%, and the addition of a co-precipitant(tRNA) may increase the recovery six fold.

With the optimized protocol we soughtto maximize the yield of DNA whilemaintaining a quality suitable for real-timePCR. Combined with an internal DNAstandard, the protocol offers quantitative andefficient DNA extraction, which is aprerequisite for quantitative methods inaquatic microbial ecology.

A major goal in aquatic microbialecology is to link the identity and functionfor the predominant bacterioplanktonspecies. One approach is to cultivatebacterioplankton and try to establish acoupling between laboratory-based analysesand in situ function. In Paper II, we usedthis approach and combined cultivation andin situ dynamics in an effort to examine therole of heterotrophic bacteria in pelagic N2-fixation in the Baltic Sea Proper.

The ecological balance of the Baltic Seais severely threatened by eutrophication.Therefore, costly measures are taken toreduce the output of in particular nitrogen.However, N2-fixation has been estimated toprovide a yearly nitrogen input in the sameorder as the entire riverine load to the BalticSea (Larsson et al. 2001), and is therefore aprocess that may overthrow the efforts toreduce anthropogenic nitrogen input. Adetailed understanding of N2-fixation andthe organisms involved in the process istherefore of particular interest in the BalticSea area. In summer, when primaryproduction in the Baltic Proper is limited bynitrogen, massive blooms of diazotrophicheterocyst-forming cyanobacteria appearand so far, N2-fixation in the Baltic has beensolely attributed to filamentous cyano-bacterial activity. However, sinceheterotrophic bacteria with a geneticpotential for N2-fixation recently have beenfound in several oceans around the world, amajor goal of this thesis was to investigatewhether heterotrophic bacteria play a rolefor pelagic N2-fixation in the Baltic Sea.This was addressed in Papers II and III.

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At the onset of this thesis work, thepublished and widely acclaimed nested nifHPCR approach was intended as the primarymethod to detect and identify organismswith genetic potential to fix nitrogen.However, despite much effort and extensiveprecautions to avoid PCR contamination,this approach often yielded variable andincoherent results. Consistent with ourspurious results, Zehr et al. (2003b) showedthat PCR reagents might contain traceamounts of nifH-containing genomic DNAproducing unwanted amplification productsin nested PCR; hence, emphasizing that thiswas a general problem to the research field.This insight partly shifted the focus of thestudy towards the development of newmethods for isolating diazotrophicbacterioplankton.

One reason for the historicalunderestimation of bacterial abundance isthe inability to cultivate most marinebacteria on nutrient agar plates. Thediscrepancy between colony forming unitsand the several orders of magnitude highernumber of bacterioplankton countable byepifluorescence microscopy has been termed“the great plate count anomaly” (Staley1985). This discrepancy is still not fullyresolved. Comparisons between sequencesfrom cellular clones and environmentalclone libraries obtained from the sameseawater sample suggest that many of themost abundant bacterioplankton species arenot readily culturable on conventional agarplates (Suzuki et al. 1997, Eilers et al.2000). In the Baltic Sea however, culturablebacteria make up a significant fraction of

bacterioplankton (Pinhassi et al. 1997).Recently, Simu et al. (2005) showed theratio of colony-forming to non-colony-forming bacteria being 10-20 fold higher inthe brackish, eutrophic Baltic Sea relative tothe North Sea. Thus, the prospects ofisolating N2-fixing heterotrophic bacteriawith the relatively simple means of agarplates and agar tubes seemed feasible in theBaltic environment.

In Paper II, we successfully isolated N2-fixing heterotrophic bacteria from the BalticSea Proper by using low nitrogen agar platesand semi-solid diazotrophic medium tubes(SSDM). The isolates, which were related toγ-proteobacteria, carried the nifH gene andwere capable of N2-fixation as measured byacetylene reduction. By means ofmicroelectrode measurements, it was shownthat they required microaerophilic toanaerobic conditions for active N2-fixation.To determine the in situ abundance of one ofthe isolates (BAL281), a specific primer-probe set was designed for quantitative real-time PCR. Community DNA was extractedusing the protocol developed in Paper I.BAL281 nifH gene copies were found intwo of eleven locations along a transect inthe Baltic Proper with relatively highabundance (3±0.4 x 104 nifH gene copies l-1)only in the sample from which the bacteriumwas originally isolated.

