Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | . , and , the gene , Discussions pufM 1,2 ´ e Blaise Pascal, . The most abun- Biogeosciences , Groupe Plancton ff , L. Garczarek 1,2 liated to the ´ e en Milieu Marin, Station Biologique ffi .fr) erent agar media were screened for Citromicrobium bathyomarinum ff ff ), the light-harvesting pigment of AAP a Gammaproteobacteria , F. Le Gall 4422 4421 ´ (BChl- eanologique de Rosco 1,2 ´ a enome et Environnement, Universit 3 gene transcripts revealed that most phylotypes were . Most other isolates were related to species and/or may represent novel taxa. Direct extraction of liated with the a ffi pufM , O. Dahan 1,2 , France , France ff ff Roseovarius transcripts observed in this ultra-oligotrophic region. To our knowl- 94%) was a -containing colonies represented a low part of the cultivable fraction. genes showed that they were all a a , D. Boeuf , and A.-C. Lehours ∼ and 1,2 1,2 pufM pufM ` ere Cedex, France , 29680 Rosco ff ´ eanique, 29680 Rosco This discussion paper is/has been under review for the journal Biogeosciences (BG). Please refer to the corresponding final paper in BG if available. UPMC Univ Paris 06, UMR 7144, Adaptation et Diversit CNRS, UMR 6023, Microorganismes: G INSU-CNRS, UMR 7144, Observatoire Oc dantly detected phylotypes occurred in theHowever, western some and were eastern exclusively Mediterranean detecteddiversity basins. in of the easternedge, basin, this reflecting is the the highest to first unveil study the active to AAP documentout community extensively in the the an discrepancies oligotrophic diversity between marine of culture-basedlights environment. and AAP the By molecular existing isolates pointing gaps methods, and in thisthe the study Mediterranean understanding high- Sea of and the likely AAP globally. ecology, especially in RNA from seawater samples enabledcoding the for analysis the of the M expressionsynthesis. subunit of Clone of libraries the of reactionhighly centre similar complex of to aerobic sequenceslarge anoxygenic previously majority photo- recovered ( from the Mediterranean Sea and a 2008. Colony-forming units obtainedthe on production three of di bacteriochlorophyll- bacteria. BChl- In total, 52 AAP strains wererRNA isolated and and the phylogenetic analyses basedThe on most their 16S frequently isolatedErythrobacter strains belonged to not reported to produce BChl- Aerobic anoxygenic phototrophic (AAP)plankton bacteria productivity play and significant biogeochemical cycles roles ofapplied in the both the surface cultivation ocean. and bacterio- mRNA-based In molecular thisAAP methods study, bacteria to we along explore the an diversity oligotrophic of gradient in the Mediterranean Sea in early summer Abstract Biogeosciences Discuss., 8, 4421–4457, 2011 www.biogeosciences-discuss.net/8/4421/2011/ doi:10.5194/bgd-8-4421-2011 © Author(s) 2011. CC Attribution 3.0 License. Received: 21 April 2011 – Accepted:Correspondence 29 to: April 2011 C. Jeanthon – (jeanthon@sb-rosco Published: 5 MayPublished 2011 by Copernicus Publications on behalf of the European Geosciences Union. 63177 Aubi Diversity of cultivated and metabolically active aerobic anoxygenic phototrophic bacteria along an oligotrophic gradientthe in Mediterranean Sea C. Jeanthon Oc 2 3 de Rosco E. M. Bendif 1 5 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , a has erent ff pufM ` a et al., 2002; ´ ej content per cell was a ) synthesis. Given their a er a unique context to link ff (Bchl- a synthesis. a gene encoding the M subunit of the photo- 4424 4423 pufM (Jiao et al., 2007). a ` a et al., 2002; Yutin et al., 2005; Jiao et al., 2007), de- ´ ej and AAP bacterial abundance decreased from the western to the a measurements suggested that AAP bacteria may constitute a very dy- a ` a et al., 2002; Karr et al., 2003). Up to now, several DNA-based molecular ´ ej The aim of this study was to identify the cultivable fraction and active members of Molecular analyses based on the The Mediterranean Sea is one of the most oligotrophic oceanic systems and is char- the stratification period through cultivation and mRNA-based techniques. used to investigate gene expressionment and to (B identify active AAPstudies bacteria have in explored the the environ- diversity(Oz patterns et of al., AAP 2005; bacteria in2010). Yutin et the al., Mediterranean However, only Sea 2005, 2008; aits Lehours very surface waters et small so al., number far 2010;never of and been Martinez-Garcia the AAP conducted. et examination bacteria al., of have the been taxa actively isolated expressing from the AAP bacterial community along a large transect in the Mediterranean Sea during having a half-life ofargued only that a analysis few of minutestentially sequences provide (Von derived a Gabain more from et representative RNA indicationcommunity al., of rather and 1983). the than a active strong DNA members Thus, of indication templates(Pichard it the of and po- bacterial has Paul, specific 1993). gene been Reverse expression transcription at PCR the (RT-PCR) approaches time have been of sampling Lehours et al.,(Ranchou-Peyruse 2010), et al., terminal 2006)though restriction these and DNA-based fragment metagenomic approaches length have analysisteria, given polymorphism insight analysis (Yutin into of et (T-RFLP) the relevant al., diversity functionalstable of communities 2007). in AAP is bac- resting still Al- cells aDNA. challenge, and In as even contrast, DNA dead RNA couldbers, is cells be have highly and been labile may correlated and alsoMoreover, rRNA with studies be levels, of cellular cultured and present activity bacteria therefore as (Kramer have ribosome identified extracellular and num- that Singleton, mRNA is 1992, typically 1993). short-lived nutrient and/or carbon limitation favored BChl- synthetic reaction center havechain explored AAP reaction bacterial (PCR) diversitynaturing/temperature (B using gradient the gel polymerase electrophoresis (DGGE/TGGE) (Yutin et al., 2008; suggesting that reliance on phototrophy varied along the oligotrophic gradient and that significantly higher in the ultra-oligotrophic eastern basin than in the two other basins nutrient availability, trophic status andphotoheterotrophic functioning populations. of The thegeochemistry food 3000 km from web the transect to Oligotrophic surveyed the toing by dynamics the the Ultraoligotrophic the of Mediterranean) stratification BOUM cruise periodunderstand (Bio- dur- the in distribution early patternenvironment. summer of abundance 2008 and In provided diversity a oftrations an companion AAP of opportunity paper, bacteria BChl- to Lamy ineastern this better et basins al. of the (2011) Mediterranean demonstratednutrient Sea and and that were dissolved concen- linked with organic concentrations carbon. of Chl- Interestingly, the BChl- whereas AAP bacterial abundance followedthe the concentration opposite of trend, chlorophyll- positively correlated to acterized by a longitudinal gradienttrophic of conditions oligotrophy available increasing eastwards. in The the di Mediterranean Sea o Cottrell et al., 2006;al., Jiao 2009). et AAP bacteria al., wererepresenting 2007; found about to Yutin 1.5–5 be et % widely al., of distributedet total 2007; in al., bacteria the Masin 2000; in marine et the Kolber environment hand, al., euphotic et zone al., BChl- 2008; of 2001; Jiang the Sieracki et oceannamic et (Kolber part al., 2006; of Jiao theof et marine organic al., bacterial carbon 2007). community inshowed On contributing the the that significantly other upper to the ocean the maximum (Koblizek cycling AAP et bacterial al., diversity 2007). was A found global in ocean oligotrophic study areas, Aerobic anoxygenic phototrophicoxygen (AAP) for bacteria their are growthcapability photoheterotrophs and of that for harvesting require bacteriochlorophyll- lightterioplankton energy, in they represent freshwater an and important marine fraction illuminated of environments the (B bac- 1 Introduction 5 5 25 20 10 15 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Isochrysis shore dur- ff C, standard ◦ (for DCM sam- (1 µM), nicotinic 1 4 C aboard the R/V − ◦ s 2 PO 80 − 2 (Fig. 1). The sampled − ). In order to promote the growth L’Atalante C until processed (within one hour). M), 1 ml of K medium trace elements ◦ Cl (10 µM), KH 4 10 − (4.10 4426 4425 1 l) were filtered as rapidly as possible, imme- 12 Synechococcus ∼ and M) vitamin B 9 C for 7 days in the dark, MAD agar plates were transferred into ◦ − -containing colonies was performed using a CCD camera based a (for surface samples) and 25 µmol photons m 1 Prochlorococcus − s (RCC 179) freshly grown in K medium (Keller et al., 1987). After the cruise, 2 − . Samples were transported frozen in dry ice to the laboratory and stored at C until RNA extraction procedures. ◦ ort to provide more tight size fractionation) Nuclepore filters of 47 mm diameter, with . galbana After incubation at 18 Detection of BChl- 80 ff ff fluorescence imaging. Briefly, after up to 7–8 weeks of incubation at 18 eukaryotes or 1/5 ofcyanobacteria full ( strength Pro2 mediumof (Moore nitrate-assimilating and cyanobacteria, Chisholm, the 1999) latteralso medium for used. amended pico- with 10 µ nitrate was thermostatic cabinets illuminated by luminescentphotons tubes at m irradiance levels ofples) 120 µmol in 12:12 light-dark cycle. Briefly, seawater sample was filterede by simple gravity througheither two 0.6 superposed µm or (in 3 an µmwas porosity partitioned (Whatman into International 50 Ltd, ml Maidstone,either culture UK). 1/100 flasks The of (Sarstedt, filtrate full Orsay, strength France) to Jaworski medium which (Jaworski was et added al., 1981) for photosynthetic peptone (Difco), 0.1 g yeastacid extract (2 µM), (Difco), biotin NH (2.10 (Keller et al., 1987)ing and the 15 g cruise. agaromitted MiA (Difco) and medium per local was liter seawater a of wasa modified replaced seawater by sampled version the of o supernatant theAAP of isolates latter a were where culture also peptone of obtainedcyanobacterial was cultures by enriched streaking onboard on as previously MAD described medium (Le 3–5 Gall µl et al., of 2008). microalgal or Seawater samples collected at the(DCM) surface were (3 m) used and to at isolate100 the µl AAP deep bacteria. of chlorophyll maximum Colonies seawater were samplesagar obtained on media by Marine spreading referred Agar 50 here to (Difco, as Detroit, MAD MI) and and two MiA. low MAD strength medium consisted in 0.5 g Bacto 2.2 Culture media, strain isolation and identification diameter and 3 µmfinal pore collection size of polycarbonate bacterioplanktonpolycarbonate filters cells filters (Cyclopore, onto (Isopore, Whatman) Millipore) 25 mm before using(Cole diameter a the Parmer four-head and Instrument Masterflex 0.22 peristaltic µmimmediately Company). pump transferred pore individually size to Three a filters screw-capbion tube per Inc., containing 1 Austin, sample ml RNAlater USA), wereL’Atalante (Am- frozen processed in and liquid− nitrogen and kept at cultures, samples were kept inBacterioplankton cells the for dark total and RNA extraction atthe were 4 same collected by water filtering sample seawatermRNA, from used small for volumes the of culturediately seawater experiments. upon ( To retrieval limitRNAlater of the was the degradation 15–20 of min. CTD. The Briefly, the time seawater from was the prefiltered in start line of through filtration 47 mm to storage in July 2008 during thestations BOUM included fourteen cruise short aboard duration the17, (4 h) 19, R.V. stations 21, (stations 24, 1, 25, 3,tative 27) 5, of and 7, three low 9, 4-day 11, nutrient drifting 13,These low stations 15, chlorophyll three (stations (LNLC) A, stations B conditions andern were (Moutin C) basin et located represen- (station al., at A), inC). the preparation). in Water center the samples of Ionian werepler Sea anticyclonic retrieved equipped (station eddies using with B) in conductivity, 12 and l temperature the in Niskin and west- the depth bottles Levantine (CTD) fitted Sea sensors. on (station For a bacterial Rosette sam- 2.1 Sampling Seawater samples were collectedranean Sea from (from 16 the stations North of along the a western transect Basin to in the the Levantine Mediter- Basin) in June and 2 Material and methods 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | - 0 C for 1.5 min. ◦ C for 30 s, and ◦ -specific primer 8F (5 C for 30 s, 58 ◦ was determined by using PCR Bacteria pufM -containing colonies was registered C using the PufWAW reverse primer a ◦ C for 10 min. The PCR products were ethidium bromide, and electrophoresed C for 10 min. PCR products were loaded ◦ ◦ 1 , 10 pmoles of each deoxyribonucleotide ) and 0 − 2 4428 4427 ` C for 1 min and extension at 72 a et al., 2002)) and PufWAW reverse primers ◦ ´ ej , (Yutin et al., 2005)) as gene-specific primer. On 0 , (B ). Reaction mixtures (25 µL) contained the following 0 0 er, and total RNA extraction was performed following ff ciency (e.g. yield) results, we selected PufMF forward site). C). The presence of ffi ◦ ff er (5 µl), 2 mM MgCl mRNA from total RNA samples as recommended by the manu- ff signal was evaluated using an infra-red kinetic fluorometer (model a C for 5 min, followed by 35 cycles of 95 genes in cDNA samples. Reaction mixtures (25 µL) prepared as pre- ◦ pufM 850 nm glass filter (Oriel 51360, USA). The purification procedure on > -CGGTTACCTTGTTACGACTT-3 C for 1 min, annealing at 50 pufM 0 ◦ C for 10 min, and the thermal profile then consisted of 30 cycles of denatura- ◦ C for 30 s, with a final extension step at 72 ◦ -AYNGCRAACCACCANGCCCA-3 -TACGGSAACCTGTWCTAC-3 Amplified PCR products were purified using the ExoSap purification kit (ExoSap- For DNA extraction, cell pellets from 1.5-ml cultures were suspended in MilliQ water, Small-subunit rRNA genes were amplified by PCR using universal reverse primer 0 0 72 in a 0.8 % agarose gel containing 0.5 µg ml of 10 µl. cDNA(5 synthesis was performedthe at basis 55 of specificity(5 and e to amplify viously contained 1 toneAmp 3 µl PCR of system cDNA 9700parameters: sample. (Applied 95 Amplifications Biosystems, were Foster carried City, out USA) in with a the Ge- following 2.4 Reverse transcription and cDNA amplification ThermoScript RT-PCR system (Invitrogen,transcription Carlsbad, of USA) was usedfacturer. for All the reverse transcription reverse reactions were performed in a total reaction volume Billerica, USA) for desalting50 µl and of concentrating RNAlater were by added(100 centrifugal µM) back to filtration. and the 350 sample µl The tubesthe of containing NucleoSpin resulting RNA 50 RA1 µl II bu manual. of glass Genomic(Ambion). beads DNA DNA was removed removal using in a RNA Turbothat DNA-free samples kit contained was RNA confirmed and by were not controlNo previously PCR amplification subjected was amplifications to detected the in reverse these transcriptiondescribed controls step. performed below. following the PCR conditions Total RNA was extractedwith using several a modifications to NucleoSpin recoverto RNA RNA the II possibly sample isolation released freeze to kitice, and the (Macherey-Nagel), and 1 thaw ml the (Frias-Lopez RNAlater et 1 due al., ml 2008). of RNAlater Samples was were loaded thawed on on a Microcon YM-50 column (Millipore, 2.3 RNA isolation Genotyping Platform (Rosco at 95 tion at 94 A final extension stepgrouped was initially by carried restriction out fragmentMnlI at length restriction polymorphism patterns. 72 (RFLP) using HaeIII and it, GE Healthcare,randomly Uppsala, chosen strains Sweden). from eachan 16S RFLP ABI group 3130 rRNA (if POP7 gene applicable) sequencer were sequences (Applied sequenced Biosystems) of using at at the Biogenouest least Sequencing- two AGAGTTTGATYMTGGCTCAG-3 components: 5 Xtriphosphate bu (dATP, dCTP, dGTP,cleotide dTTP; primer, 1.25 U Eurogentec), of GoTaq Flexi1 10 µl DNA pmoles polymerase of (Promega, of extract. Madison, USA) each The and initial oligonu- denaturation step consisted of heating the reaction mixture FL3500, Photon Systems Instrumentsbroth Ltd.) under a in 14 pure h–10 h cultures light-dark cultivated regime. in 1/10boiled marine for 10centrifuged min, (5000 g, left 5 min, at 4 amplification room with specific temperature primers for (Lehours et 10 min, al., 2010). boiled1492R again for (5 10 min and (470 nm) and infra-redby fluorescence a from CCD BChl- a camera long (Photon pass SystemsMAD Instruments plates Ltd., was repeated Brno, twopresence to Czechia) of three BChl- protected times by until cultures were considered pure. The Petri dishes with colonies were illuminated by a set of Nichia blue light emitting diodes 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | er. The DNA ff liations. A conser- ffi http://www.ncbi.nlm.nih. gene sequences of the isolates er (1 mM NaEDTA (pH 8.0), 40 mM ff pufM gene sequences obtained in this study are 4430 4429 pufM old nucleotide sequences was constructed using ARB ff site). Clone libraries were screened for chimeric se- ff Check program available on the RDP website (Cole et al., ) and the GOS sca database containing more than 700 aligned sequences of cultured species pufM A The oceanographic context, themasses physical in and the Mediterranean hydrologic Sea characteristics during of BOUM the cruise water are extensively described by and JF421730-JF421749, respectively.have been The depositedFR852767. in EMBL database under the accession numbers FR852756- 3 Results 3.1 Oceanographic context the branching pattern was(DNAPARS) methods tested included with in the the PHYLIP neighbor-joining package and as maximum implemented in parsimony and ARB. environmental clones retrieved fromgov/Genbank GenBank database ( Bork, 2007). 