The paper shows that N2-fixingheterotrophic bacteria may be isolated fromthe Baltic Sea and that these are at leastlocally abundant in situ. This findinghighlights the potential importance of

19

heterotrophic bacteria for N2-fixation in theBaltic Sea and emphasizes the need forfurther research to quantify their importance.The study illustrates how the combination ofextensive cultivation efforts and molecularquantification of specific nitrogenase genescan provide insights to the role and ecologyof N2-fixing heterotrophic bacterioplanktonin the sea.

While the results of Paper II showedthat some heterotrophic bacteria in the BalticSea have the genetic potential for N2-fixation, as well as the capability to fixnitrogen, Paper III focused on the in situexpression of the nifH gene. A large clonelibrary (84 clones) was constructed fromnifH transcripts in a sample integrating 4surface waters from the Baltic Proper duringthe summer season. The clone libraryconsisted exclusively of nifH transcriptsfrom the heterocystous cyanobacteriaNodularia sp. and Aphanizomenon sp. ThenifH gene of an Aphanizomenon sp. isolatewas sequenced in this study. No genesrelated to heterotrophic bacteria were found,suggesting that they were non-existing ormore likely too few to be detected in theclone library. Hence, indicating that thecontribution of heterotrophic bacteria to N2-fixation was of limited importance at thetime of sampling.

To determine the temporal and spatialvariability of N2-fixation, a specific primer-probe set targeting the Nodularia sp. nifHgene cluster was designed. Real-time PCRshowed that n i fH gene expression byNodularia was highly variable between

stations but was one order of magnitudehigher during mid summer relative to earlysummer and fall. The patchy distribution ofexpressed Nodularia nifH genes suggeststhat high-resolution sampling is required toassess N2-fixation in the Baltic Sea.

As exemplified in Papers II and III, thecombination of cultivation, to examinephysiology and metabolism of singlebacterial species, and monitoring of in situdynamics, is one way to elucidate thefunctional role of bacterioplankton speciesin the sea. Another option is to examine theoverall diversity of bacterioplankton and thelarge-scale processes that regulate globalspecies distribution. The establishment ofsuch patterns may help reveal the ecology ofcertain bacterial species or groups. Anexample of this was provided by Hagströmet al. (2002) in their efforts to distill theinformation available in GenBank. Toaccess this vast amount of information,Hagström et al. (2002) retrieved all marinebacterioplankton 16S rDNA sequences,merged fragments of these, and sorted outunique contigs (ribotypes) based on ≥ 97%similarity for identical ribotypes. In total,they found only 1117 different ribotypes,which is in contrast to the great biodiversitypreviously anticipated for marine bacterio-plankton. This in silico finding of a limiteddiversity prompted the study presented inPaper IV, in which we sought to get aglobal view of bacterioplankton diversity insitu. In this single study, covering samplesfrom 9 locations in polar, tropical andtemperate oceanic regions, we found 562distinct ribotypes, which is about half of the

20

diversity found by Hagström et al. (2002)and ~1/4 of the diversity found by Pommieret al. (2005) in an updated search inGenBank. Of the 562 ribotype sequences,~80% were similar to sequences in GenBankindicating that a large fraction of the marinebacterioplankton has already been sampled.The results show that only few ribotypes arecosmopolitan and that the number ofendemic ribotypes is high. In addition, thestudy suggests a latitudinal gradient ofincreasing number of ribotypes withdecreasing latitude, which is a patternknown from macroorganisms. This studyprovides a unique view of global marinemicrobial diversity and suggests a limitednumber of 16S rDNA ribotypes in the globalbacterioplankton community.