2.7 Nucleotide sequence accession numbers The 16S rRNA andavailable in environmental GenBank database under the accession numbers HQ871842-HQ871863 method implemented in ARB, using only almost full-length sequences. The stability of (Ludwig et al., 2004).ment was then Sequences used were towas translated manually realign to first nucleotide protein sequences. constructed A and withof neighbor-joining the inferred tree all resulting tree the topologies align- sequences wasPHYLIP longer tested (Felsenstein, by than 1993). bootstrap 600 analysisto bp Shorter (1000 the and sequences resamplings) tree the using were using robustness sequences aligned ADD-BY-PARSIMONY to algorithm, as existing which phylogenetic above trees allows without andlogenetic the changing tree added global addition display tree and of topologies. annotation short Phy- were performed with iTOL software (Letunic and with high similarities to thosealignment. determined in Framework this trees study were were calculated retrieved and with added fastDNAmL, to a the maximum-likelihood 2.6 Phylogenetic analyses Sequence data were analyzedThe with the new ARB sequences softwareFast were package Aligner (Ludwig added tool. et to al.,sary. 2004). the Alignments 16S SSU were rRNA checkedpublic ARB gene and databases sequences database from corrected with and the manually BLASTn aligned isolates where (Altschul with were neces- et compared the al., to 1997). sequences in 16S rRNA gene sequences 2003). The remainingavailable sequences sequences to were determine their subjected approximatevative phylogenetic to value a of BLAST 94 % search nucleicgrouping acid against sequence sequences publicly similarity into (Zeng Operational et Taxonomicet al., Units 2007) al., (OTUs) was 2009). chosen using for Mothural., Coverage (Schloss 1995). value (C) was calculated as previously described (Mullins et 2.5 Library construction and analyses Fresh RT-PCR products were clonedmanufacturer’s instructions using (Invitrogen). the Recombinant TOPO-TA clones cloning werecontaining screened kit plasmids for by according insert- direct to PCR amplification the Clones with M13 were forward and sequenced reverseGenotyping primers. using Platform the (Rosco quences latter with primers Chimera at the Biogenouest Sequencing- procedure, modified Tris-acetate EDTA (TAE)Tris acetate) bu was used toband prepare the of agarose the gel targeted andDNA as size isolated the from was running gel carefully bu using cut an from Ultrafree-DA centrifuge the filter gel (Millipore). using a transilluminator and at 80 V for 1.5 h. Since the amplified DNA was later released from gel for the cloning 5 5 25 15 20 10 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | a Ery- (Zhang Tateya- as clos- species, a C (station as closest ◦ M. litoreum liated to . Within the ffi Marivita Thalassobaculum 40 to 80 m) to east R. tolerans , the only species of (Table 2, Fig. 2 and and had ∼ has been detected only a 98 %), respectively. Although Marivita Sulfitobacter dubius > S. stellata (94 %). Both species of the re- . , BChl- gene by PCR amplification. BChl- (Gonzalez et al., 1997). Finally, we . Strains RCC 1906 and RCC 1923 gene sequences demonstrated that a pufM Sagittula pufM were obtained from microalgal cultures. A were not reported to produce BChl- Tateyamaria Alpha-4- 4432 4431 Citromicrobium bathyomarinum . Among them, RCC 1920 and RCC 1921 rep- a and . Interestingly, all strains having (Kurahashi and Yokota, 2007; Sass et al., 2010). Within R. halotolerans C (station B). Salinity was higher in the eastern basin and Alpha-3 . The numbers of colonies showing infra-red fluorescence , RCC 1910 was highly related (99 %) to ◦ a , Thalassobaculum salexigens Thalassobaculum Roseibacterium elongatum as closest cultivated neighbors ( , two other clusters representing 8 strains were a was not detected in their cultures (Hwang et al., 2009). Two 98 %). Among the most frequently isolated strains, twelve be- Roseovarius > a liated to Alpha-1 ffi production in strain RCC1888 that had genes were amplified from the type strains of both (97–98 %). In the genus T. pelophila a species (Labrenz et al., 2000; Park et al., 2007; Yoon et al., 2007). 100 m). > species ( pufM while four strains (RCC 1908, RCC 1918, RCC 1925 and RCC 1926) were 99.8 % 16S rRNA identity) to ∼ and M. cryptomonadis Alpha-1-proteobacteria Additionally to strains RCC 1908, RCC 1918, RCC 1925 and RCC 1926 that ex- Phylogenetic analysis of 16S rRNA and Sulfitobacter hibited marked novelty (Tablesequence 2), identity to other taxonomically characterized strains(Stackebrandt species may bearing and also 96 represent Goebel, % new taxa most 1994). or closely less related Among to 16 themmay rRNA represent was gene a categorized new strainthis species genus, RCC of was 1927 the not genus reporteddetected BChl- to synthetize BChl- characterized relative. This phenotypic trait has been reported only in a few described the litoreum more distantly related to cently described genus et al., 2008; Urios et al., 2010). were not known toresented synthetize two BChl- strainsand of the recentlypufL described genus production of BChl- isolates (RCC 1909 andmaria RCC omphalii 1919) were identifiedin as the very close cells relatives of of throbacter longed to the genus est cultivated species (98whereas %) those were a isolatedportion after of direct the isolates plating was of highly related seawater to samples recently described genera, whose species Alpha-3-proteobacteria Fig. 3a). Twentytical isolates recovered ( from surface or DCM waters were almost iden- cultivated after direct platingcarbon were content. recovered Twenty on four strains the13 were from two isolated the media from central with theisolates basin western low were (3 basin positive nutrient for (4 stations) and the stations), andfluorescence presence one of signals the from were the detectedmarine eastern in broth. basin the (1 cultures station). of all All isolates whenall grown in isolates 1/10 weremembers exclusively of alphaproteobacterial the and were more closely related to under blue light illumination varied greatlyonly between samples. a small AAP percentage bacteria represented (less54 than AAP 1 %) strains of were the isolated; cultivableseawater bacterial 38 samples community. strains and A were 16 total obtained were of terial after retrieved cultures direct after enriched plating streaking onboard of of the from primary fresh seawater algal samples or cyanobac- (Table 1). All but one strains fined Deep Chlorophyll Maximumdetails, (DCM), see more pronounced Crombet in et(from the 80 al. western to (2011)), basin and (for deepening from west3.2 ( AAP isolates Colonies grown on the agar platesfor of the the presence three of media used BChl- in this study were screened 25) to in theespecially western from 26.9 station 5 whereconcentrations it were remained above close 39 or ‰in down below the to the eastern 200 detection m basin depth. limit wherewere Nutrient in lower measured pigment the concentrations (Pujo-Pay upper and et layers, primary al., decreased production 2011). rates Chlorophyll concentration showed a well de- Moutin et al. (2011). Briefly, sea surface temperatures ranged from 21.4 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | con- ered ff a Methy- sequences -containing and 94 % of the a ∼ concentrations representatives pufM a 85 %) indicated that > sequences were more transcripts directly from pufM pufM KT71 (Eilers et al., 2001), strains genes were amplified from sam- 97%) to sequences recovered from ≥ Roseobacter denitrificans pufM Gammaproteobacteria in the cultivable community 10) were recovered in both basins. BOUM − 4434 4433 transcripts was higher at station C (Shannon- erent trophic regimes (Lamy et al., 2011). To com- 3 and ff − pufM mRNA transcripts, using reverse transcription-PCR to Congregibacter litoralis 6). . 1 , respectively. Thirteen OTUs, representing = pufM 0 ` a et al., 2002), all related to the NOR5/OM60 clade (Rappe et Alphaproteobacteria 1), located in the most oligotrophic region of the Mediterranean ´ ej . 2 inserts were sequenced; 109 were recovered from station A and 11 = 0 gene sequences of the isolates did not cluster with that of cultivated pufM in the Mediterranean Sea. We amplified by PCR pufM pufM Only two OTUs, namely BOUM 13 (3 sequences) and BOUM 14 (2 sequences) were Using an operational taxonomic unit definition of 94 % nucleic acid sequence similar- Most infrared microscopy data ofcentrations Lamy et and al. AAP (2011) bacterial revealing the abundance decrease from of west BChl- to east. All BChl- plement these detailed data on theirat abundance and identifying distribution, the we aimed diversitybacteria in and this along study the distribution two of major cultivable basins and of the metabolically Mediterranean4.1 active Sea. AAP Dominance of Our data report aeastern decrease basin of of the numbers the of Mediterranean AAP Sea. colonies from This the is western to in the agreement with the HPLC and a large range of trophicoligotrophic conditions western from basin the during ultra-oligotrophicduring the eastern stratification the basin period. to same the cruise Ain less reported parallel the study the Mediterranean carried largest Sea out trends spatial of and dataset AAP provided of bacteria a BChl- along comprehensive its di picture of biogeographical 2 and BOUM 10predominated in were surface uniformly samples. distributedquences BOUM at along station the 4, C, was euphotic representingin absent more layer DCM in samples than while other predominated 20 stations. BOUM % at In of station 3 contrast, A. the BOUM se- 5 only detected 4 Discussion The BOUM cruise constituted a unique opportunity for ecological studies as it o NOR5-3, NOR51B and HTCC2080and (Cho EBAC29C02 et (B al.,al., 2007) 1997). and Most of BAC themthe clones were Mediterranean very EBAC65D09 Sea highly (Lehours similar ( etterial al., OTUs (i.e., 2010). OTUs The BOUM most 2, dominant gammaproteobac- including few isolates such as lobacterium radiotolerans sequences, fell into group K which contained Mediterranean Sea (93.7 %) (Tableisolates. 3). The phylogenetic None analysis were demonstratedwere that closely distributed the related into expressed to 4 that(Fig. of of 3a the our 12 and phylogroups AAP b).Wiener previously defined index The by H diversity Yutin of etSea, al. than at (2007) station A (H most similar to sequences of known organisms, from station 21 infrom the station western 1 basin in the whereas eastern 83 basin. were retrieved fromity, 20 station distinct C OTUs and weremost 4 identified of (Table 3). the Coverage diversity valuesthan was ( 94 detected % identical in to the known libraries. sequences and Most were related to clones retrieved from the Functional expression of produce cDNA, was performed toing determine which phylotypes wereRNA actively (cDNA) transcrib- extracted from mixedand picoplankton assemblages eastern collected Mediterranean in Sea. theples western Interestingly, collected during night timeretrieved but no during amplification products the were day obtainedcontaining from (stations those 25 and B). A total of 207 randomly picked clones organisms (Fig. 3a).sequences They recovered from exhibited environments generally other highest than the sequence Mediterranean levels Sea. 3.3 of identity to Diversity of active AAP bacteria 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | a Al- pufM and uent com- ffl R. halotoler- pufL spp. are among C. bathyomarinum in the upper waters of are among those whose cul- (Eilers et al., 2001; Fuchs et and to show a strain-dependent erent genera reflects a high cul- ff a , with one known betaproteobacte- (Suyama et al., 1999), one described -like bacteria, enhanced their growth or Roseovarius C. bathyomarinum (Allgaier et al., 2003; Park et al., 2007; Hwang 4436 4435 gene was amplified from all strains and BChl- a -containing bacteria recovered from marine envi- , originally isolated from deep-sea hydrothermal a pufM set from hydrothermal vents, they suggest that it may Congregibacter litoralis ff R. halotolerans o Alphaproteobacteria species is not surprising since -like bacteria associated with algal cultures did not appear to strains representing two clades were identified. Although strains synthetized. In this study, we cultivated our strains in low carbon Roseateles depolymerans . This finding is not very surprising because cultivated AAP bacteria C. bathyomarinum a . It is even not uncommon that strains containing the erent associated bacterial species that may rely on organic compounds (Biebl et al., 2005a) and other AAP bacteria isolated from algal cultures a ff C. bathyomarinum Erythrobacter R. halotolerans production (Labrenz et al., 1999; Allgaier et al., 2003; Biebl et al., 2005a). Roseovarius a were only isolated from microalgal cultures obtained during the cruise. Similarly R. mucosus Several of our isolates belonged to species whose type strains were not reported to The distribution of our isolates within at least 9 di direct plating. Therefore, this study demonstrates that primary cocultivation with other nated by specific bacterialto taxa. select for On di theavailable other in hand, algal phytoplankton exudates cultures andpionate are production (DMSP). likely of osmolytes It suchculture is, as conditions however, dimethyl selected sulfoniopro- not for possiblepromoted their to cultivability conclude ordence whether ability of algal to form growth coloniesbe and on restricted agar to medium. thisisolation The putative of high new three inci- species. taxa -including Indeed, an this additional new culture species- approach that allowed were the not obtained by ans to (Biebl et al., 2005b;benefit Biebl from et algal al., extracellularused 2006; products. for Biebl It algae et is may al., also also 2007), conceivable contribute that this to culture species the conditions may in probably vitro development of communities domi- media under natural light-dark cycle andthat several all independent our lines isolates of are evidence AAPs. indicate signals The and fluorescence wereimaging, recorded respectively. in Species all of cultures thetures genus using are fluorometry reported and to infrared BChl- contain low amounts ofTwelve BChl- from both clades were recovered from the same sample, those related to et al., 2009),genes pointing seems out to that bemay the probably highly expression be dependent of considered on(Biebl as the environmental a and photosynthetic parameters. result Wagner-Dobler, reaction of 2006)amounts These centre inappropriate of and culture failures BChl- possibly conditions employed inadequate methods to detect trace genes were found not to produce BChl- produce BChl- be endemic to deep ventponents. plumes and However, possibly the benefit wide fromthe hydrothermal Mediterranean distribution e Sea of (3 stationssurface within and the up western to(Koblizek basin et and 85 m al., the depth) Ionian 2003;isolation Sea and Jiao of at et their the al., occurrence 2010)the in does more other not commonly surface favor cultured this marineronments BChl- hypothesis. (Shiba waters et The al., frequent 1991; Kolber et al., 2001; Koblizek et al., 2003) . AAP isolate was vent plume waters (Yurkovwere and isolated Beatty, from 1998a). deepOcean ocean (Rathgeber Other waters et strains abovecolonies the al., of of Juan 2008). de Fuca Since Ridge these in authors the Pacific were unsuccessful to obtain phaproteobacteria are dispersed throughout the rial representative, gammaproteobacterial species, al., 2007) and three unassigned2004; gammaproteobacterial Cho isolates et (Cho al., and 2007; Giovannoni, Thrash et al., 2010). tivable diversity for this functional group in the Mediterranean Sea. The most frequent assessing cultivable diversity although AAPtivable bacteria fraction. represented a low To part ourdiversity of of knowledge, marine the although AAP cul- bacteria severalet (Allgaier studies al., et 2008; examined al., Salka 2003; the et al., Koblizekisolated cultivable et 2008), this in al., work 2003; the presents Rathgeber the frame largest of collection of an AAP oceanographic bacteria cruise. All our isolates belonged to the bacteria that formed visible colonies on plates were identified phylogenetically, thus 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | to .A ex- RT- cul- mRNA pufM pufM shore re- pufM transcripts transcripts ff pufM n et al., 2006; ` ı a et al., 2002). ´ pufM pufM ej sequences also in- R. denitrificans mRNAs in daytime sea- pufM Gammaproteobacteria pufM transcripts in mRNA library, namely BOUM 2 was puf 10, well represented in the DNA library in the metabolically active AAP − pufM 4438 4437 5 and 19), previously undetected in the Mediterranean − − mRNAs from the stations harvested in both cruises, pufM 17 and Moreover, although the four strains isolated from marine . − 1 l of seawater in all stations sampled during the night time. ∼ Gammaproteobacteria cult the comparison between both data sets. However, comparison between synthesis by high levels of irradiance in cultivated AAP bacteria (Yurkov and ffi gammaproteobacterial sequences were detected in coastal and o a sequences were similar to sequences recovered previously in DNA libraries from community pufM Previous studies have also revealed a high contribution of RT-PCR analysis has been used to detect the expression of functional genes in the relative natural abundance 2010) and suggest theirNOR5/OM60 dominant clade photosynthetic activity. also Our wellMediterranean sequences represented Sea fall in waters into a (Feingersch the to metagenomic et have library al., a 2010). from the cosmopolitanence This eastern for occurrence clade coastal in was waters recentlyserved the (Yutin shown this et marine habitat al., environment, preference 2007;of in with Yan the a et Mediterranean al., clear Seagions 2009). since prefer- (Lehours equivalent We proportions et didbe al., not linked 2010). clearly to ob- the However, fact we that relative acknowledge clone that library this abundance does discrepancy not may necessarily reflect AAP populations in variousYutin marine et environments al., (Hu 2007) et butThe al., their 2006; data activity Mas obtained has in onlyof been this the poorly work gammaproteobacterial evaluated support AAP (B the bacteria in previous the observations Mediterranean on Sea the (Lehours abundance et al., curred alone. Phylotypes BOUM(as 3, PROSOPE 14, 34tion of and their 20), mRNA,most are are frequently presumably clearly photosynthetically detected present active phylotypenot and, as in abundantly well. as the recovered in indicated In the contrast,cluded by DNA the OTUs the library. (BOUM The detec- 12, expressed Sea. These phylotypes mayhighly represent active. bacteria that are uncommon but metabolically thetic groups detectable bysimilar trend direct has PCR been of observedpression in DNA analysis other could in studies lake be (Zanioverlap Fryxell et shown between (Antarctica) al., our to (Karr 2000) RNA et including express library andet al., the 2003). al. PROSOPE DNA (2010), We library found but produced substantial by also Lehours many cases in which a particular DNA or RNA sequence oc- stations C and MIO, both located in the eastern basin, indicated that fewer photosyn- we