21

CONCLUDING REMARKS

The results in this thesis have been obtained by traditional microbiological methodscombined with novel high-technology molecular-based techniques, such as real-time PCRand extensive cloning/sequencing. The presented conclusions demonstrate that the appliedintegration of methods provides powerful means of approaching a mechanistic understandingof aspects of bacterioplankton function.

The thesis contributes some information of direct value to our ultimate understanding ofthe biogeochemical role of bacterioplankton in marine waters. However indirectly, theobtained knowledge on methodology, bacterial N2-fixation, and global bacterioplanktondiversity constitute a valuable platform for future scientific progress. In a near-future context,I find that the main challenges will be:

I. To optimize the SSDM tube isolation method used in Paper II to exclusivelyselect for N2-fixers. This would allow for extended analysis of the genetic andfunctional diversity of marine heterotrophic N2-fixing bacteria.

II. To develop nifH gene expression analyses, as used in Paper III, into a meansof quantifying N2-fixation. This goal, which would allow distinguishing therelative importance of heterotrophic bacteria and cyanobacteria in planktonicN2-fixation, would have to be preceded by an establishment of the relationshipbetween nifH gene expression and actual N2-fixation. For instance, a tentativecalibration of nifH gene expression relative to N2-fixation measured by the15N2 assay would be a first step. Absolute quantification of nifH geneexpression would also require knowledge of RNA extraction efficiency andcDNA synthesis efficiency (e.g. by using RNA standards).

III To investigate the large-scale distribution of functional genes such as nifH. Theuse of 16S rRNA gene information in studies of bacterial distribution hasgenerated new knowledge about bacterioplankton biogeography (Paper IV). Asimilar analysis focusing on functional genes could constitute a rewarding task.

A more general future challenge will be to establish the relationship between 16S rDNAhomology and genomic variability. Today, 16S rDNA is widely used to delineate species andto constrain bacterial population in situ (Paper IV is an example). Though, whether bacteriawith a specific function (ecotypes) may be resolved by 16S rDNA identity is, to myknowledge, an open question.

22

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26

ACKNOWLEDGEMENTS

This thesis would not have been completed without the help and support from numerouspeople. Thank you all!!!

In particular, I would like to thank:My supervisor Lasse Riemann, who guided this work and helped whenever I was inneed. Your encouragement, enthusiasm and energy have been invaluable!My co-supervisor Åke Hagström, for giving me the opportunity to work in themicrobiology group. Your always positive attitude and support kept me going, whentimes were tough.My first supervisor Ulla Li Zweifel, who was my supervisor in the initial stages ofmy study until she changed job, for introducing me to this field of research.

Colleagues in the ÅH-group: Karin Simu for sharing her large lab-experience andfor being such a good friend, and Cissi, Sabina, Karin, Laura and Jarone for all thefun chats, not always about work…

Heléne Werthwein, for not giving up with the endless isolation experiments.Cecilia Petersen, for helping me out with some of the Gotland cruises.Anders, Conny and Sven, I am especially grateful for your help fixing “things”.

Gunilla Åkesson-Nilsson for all help with the acetylene reduction assay, even if theresults didn’t become as superb as expected.The “Norrgård people” for letting me use some of your equipment and for nicechats in the coffee room every now and then.

The crew at M/S Tjelvar for help and company at the many sampling trips betweenOskarshamn and Visby.

Friends, for being around talking about something else than science. Sofia andPelle, I hope the wine discussions will continue.

My family, for always being there for me.My wonderful girls Frida and Emma, I love you!My wonderful “extra” girls Jessika and Josefin, for contributing to the joyfulatmosphere in our home, love you!

My Mats, I know you have had a hard time not knowing whether to support me togo on with my Ph.D. studies, or to recommend me to quit.You made the right choice!Tack Mats, jag älskar dig!

Kjärstin