were not able tomaking amplify di water samples was likelytranscripts to in be the total amay RNA consequence have extracts. been of obtained However, the we from low acknowledge larger that volumes relative of amounts seawater. ofenvironment these and several studiesbased have assemblages (Zani compared et the al., 2000;pufM composition Labrenz et of al., DNA 2010). and Thethe majority RNA- PROSOPE of cruise expressed in the Mediterranean Sea (Lehours et al., 2010). Unfortunately, PCR products could be obtainedThis from finding stations is 25 and consistenttures B with shifted sampled from the during darkness decrease the toof day light of BChl- time. (Nishimura et al., 1996)Beatty, and 1998b). the complete inhibition Sincesame we RNA succeeded extracts in in amplifying RT-PCRs, the other failure functional to genes amplify using the The isolation and analysis ofis mRNA an transcripts important from step environmental tobial microbial increase ecology. samples our understanding In of order theat complex the to processes time investigate of of the micro- of sample diversity collection the of and AAP bacteria thus community, expressing coding likely RT-PCR was for the the performed photosynthetically photosynthetic to active reaction detect center. members could Our the be results expression detected showed of from that Using this the gene same experimental conditions and the same seawater volume, no previously uncultured microorganisms. 4.2 Dominance of organism(s) may be a fruitful approach to extend the cultivable diversity and to isolate 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | per cell a grow optimally in synthesis. Our results a 0.8 µM) of the inoculum to transcripts bring two major < C. litoralis 0.8 µm fraction) (Lamy et al., < pufM 7) were exclusively detected in the , is an obligate aerobe (Csotonyi et − liated with any cultivated organisms. ffi 6, and C. litoralis − 4440 4439 synthesis, resulting in higher species diversity. a transcripts was present in the most oligotrophic basin pufM nity and increased BChl- ffi genes (Fuchs et al., 2007; Cho et al., 2007), it cannot be taken for granted that RT-PCR analysis to identify theprevious active observations populations on of the AAP bacteria dominant supports role the of the gammaproteobacterial AAP bacteria in the Mediterranean Sea during the stratificationmost period. environmental sequences were not a In the latter metagenomic analysis, Feingersch et al. (2010) showed that strain HTCC The highest diversity of (i) (ii) 5 Conclusions The present data based onconclusions: AAP culture isolation and tempt to favor colony formation belowHowever, strain the EG19, air-solid a interface distant could relative be to al., more 2008). appropriate. Finally, we proposecultured that AAP further bacteria cultivation could attemptsremove consider large, to filtration generally isolate (pore highly yet size, active, thesein particle-associated un- soft bacteria agar and medium colony for isolation bacteria unable to grow at the air-water interface. These data highlight the existing gapstosynthesis, in especially the in understanding the of Mediterranean aerobicfor Sea anoxygenic culture-dependent and pho- investigations likely to globally isolate and these form yet the uncultured basis groups of bacteria. We experienced this situation duringdecreased the cruise along because the bacterial oligotrophic and gradient.because AAP This gammaproteobacterial cultivability can AAP explain bacteria ourers have failure, et been but only al., isolated partly, 2000; onmicroaerophilic agar Csotonyi conditions et plates (Fuchs (Eil- al., et 2008). al., 2007; Since cultures Spring of et al., 2009), strategies that at- colonies on agar upon their first isolation from seawater (Cho and Giovannoni, 2004). that obligately oligotrophic gammaproteobacterial AAP bacteria are not able to form 2080, one of the fourhits gammaproteobacterial displaying AAP only bacteria currently agenome isolated, 70 sequences recruited % are identity good average.ranean references Sea. This for We highlights organisms attempted in that livingAAP this in none diversity. study the to of By vary eastern the using cultureyeast Mediter- current conditions low extract, local to concentrations seawater isolate or of the supernatant undefinedgammaproteobacterial largest of AAP substrates an colonies algal like although culture, they peptone we wereIn were and active unable the at to the Mediterranean grow time Sea, of2011) sampling. most are of free-living, the the common AAP lifestyle bacteria of ( oligotrophic bacteria. It has been shown gradient and that nutrient and/ortogether carbon with limitation favors this BChl- lastcompetition study due suggest to that nutrient insubstrate limitation the a may most favor extreme less oligotrophic abundant conditions, AAP bacteria with high and diversity of AAP assemblages suggestedsis previously of by their a distribution large-scale survey patternslotypes analy- (Jiao occurred et in al., the 2007).photosynthetically western The active and OTUs most (BOUM eastern abundantly 4, detected Mediterraneanultra-oligotrophic phy- basins. eastern basin However, several confirming oura previous companion study paper, (Lehours Lamy et et al., al. 2010).in (2011) showed AAP In that bacterial the concentration communities of waswestern BChl- significantly basin, higher suggesting in that the reliance eastern basin on than phototrophy in varied the along the oligotrophic pufLM all members of NOR5/OM60 clade are AAP bacteria. where abundance of AAPet bacteria al., is 2011). the These lowest findings during are consistent the with stratification the period contrasting (Lamy variation of abundance surface waters KT71, HTCC2080, HTCC2246 and HTCC2148 were shown to contain 5 5 15 10 20 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ) as and Roseo- ´ ebastien comb. nov. and ´ eane Dahan are Stappia aggregata formation in taxonomically a Roseibium denhamense clade with trace amounts of http://www.com.univmrs.fr/BOUM Labrenzia marina as cient help in CTD rosette management ffi Roseobacter for outstanding shipboard operation. Claudie , Appl. Environ. Microbiol., 70, 432–440, 2004. , Environ. Microbiol., 9, 1456–1463, 2007. 4442 4441 gen. nov., sp. nov., a novel alphaproteobacterium , and of the species Stappia marina cultures. This work was financially supported by the L’Atalante comb. nov., and emended descriptions of the genera er, A. A., Zhang, J. H., Zhang, Z., Miller, W., and Lipman, D. ff , and a proposal for reclassification of a Roseibium gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated Isochrysis and , Int. J. Syst. Evol. Microbiol., 57, 1095–1107, 2007. sp. nov., a novel marine aerobic alphaproteobacterium that forms comb. nov., of , Int. J. Syst. Evol. Microbiol., 55, 2377–2383, 2005a. , Int. J. Syst. Evol. Microbiol., 56, 821–826, 2006. Labrenzia alba Gammaproteobacteria Gammaproteobacteria a a This is a contribution of the BOUM (Biogeochemistry from the Olig- Labrenzia alexandrii sp. nov., a member of the as , H., Felske, A., and Wagner-Dobler, I.: Aerobic anoxygenic photosynthesis clade bacteria from diverse marine habitats, Appl. Environ. Microbiol., 69, Stappia ff , Stappia alba Dinoroseobacter shibae Roseobacter ` a, O., Suzuki, M. T., Heidelberg, J. F., Nelson, W. C., Preston, C. M., Hamada, T., Eisen, J. Barofsky, D. F., and Giovannoni,oligotrophic S. marine J.: Polyphyletic photosynthetic reaction centre genes in D. M., Schmidt, T.(RDP-II): M., previewing Garrity, a G. new autoaligner M.,, Nucleic that and Acids allows Res., Tiedje, regular 31, updates J. 442–443, and M.: 2003. the The new prokaryotic Ribosomal Database Project I.: Description of containing bacteriochlorophyll- Labrenzia aggregata of Pannonibacter Roseibium hamelinense oligotrophic marine Hoeflea phototrophica bacteriochlorophyll- diverse aerobic anoxygenicregimen phototrophic and bacteria starvation, Process in Biochem., chemostat 41, 2153–2159, culture: 2006. Influence of light varius mucosus bacteriochlorophyll- I.: from dinoflagellates, Int. J. Syst. Evol. Microbiol., 55, 1089–1096, 2005b. A., Fraser, C. M.,genic and phototrophs, DeLong, Nature, E. 415, F.: 630–633, 2002. Unsuspected diversity among marine aerobic anoxy- J.: Gapped BLAST and PSI-BLAST:Nucleic a Acids new Res., generation of 25, protein 3389–3402, database 1997. search programs, 5051–5059, 2003. in ´ ej Cho, J. C., Stapels, M. D., Morris, R. M.,Cole, J. Vergin, R., Chai, K. B., Marsh, L., T. L., Schwalbach, Farris, R. M. J., Wang, S., Q., Kulam, Givan, S. A., S. Chandra, S., A., McGarrell, Cho, J. C. and Giovannoni, S. J.: Cultivation and growth characteristics of a diverse group of Biebl, H., Pukall, R., Lunsdorf, H., Schulz, S., Allgaier, M., Tindall, B. J., and Wagner-Dobler, Biebl, H., Tindall, B. J., Pukall,Biebl, R., H. and Lunsdorf, Wagner-Dobler, I.: H., Growth and Allgaier, bacteriochlorophyll- M., and Wagner-Dobler, I.: Biebl, H., Allgaier, M., Tindall, B. J., Koblizek, M., Lunsdorf, H., Pukall, R., and Wagner-Dobler, Biebl, H., Allgaier, M., Lunsdorf, H., Pukall, R., Tindall, B. J., and Wagner-Dobler, I.: B Altschul, S. F., Madden, T. L., Scha Allgaier, M., Upho The publication of this article is financed by CNRS-INSU. References otrophic to the Ultraoligotrophic Mediterranean) experiment ( Acknowledgements. Marec and Louis Prieur areand warmly data thanked processing. for their e We wish to thank Thierryare Moutin for grateful coordinating to the Michal BOUM Koblizekred cruise for and help system inviting with us available detection onboard. ofSequencing in We AAP Platform his bacterial colonies laboratory, at usingHenry Morgan the the infra- for Perennou Station and the Biologique)program Gwenn fresh for LEFE-CYBER Tanguy grown help (Biogenouest PANAME with (CNRS-INSU).supported by sequencing Dominique grants and from Boeuf the to and Ministry of S Oc Higher Education and Research. of the frenchtional national IMBER project. LEFE-CYBER The program,l’Univers BOUM (INSU) experiment the was and european coordinated managedThe by IP by the authors the SESAME thank Institut Centre the des and National crew Sciences of de de the the la interna- R/V Recherche Scientifique (CNRS). 5 5 30 25 20 15 10 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ` a, sp. ´ Cit- ej spp. during Sagittula stellata Erythrobacter Vibrio gen. nov., sp. nov. , isolated from marine habitats, erent photosynthetic operons in ff Marivita cryptomonadis . 4444 4443 JL354 as revealed by whole-genome sequencing, J. Bacteriol., ´ eguiner, B., Moutin, T., Rimmelin, P.,Ras, J., Claustre, H., Leblond, sp. nov., of the family ` a, O., Bidigare, R. R., Christensen, S., Benitez-Nelson, B., Vetriani, C., Kolber, ´ ej gene in oxic seawater, FEMS Microbiol. Lett., 263, 200–206, 2006. Marivita litorea Vetriani, C., Koblizek, M., Rathgeber,toheterotrophic C., bacteria and to Falkowski, the P. carbon G.: cycle Contribution in of the aerobic ocean,starvation-survival pho- Science, and 292, recovery, 2492–2495, Appl. 2001. Environ. Microbiol., 58, 201–207, 1992. microorganisms on the Southwestern U. S. continental shelf, Appl. Environ. Microbiol., 59, anoxygenic phototrophs in the ocean, Environ. Microbiol., 9, 2401–2406,in 2007. surface waters of the open ocean, Nature, 407, 177–179, 2000. ultraphytoplankton, J. Phycol., 23, 633–638, 1987. M. K., Falkowski, P. G., andstrains Kolber, from Z. the S.: upper Isolation ocean, and Arch. characterization Microbiol., of 180, 327–338, 2003. pufM romicrobium bathyomarinum 192, 1169–1170, 2010. diversity of phototrophic purple bacteria inMicrobiol., a 69, permanently frozen 4910–4914, Antarctic 2003. lake, Appl. Environ. S., Johnson, J., Glockner,teobacterium F. O., capable and of Amann,104, aerobic R.: 2891–2896, anoxygenic 2007. Characterization photosynthesis, of Proc. a marine Natl. gammapro- Acad.gen. Sci. nov, USA, sp. nov,Bacteriol., a 47, lignin-transforming 773–780, bacterium 1997. from a coastal environment, Int. J. Syst. diversity of aerobic anoxygenic phototrophicFEMS bacteria Microbiol. in Ecol., saline 67, lakes 268–278, on 2009. the Tibetan plateau, pattern of abundance and diversityocean, of Environ. aerobic Microbiol., anoxygenic 9, phototrophic 3091–3099, bacteria 2007. in the global on growth and sinking1981. rate of 2 planktonic diatoms in culture, Br. Phycol. J., 16, 395–410, University of Washington, Seattle, 1993. DeLong, E. F.: MicrobialAcad. community Sci. USA, gene 105, expression 3805–3810, in 2008. ocean surface waters, Proc. Natl. and Int. J. Syst. Evol. Microbiol., 59, 1568–1575, 2009. novel pelagic bacteria frompicoplankton, Appl. the Environ. German Microbiol., 67, bight 5134–5142, and 2001. their seasonalO.: contributions Microbial community to genomics surface 4, in 78–87, eastern 2010. Mediterranean Sea surface waters, ISME J., anoxygenic phototrophs from529–539, a 2008. Canadian hypersaline spring system, Extremophiles,pelagic bacteria 12, from the North Sea, Appl. Environ. Microbiol., 66, 3044–3051, 2000. N., Oriol, L., andranean Sea, Pujo-Pay, Biogeosciences, M.: 8, 459–475, Deephttp://www.biogeosciences.net/8/459/2011/ 2011, silicon maxima in the stratified oligotrophic Mediter- the Mid-Atlantic Bight and2006. the North Pacific Gyre, Appl. Environ. Microbiol., 72, 557–564, Kramer, J. G. and Singleton, F. L.: Measurement of rRNA variations in natural communities of Kolber, Z. S., Plumley, F. G., Lang, A. S., Beatty, J.Kramer, T., J. Blankenship, G. R. E., and VanDover, Singleton, C. F. L., L.: Variations in rRNA content of marine Kolber, Z. S., Van Dover, C. L., Niederman, R. A., and Falkowski, P.G.: Bacterial photosynthesis Koblizek, M., Masin, M., Ras, J., Poulton, A. J., and Prasil, O.: Rapid growth rates of aerobic Koblizek, M., B Hwang, C. Y., Bae, G. D., Yih, W., and Cho, B. C.: Keller, M. D., Selvin, R. C., Claus, W., and Guillard, R. R. L.: Media for the culture of oceanic Hu, Y., Du, H., Jiao, N., and Zeng, Y.: Abundant presence of the gamma-like proteobacterial Jiao, N., Zhang, R., and Zheng, Q.: Coexistence of two di Karr, E. A., Sattley, W. M., Jung, D. O., Madigan, M. T., and Achenbach, L. A.: Remarkable Gonzalez, J. M., Mayer, F., Moran, M. A., Hodson, R. E., and Whitman, W. B.: Jiao, N., Zhang, Y., Zeng, Y., Hong, N., Liu, R., Chen, F., and Wang, P.: Distinct distribution Fuchs, B. M., Spring, S., Teeling, H., Quast, C., Wulf, J., Schattenhofer, M., Yan, S., Ferriera, Jiang, H., Dong, H., Yu, B., Lv, G., Deng, S., Wu, Y., Dai, M., and Jiao, N.: Abundance and Jaworski, G. H. M., Talling, J. F., and Heaney, S. I.: The influence of carbon dioxide-depletion Frias-Lopez, J., Shi, Y., Tyson, G. W., Coleman, M. L., Schuster, S. C., Chisholm, S. W., and Felsenstein, J.: PHYLIP (Phylogeny Inference Package). Version 3.5., Department of Genetics, Eilers, H., Pernthaler, J., Peplies, J., Glockner, F. O., Gerdts, G., andFeingersch, Amann, R., R.: Suzuki, Isolation M. of T., Shmoish, M., Sharon, I., Sabehi, G., Partensky, F., and B Eilers, H., Pernthaler, J., Glockner, F. O., and Amann, R.: Culturability and In situ abundance of Csotonyi, J. T., Swiderski, J., Stackebrandt, E., and Yurkov, V. V.: Novel halophilic aerobic Crombet, Y., Leblanc, K., Qu Cottrell, M. T., Mannino, A., and Kirchman, D. L.: Aerobic anoxygenic phototrophic bacteria in 5 5 30 25 20 30 15 10 25 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | a from Roseo- Staleya zek, M.: ı Prochloro- , Plant Cell , B., Reichel, ff -like bacteria in Proteobacteria doi:10.5194/bg-8-883- , Syst. Appl. Microbiol., genes, involved in aero- Nitrosopumilus maritimus eri ff Roseobacter , 2011. pufM eri pfei gen. nov., sp. nov., an alpha- ff ` a, O.: Roseobacter denitrificans sp. nov., alpha-3- ´ ej 4446 4445 Omphalius pfei , 2008. doi:10.5194/bg-8-973-2011 Sulfitobacter brevis Tateyamaria omphalii erences among cultured isolates, Limnol. Oceanogr., 44, 628–638, sp. nov., a marine bacterium isolated from the East Sea, Korea, Int. J. ff gen. nov., sp. nov., a budding bacterium with variable bacteriochlorophyll- gen. nov., sp. nov. and doi:10.5194/bg-5-203-2008 operon in an aerobic photosynthetic bacterium, : Ecotypic di , 2011. puf n, M., Zdun, A., Ston-Egiert, J., Nausch, M., Labrenz, M., Moulisova, V., and Kobl ı 2011 Moutin, T., and Prieur,western L.: to eastern Integrated Mediterranean survey Sea, of Biogeosciences, 8, elemental 883–899, stoichiometry (C, N, P) from the Sulfitobacter litoralis Syst. Evol. Microbiol., 57, 692–695, 2007. expression in aquatic microorganims, Appl. Environ. Microbiol., 59, 451–457, 1993. the Physiol., 37, 153–159, 1996. Red and Mediterranean Sea aerobicMicrobiol., anoxygenic 71, photosynthetic 344–353, populations, 2005. Appl. Environ. Oligotrophic to the Ultraoligotrophic Mediterraneanprep., (BOUM) 2011. experiment, Biogeosciences, in the same unknown bacterialLimnol. lineages Oceanogr., in 40, Atlantic 148–158, 1995. and Pacific bacterioplankton communities, display and annotation, Bioinformatics, 23, 127–128, 2007. Steppi, S., Jobb, G., Forster, W.,S., Brettske, I., Hermann, Gerber, S., S., Ginhart, Jost,B., A. R., W., Strehlow, Gross, Konig, R., O., A., Stamatakis, Grumann, Liss, A.,and T., Stuckmann, Schleifer, Lussmann, N., K. R., Vilbig, H.: May, A.,32, M., Lenke, ARB: 1363–1371, Nonho M., 2004. a Ludwig, software T., environment Bode, for A., sequence data, Nucleic Acids Res., Seasonal changes and diversity ofMicrob. aerobic Ecol., anoxygenic 45, phototrophs 247–254, in 2006. the Baltic Sea, Aquat. coccus 1999. tion and diversity ofrelation aerobic to anoxygenic environmental variables, phototrophic FEMS bacteria Microbiol. in Ecol., the 74, 397–409, Mediterranean 2010. Sea in totrophs in temperate freshwater systems, Environ. Microbiol., 10, 1988–1996, 2008. D.: Picoplankton diversity in the203–214, South-East Pacific Ocean from cultures, Biogeosciences, 5, bic anoxygenic photosynthesis, in theans, bacterial FEMS communities Microbiol. associated Ecol., with 71, colonial 387–398, ascidi- 2010. Pujo-Pay, M., Oriol, L., Bariat, L.,aerobic Catala, anoxygenic P.,Cornet-Barthaux, phototrophic bacteria V., and along Lebaron, anSea, P.: oligotrophic Ecology Biogeosciences, gradient of in 8, the 973–985, Mediterranean guttiformis hypersaline, heliothermal and meromictic50, antarctic 303–313, Ekho 2000. Lake, Int. J. Syst. Evol. Microbiol., Relevance of a crenarchaeotalto subcluster ammonia related oxidation in to the2010. Candidatus suboxic zone of the central Baltic Sea, ISME J., 4, 1496–1508, varius tolerans production from hypersaline Ekho Lake, Int. J. Syst. Bacteriol., 49, 137–147, 1999. 2430–2436, 1993. proteobacterium isolated from a top30, 371–375, shell 2007. Ranchou-Peyruse, A., Herbert, R., Caumette, P., and Guyoneaud, R.: Comparison of Pujo-Pay, M., Conan, P., Oriol, L., Cornet-Barthaux, V., Falco, C., Ghiglione, J.-F., Goyet, C., Pichard, S. L. and Paul, J. H.: Gene-expression per gene dose, a specific measure of gene- Park, J. R., Bae, J. W., Nam, Y. D., Chang, H. W., Kwon, H. Y., Quan, Z. X., and Park, Y. H.: Oz, A., Sabehi, G., Koblizek, M., Massana, R., and B Nishimura, K., Shimada, H., Ohta, H., Masuda, T., Shioi, Y., and Takamiya, K.: Expression of Moutin, T., Van Wambeke, F., and Prieur, L.: IntroductionMullins, to T. D., the Britschgi, Biogeochemistry T. B., from Krest, R. the L., and Giovannoni, S. J.: Genetic comparisons reveal Moore, L. R. and Chisholm, S. W.: Photophysiology of the marine cyanobacterium Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Letunic, I. and Bork, P.: Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree Mas Lehours, A. C., Cottrell, M. T., Dahan, O., Kirchman, D. L., and Jeanthon, C.: Summer distribu- Masin, M., Nedoma, J., Pechar, L., and Koblizek, M.: Distribution of aerobic anoxygenic pho- Le Gall, F., Rigaut-Jalabert, F., Marie, D., Garczarek, L., Viprey, M., Gobet, A., and Vaulot, Martinez-Garcia, M., Diaz-Valdes, M., and Anton, J.: Diversity of Lamy, D., Jeanthon, C., Cottrell, M. T., Kirchman, D. L., Van Wambeke, F., Ras, J., Dahan, O., Labrenz, M., Sintes, E., Toetzke, F., Zumsteg, A., Herndl, G. J., Seidler, M., and Jurgens, K.: Labrenz, M., Tindall, B. J., Lawson, P. A., Collins, M. D., Schumann, P., and Hirsch, P.: Labrenz, M., Collins, M. D., Lawson, P. A., Tindall, B. J., Schumann, P., and Hirsch, P.: Kurahashi, M. and Yokota, A.: 5 5 30 25 20 15 10 30 25 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | sp. Pro- ` a, O.: ´ ej Labrenz Tateyamaria gen. nov., sp. nov., and of Cape Hatteras, North – producing bacteria in , Syst. Appl. Microbiol., ff Thalassobaculum salex- a comb. nov. and emended gen. nov., sp. nov., a new Staleya guttiformis Sulfitobacter donghicola from the NW Mediterranean (vol 60, pg 209, 2010), Int. J. on the east and west coasts of Tateyamaria a Congregibacter litoralis Gammaproteobacteria Rhodospirillaceae Thalassobaculum 4448 4447 Sulfitobacter guttiformis as Roseateles depolymerans , Int. J. Syst. Evol. Microbiol., 57, 1788–1792, 2007. : investigation of the fate of specific segments of tran- ` a, O.: Novel primers reveal wider diversity among marine ´ ej Sulfitobacter Sulfitobacter , J. Bacteriol., 192, 3842–3843, 2010. sp. nov., a facultatively anaerobic alphaproteobacterium isolated from – containing obligate aerobe belonging to the beta-subclass of the Escherichia coli a , Int. J. Syst. Bacteriol., 49, 449–457, 1999. , Int. J. Syst. Evol. Microbiol., 60, 1770-1777, 2010. sp. nov., a new member of the family aerobic anoxygenic phototrophs, Appl. Environ. Microbiol., 71, 8958–8962, 2005. Assessing diversity and biogeographyface waters of of aerobic the anoxygenic Atlanticmetagenomes, phototrophic and Environ. Pacific Microbiol., bacteria Oceans 9, in using 1464–1475, 2007. the sur- Global Ocean Sampling expedition smoker plume waters ofbiol., the 64, Juan 337–341, de 1998a. Fuca ridge in the PacificRev., ocean, 62, 695-724, Appl. 1998b. Environ. Micro- nov., isolated from seawater of theet East Sea al. in 2000 Korea, to transfer of description the of genus the genus planktonic aerobic anoxygenic photoheterotrophic bacteriaOceanogr., in 51, the 38–46, northwest 2006. Atlantic, Limnol. regulation in the aerobic gammaproteobacterium PLoS One, 4, 2009. quence analysis in the present species definition in bacteriology, Int. J. Syst. Bacteriol., 44, Syst. Evol. Microbiol., 60, 2507–2507, 2010. messenger-RNA in scripts, Proc. Natl. Acad. Sci. USA, 80, 653–657, 1983. and phylogeny of the32, NOR5/OM60 124–139, 2009. clade of iology of aerobic bacteriaAustralia, Appl. containing Environ. bacteriochlorophyll- Microbiol., 57, 295–300, 1991. sequences of strains HTCC2148 andGammaproteobacteria HTCC2080, belonging to the OM60/NOR5 clade of the igens Sea, and emended description of the genus R. A., Oakley, B.Van B., Horn, Parks, D. D. J., H., andcommunity-supported Robinson, Weber, C. C. software F.: J., for Introducing Sahl, describing Mothur:Environ. J. and Microbiol., Open-source, W., 75, comparing platform-independent, Stres, 7537–7541, microbial B., 2009. communities, Thallinger, G. Appl. G., 846–849, 1994. Y., Kanagawa, T., andbacteriochlorophyll- Hanada, S.: teobacteria Tateyamaria pelophila tidal-flat sediment, and emended descriptionsomphalii of the genus A., Plumley, F. G.,characterization Van Dover, of C. aerobic L., phototrophicOcean, Beatty, Photosynth. bacteria J. Res., at 97, T., the 235–244, and 2008. Juan Yurkov, V.: de Vertical Fuca distributiondepth Ridge distribution, and in and the compositionfour Pacific of basins aerobic of bacteriochlorophyll- the central Baltic Sea, Appl. Environ. Microbiol., 74, 4398–4404, 2008. picoplankton 16S rRNA genesCarolina, Limnol. cloned Oceanogr., from 42, 811–826, the 1997. continental shelf o cultivation-dependent and molecular methods forphototrophs studying in the sediments diversity of of an anoxygenic eutrophic2006. purple brackish lagoon, Environ. Microbiol., 8, 1590–1599, Yutin, N., Suzuki, M. T., andYutin, B N., Suzuki, M. T., Teeling, H., Weber, M., Venter, J. C., Rusch, D. B., and B Yurkov, V. V., and Beatty, J. T.: Aerobic anoxygenic phototrophic bacteria, Microbiol. Mol. Biol. Yurkov, V. and Beatty, J. T.: Isolation of aerobic anoxygenic photosynthetic bacteria from black Yoon, J. H., Kang, S. J., Lee, M. H., and Oh, T. K.: Description of Stackebrandt, E. and Goebel, B. M.: A place for DNA-DNA reassociation and 16S rRNA se- Spring, S., Lunsdorf, H., Fuchs, B. M., and Tindall, B. J.: The photosynthetic apparatus and its Von Gabain, A., Belasco, J. G., Schottel, J. L., Chang, A.Yan, C. S., Fuchs, Y., and B. M., Cohen, Lenk, S. S., N.: Harder, J., Decay Wulf, of J., Jiao, N. Z., and Amann, R.: Biogeography Sieracki, M. E., Gilg, I. C., Thier, E. C., Poulton, N. J., and Goericke, R.: Distribution of Urios, L., Michotey, V., Intertaglia, L., Lesongeur, F., and Lebaron, P.: Shiba, T., Shioi, Y., Takamiya, K., Sutton, D. C., and Wilkinson, C. R.: Distribution and phys- Thrash, J. C., Cho, J. C., Ferriera, S., Johnson, J., Vergin, K. L., and Giovannoni, S. J.: Genome Suyama, T., Shigematsu, T., Takaichi, S., Nodasaka, Y., Fujikawa, S., Hosoya, H., Tokiwa, Schloss, P.D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M., Hollister, E. B., Lesniewski, Sass, H., Kopke, B., Rutters, H., Feuerlein, T., Droge, S., Cypionka, H., and Engelen, B.: Salka, I., Moulisova, V., Koblizek, M., Jost, G., Jurgens, K., and Labrenz, M.: Abundance, Rathgeber, C., Lince, M. T., Alric, J., Lang, A. S., Humphrey, E., Blankenship, R. E., Vermeglio, Rappe, M. S., Kemp, P. F., and Giovannoni, S. J.: Phylogenetic diversity of marine coastal 5 5 30 25 20 30 15 10 25 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | b MAD MAD MAD MAD MAD 3 3 NO + NO genes in natural + 3 3 3 NO NO NO gen. nov., sp. nov., a + + + nifH sequences, Lett. Appl. pufM culture obtained in PRO2/5 culture obtained in JM/100 MAD culture obtained in PRO2/5 culture obtained in PRO2/5 MAD culture obtained in JM/100 MAD Pycnococcus Ochromonas Osteococcus Synechococcus Synechococcus Thalassobaculum litoreum 4450 4449 isolated from coastal seawater, Int. J. Syst. Evol. C) Sampling date Source of isolation Isolation medium ◦ Culture Collection) numbers. ff E 5 27 27/06/2008 mixed culture obtained in PRO2/5 E 25 22 08/07/2008 mixed culture obtained in PRO2/5 MAD E 100 16 08/07/2008 EEE 93E 5E 5 80 16 80 27 27 08/07/2008 16 16 06/07/2008 seawater 06/07/2008 09/07/2008 seawater 09/07/2008 seawater seawater seawater MAD MiA MAD MiA MAD E 5 23 22/06/2008 seawater MAD E 5 22 18/07/2008 mixed culture obtained in PRO2/5 MAD E 5 22 18/07/2008 mixed culture obtained in PRO2/5 E 5 22 18/07/2008 mixed culture obtained in PRO2/5 MAD E 5 18 19/07/2008 mixed culture obtained in PRO2/5 MAD EE 5 5 18 18 19/07/2008 19/07/2008 mixed culture obtained in PRO2/5 E 5 18 19/07/2008 unidentified algal culture obtained in PRO2/5 MAD E 100 15 14/07/2008 EE 100 100 15 15 14/07/2008 14/07/2008 E 5 22 18/07/2008 seawater MiA E 90 15 16/07/2008 seawater MAD E 25 15 19/07/2008 seawater MiA E 25 15 19/07/2008 seawater MAD E 5 18 19/07/2008 seawater MA E 5 18 19/07/2008 seawater MAD E 5 22 18/07/2008 seawater MAD E 90 15 16/07/2008 seawater MAD E 85 15 11/07/2008 seawater MiA E 85 15 11/07/2008 seawater MAD 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 primers to monitor aerobic anoxygenic phototrophic assemblages, 39 06 06 42 27 27 00 00 30 03 03 03 56 56 56 56 21 21 21 03 21 56 56 56 56 03 21 55 55 ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ Rhodospirillaceae ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ N 32 N 14 N 5 N 5 N 5 N 4 N 14 NN 4 4 N 4 N 5 N 5 N 5 N 5 N 5 N 4 N 4 N 4 N 4 N 5 N 5 N 7 NN 16 N 18 N 18 N 12 N 12 7 N 24 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pufM 38 40 05 05 05 13 40 13 13 13 06 06 06 05 06 13 13 13 13 05 06 38 53 08 08 10 10 38 03 ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ Station Latitude Longitude Depth (m) Temp ( Location, depth, temperature and date of sampling, source and medium of isolation of a tosynthetic bacteria byMicrobiol., distance-based 45, grouping 639–645, 2007. analysis of member of the family Microbiol., 58, 479–485, 2008. microbial assemblages in LakeAppl. George, Environ. New Microbiol., York, 66, detected 3119–3124, by 2000. reverse transcriptase PCR, fully degenerate Limnol. Oceanogr. Methods, 6, 427–440, 2008. isolates are identified by their RCC (Rosco indicates the agar medium used to isolate the corresponding organism (see the Material and methods section). RCC 1928 C 33 RCC 1927RCC 1929 15 35 24 41 RCC 1925–1926 24 41 RCC 1924 24 41 RCC 1923 27 43 RCC 1919 15 35 RCC 1920RCC 1921–1922 27 27 43 43 RCC 1918 27 43 RCC 1917 A 39 RCC 1914 A 39 RCC 1915–1916 A 39 RCC 1912–1913 24 41 RCC 1911 A 39 RCC 1910 27 43 RCC 1909 27 43 RCC 1908 27 43 RCC 1907 27 43 RCC 1905–1906 24 41 RCC 1899–1904 A 39 RCC 1895–1898 21 38 RCC 1878–1882RCC 1883–1887RCC 1888 BRCC 1889 BRCC 1890–1894 34 34 21 17 17 38 37 37 RCC 1877 13 34 Isolate RCC 1876 5 34 a b Table 1. cultivated Mediterranean AAP bacteria. Zhang, G. I., Hwang, C. Y., and Cho, B. C.: Zeng, Y. H., Chen, X. H., and Jiao, N. Z.: Genetic diversity assessment of anoxygenic pho- Zani, S., Mellon, M. T., Collier, J. L., and Zehr, J. P.: Expression of Yutin, N., Beja, O., and Suzuki, M. T.: The use of denaturing gradient gel electrophoresis with 5 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | libraries and pufM (99) 19 (RCC 1879–87, RCC 1890–98, RCC1907) (94) 1 (RCC 1918) (94) 1 (RCC 1926) (96) (99) (99) 5 (RCC 1899–04) (97) 2 (RCC1924, RCC1929) (98) (98) (97) (98) (98) (99) 8 (RCC 1905, RCC 1911-17) (96) 4452 4451 (96) 1 (RCC 1923) (99) JCM2831 95 Japanese unhulled rice CP001002 OCh114 98 Coastal marine sediments (Australia) CP000362 a Sagittula stellata Thalassobaculum salexigens Sulfitobacter dubius Erythobacter aquamaris Tateyamaria omphalii Thalassobaculum litoreum Erythrobacter litoralis Erythrobacter litoralis Citromicrobium bathyomarinum Tateyamaria omphalii Marivita litorea Marivita cryptomonadis Roseovarius halolerans Thalassobaculum salexigens Roseibacterium elongatum Roseovarius tolerans Roseobacter denitrificans Methylobacterium radiotolerans StC7-P349 Uncultured bacterium clone PROSOPE 42 99 Mediterranean Sea GQ468972 sequence accession (% 16S rRNA sequence length (bp) no. identity) Closest phylogenetic neighbor, nucleotide accession number and 16S rRNA gene Operational taxonomic units (OTU) recovered in the RNA-based RCC 1906RCC 1908 1300 1272 HQ871851 HQ871852 RCC 1888RCC 1889 1318 1352RCC 1909 HQ871846 RCC 1910 HQ871847 1072 1349 HQ871853 HQ871854 Isolate 16S rRNARCC 1876 NucleotideRCC 1877 Closest described speciesRCC 1878 1274 1317 1315 HQ871842 HQ871843 HQ871844 Other strains with identical RFLP RCC 1919 1351 HQ871856 RCC 1920RCC 1921RCC 1922 1357RCC 1925 1361RCC 1927 1352 HQ871857 RCC 1928 1381 HQ871858 1364 HQ871859 1367 HQ871861 HQ871862 HQ871863 As determined by the program BLAST. OTUBOUM 1BOUM 2 Representative clone BOUM StC7-P236BOUM 3 Closest BOUM relativeBOUM in 4 GenBank BOUM Uncultured StA6-CJ7BOUM bacterium 5 clone PROSOPE BOUM 40 StC3-CJ3BOUM 6 BOUM StA2-P128BOUM 7 Uncultured bacterium BOUM clone StC3-P114BOUM PROSOPE 8 14 Uncultured bacterium BOUM clone StC3-CJ17 UnculturedBOUM PROSOPE bacterium 9 15 clone PROSOPE BOUM 34 Uncultured StA6-P175BOUM bacterium 10 clone PROSOPE BOUM 41 Uncultured StA6-CJ24BOUM bacterium 11 BOUM clone St213-P260 PROSOPE 2 98 UnculturedBOUM bacterium BOUM 12 clone StA2-P146 PROSOPE 45 UnculturedBOUM Uncultured bacterium 13 BOUM bacterium clone StC3-P18 clone PROSOPE PROSOPE 26 20 Identity BOUM (%) St216-P381 99 Uncultured Mediterranean bacterium Sea clone 100 2A08 Habitat of closest Uncultured 99 relative bacterium clone envSargasso 97 Mediterranean Sea Mediterranean Sea 99 Mediterranean Sea 97 Mediterranean Sea 98 97 Mediterranean Sea Accession number Mediterranean Sea Mediterranean Mediterranean Sea Sea 87 GQ468970 96 Sargasso sea GQ468954 GQ468955 Delaware Bay GQ468964 GQ468971 GQ468944 GQ468975 GQ468961 GQ468960 DQ080981 EU191269 BOUM 14 BOUM St216-P280 BOUM 15BOUM 16 BOUM St213-P354BOUM 17 BOUM StC7-CJ19BOUM Uncultured BOUM 18 proteobacterium StC3-P336 clone EBAC000-29C02BOUM 19 BOUM Uncultured StC7-P232 bacterium cloneBOUM PROSOPE 20 BOUM 50 Global StA6-P161 Ocean Sampling GS-31-01-01-1P3 BOUM Uncultured 97 StC3-CJ10 bacterium clone 2A08 Uncultured bacterium clone jl-npgw26-s7 Uncultured bacterium clone Monterey PROSOPE Bay 45 86 90 Mediterranean Sea 95 Pacific Ocean (Galapagos 93 islands) 88 China Sea Mediterranean Sea EK382594 Delaware Bay AE008920 GQ468980 GQ468975 AY652833 EU191269 Table 3. general features of their closest relatives in GenBank. a Table 2. sequence length of the AAP bacteria isolated from the Mediterranean Sea. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ) • C 11 ) indicate stations 9  - 4 3 1 - - - 7 Alpha Alpha Alpha Proteobacteria Proteobacteria Proteobacteria 5 3 (DQ269074) (EF016464) (EF471666) (AY771771) (Y16267) concentrations (June 2008, courtesy to (EF203900) (M96746) 1 a 2 (EF512125)2 SK44 (EU512919) (EU431217) (AJ534215) - (Y13155) 8 (DQ412059) 351 (DQ104407)351 (AY344411) - (DQ915626) - (AB193438) CL (X78312) (AF465835) B litoreum (DQ665839) . JL . CSQ- HQ871861) vestfoldensis . WM2 (EF044234) HQ871851) (DQ915628) HQ871860) HQ871852) proteobacterium . LOB JM1 (EU512918) JM1 proteobacterium sp . B108 (EU742628) sp - HQ871863) denitrificans sp proteobacterium bathyomarinum mucosus DG949(AY258092) sp HQ871842) sp tolerans pontiacus HQ871862) HQ871850) longus HQ871846) HQ871844) CL litoralis HQ871845) HQ871848) omphalii HQ871847) Fig. 1 Fig. bacterium HQ871843) halotolerans HQ871849) alpha alpha stellata alpha 13 4454 4453 Loktanella litorea cryptomonadis nitrireducens bacterium RCC1925 ( RCC1925 uncultured alphaproteobacterium (EF688609) Roseobacter Roseobacter RCC1906 ( RCC1906 RCC1923 ( RCC1923 ( RCC1908 Thalassobaculum Roseobacter Roseobacter Roseovarius RCC1910 (HQ871854) RCC1910 RCC1922 (HQ871859) RCC1922 Roseobacter RCC1921 (HQ871858) RCC1921 Roseovarius RCC1928 ( RCC1928 Roseovarius bacterium DG878(AY258077) Sulfitobacter uncultured alphaproteobacterium (EF471669) RCC1916 (HQ871855) RCC1916 RCC1909 (HQ871853) RCC1909 RCC1919 (HQ871856) RCC1919 uncultured Rhodobacteraceae bacterium(AF513932) unidentified Sagittula 84 62 RCC1876 ( RCC1876 Roseobacter uncultured 88 65 Tateyamaria uncultured alpha proteobacterium ML42 (AJ315683) Erythrobacter Nisea Citromicrobium RCC1927 ( RCC1927 RCC1904 ( RCC1904 RCC1888 ( RCC1888 (HQ871857) RCC1920 ( RCC1878 82 RCC1885 ( RCC1885 Roseovarius RCC1897 ( RCC1897 RCC1889 ( RCC1889 Marivita uncultured 100 RCC1877 ( RCC1877 Marivita 74 RCC1903 ( RCC1903 100 100 100 63 100 58 15 100 93 56 100 100 90 60 97 100 88 90 64 100 96 KT71 (AAOA01000004) 61 65 100 100 100 100 100 96 97 100 100 Fig. 2 Fig. 89 72 81 100 64 70 litoralis 17 100 100 libraries were constructed. 19 Congregibacter 100 pufM 21 0.02 A 24 27 25

Phylogenetic analysis based on the 16S rRNA sequences indicating the position of Track of the BOUM cruise (16 June–20 July 2008) superimposed on the composite

) m (mg a Chl

3 – Fig. 2. selected strains recovered during thejoining BOUM methods. cruise. The Percentagesabove tree of 50 was % 1000 calculated are by bootstrap indicated.tions. the resamplings neighbor- Bar that represent support the number branching of points substitutions per 100 sequence posi- E. Bosc). Symbol types represent analyses performed at sampled stations: Black spheres ( SeaWiFS composite image of the sea surface Chl- Fig. 1. indicate stations where AAPwhere isolation both RNA-based was attempted and white squares ( Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | J K I H G F E C D B A H J K I G F E A B D C Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup Phylogroup 4456 4455 Fig.3b Fig.3a 5 m 85 m 5 m 100 m 90 m 5 m 50m - - - - Station A Station Station A Station Station 1- Station C Station C Station 21- Station 21- Station 5 m 85 m 5 m 100 m 90 m 5 m 50m - - - - Station A Station Station A Station Station 1- Station C Station C Station 21- Station 21- Station Caption on next page. Fig. 3b. Fig. 3a. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | pufM gene erent liated ff ffi pufM PARISMONY. genes a pufM Phylogenetic relationships of (b) erent clone libraries. Colors used to represent ff 4457 Phylogenetic relationships of (a) 50 % for branches found in the initial NJ trees. Sequences retrieved in this > phylogenetic trees showing the inferred phylogenetic relationships of liated to phylogroups C and K and to unidentified phylogroups. Trees are based ffi pufM the clone libraries are indicated at the left of the trees. study are indicated asfrequencies BOUM of 1 the to corresponding BOUM OTU 20. in The the di multivalue bar charts represent the relative Fig. 3. Sequences used to construct NJconfidence trees values are marked in bold and black circles on nodes represent sequences from AAP isolates andgroups environmental defined samples. by Color Yutin rangesto et highlight phylogroups al. the F, G, (2007). di Igenes and a to unidentified phylogroups. on a neighbor-joining (NJ) tree to which short sequences were added by ARB