Analysis of bacterial core communities in the central Baltic by comparative RNA-DNA- based fingerprinting provides links to structure-function relationships

SUPPLEMENTARY INFORMATION

(revised version April 14, 2011)

Ingrid Brettar1, Richard Christen2 and Manfred G. Höfle1*

1Department Vaccinology and Applied Microbiology, Helmholtz Centre of Infection Research

(HZI), Inhoffenstr. 7, D-38124 Braunschweig, Germany

2Virtual Biology Lab, Centre for Biochemistry, University of Nice Sophia Antipolis & CNRS,

Campus Valrose, F-06108 Nice, France

*Corresponding author. Email: [email protected]

Subject Category: Microbial ecology and functional diversity of natural habitats

Running title: Analysis of bacterial core communities in the Baltic Sea

1 SUPPLEMENTARY INFORMATION

Fig. S1. Representative 16S rRNA- (R) and 16S rRNA gene- (D) based SSCP fingerprints from bacterioplankton samples of the central Baltic Sea. Single stations are indicated as: Bot

= Bot1, Teili =T1, G = Gotland Deep (depth in m), ST = standard with five reference species.

All samples are labeled with the appropriate depth in meters (bottom) and the respective fingerprint (R/D) on the top of the lane.

Fig. S2. Cluster analysis of SSCP fingerprints from different samples of the surface (5m) samples on stations T1, G1 and Bot1 shown in Supplementary Fig. S1.

Fig. S3. Phylogentic tree based on 16S rRNA gene comparison of the reference bacteria

(accession numbers are given in front of the species name) from the class

Alphaproteobacteria with the phylotypes found in the central Baltic. Single phylotypes (PT) are indicated in bold and labeled with their sample origin (G=Gotland Deep (depth in m),

T=T1 and L=LL12), depth in meters and nucleic acid fraction (R = RNA, D = DNA) they were first detected. Phylotype 103 is not included in the tree due its deep routing position between Alpha- and Deltaproteobacteria.

Fig. S4. Phylogentic tree based on 16S rRNA gene comparison of the reference bacteria

(accession numbers are given in front of the species name) from the phylum Actinobacteria with the phylotypes found in the central Baltic. Single phylotypes (PT) are indicated in bold and labeled with their sample origin (G= Gotland Deep (depth in m), T=T1 and L=LL12), depth in meters and nucleic acid fraction (R = RNA, D = DNA) they were first detected.

2 Fig. S5. Phylogentic tree based on 16S rRNA gene comparison of the reference bacteria

(accession numbers are given in front of the species name) from the phylum Bacteroidetes with the phylotypes found in the central Baltic. Single phylotypes (PT) are indicated in bold and labeled with their sample origin (G= Gotland Deep (depth in m), T=T1 and L=LL12), depth in meters and nucleic acid fraction (R = RNA, D = DNA) they were first detected.

Table S1. Taxonomic identification of all phylotypes detected above the detection limit of

0.1% relative abundance and sorted according to their phylum, including the site and depth of detection and the type of fingerprint. Most of the phylotypes represent single bands of the

SSCP gel given in Supplementary Figure S1.

3 Fig. S1. Representative 16S rRNA- (R) and 16S rRNA gene- (D) based SSCP fingerprints from bacterioplankton samples of the central Baltic Sea. Single stations are indicated as: Bot = Bot1, Teili =T1, G = Gotland Deep (depth in m), ST = standard with five reference species. All samples are labeled with the appropriate depth in meters (bottom) and the respective fingerprint (R/D) on the top of the lane.

4 DNA

RNA

Fig. S2. Cluster analysis of SSCP fingerprints from different samples of the surface (5m) samples on stations T1, G1 and Bot1 shown in Supplementary Fig. S1.

5 Fig. S3. Phylogentic tree based on 16S rRNA gene comparison of the reference bacteria (accession numbers are given in front of the species name) from the class Alphaproteobacteria with the phylotypes found in the central Baltic. Single phylotypes (PT) are indicated in bold and labeled with their sample origin (G= Gotland Deep (depth in m), T=Teili 1 and L=LL12), depth in meters and nucleic acid fraction (R = RNA, D = DNA) they were first detected. Phylotype 103 is not included in the tree due its deep routing position between Alpha- and Deltaproteobacteria.

6 Fig. S4. Phylogentic tree based on 16S rRNA gene comparison of the reference bacteria (accession numbers are given in front of the species name) from the phylum Actinobacteria with the phylotypes found in the central Baltic. Single phylotypes (PT) are indicated in bold and labeled with their sample origin (G= Gotland Deep (depth in m), T=Teili 1 and L=LL12), depth in meters and nucleic acid fraction (R = RNA, D = DNA) they were first detected.

7 Fig. S5. Phylogentic tree based on 16S rRNA gene comparison of the reference bacteria (accession numbers are given in front of the species name) from the phylum Bacteroidetes with the phylotypes found in the central Baltic. Single phylotypes (PT) are indicated in bold

8 and labeled with their sample origin (G= Gotland Deep (depth in m), T=Teili 1 and L=LL12), depth in meters and nucleic acid fraction (R = RNA, D = DNA) they were first detected.

9 Table S1. Taxonomic identification of all phylotypes detected above the detection limit of 0.1% relative abundance and sorted according to their phylum, including the site and depth of detection and the type of fingerprint. Most of the phylotypes represent single bands of the SSCP gel given in Supplementary Fig. S1.

PT Accession Samplea / DNA/ phylum/class Closest 16S rRNA gene Accession Sim. Closest described species Accession Sim. No. No. depth RNAb sequence No. % c No. % c 116 FR715020 G120m R Acidobacteria uncultured acidobacterium FJ529316 96.2 Candidatus Solibacter usitatus GQ287461 91.6 5 FR714945 T,L,G5m D Actinobacteria uncultured actinobacterium GU127254 99.5 Planktophila limnetica FJ428831 95.6 6 FR714946 T,L,G5m D Actinobacteria uncultured actinobacterium FN668296 99.2 Planktophila limnetica FJ428831 93.6 15 FR714951 G5-30m D Actinobacteria uncultured actinobacterium FJ916152 92.8 Planktophila limnetica FJ428831 90.0 20 FR714955 G30m D Actinobacteria uncultured bacterium FN297464 97.4 Planktophila limnetica FJ428831 94.3 22 FR714956 G175m R Actinobacteria uncultured bacterium EU362277 90.3 NA 68 FR714987 L D Actinobacteria uncultured bacterium GQ340166 98.5 Gordonia sputi X80634 84.4 8 FR714948 L G5-80m R Bacteroidetes marine sponge bacterium EU346449 99.3 Roseivirga ehrenbergii AY739663 99.3 liquidOTU3 28 FR714961 G120-150m D/R Bacteroidetes uncultured Bacteroidetes EU626662 98.8 Lutibacter litoralis AY962293 98.5 32 FR714964 G80-120m D/R Bacteroidetes uncultured flavobacterium FJ745043 98.3 Gilvibacter sediminis AB255368 95.3 40 FR714969 G80-175m D/R Bacteroidetes uncultured bacterium FJ497462 98.0 Polaribacter glomeratus EU000227 92.1 44 FR714972 T,G5-110m D/R Bacteroidetes uncultured Cytophaga sp. AB015543 95.8 Rhodothermus marinus Y14143 93.6 46 FR714974 T,L,G5-120m D/R Bacteroidetes uncultured flavobacterium FN433334 98.5 Owenweeksia hongkongensis AB125062 88.9 47 FR714975 T,L,G5m D/R Bacteroidetes uncultured bacterium EU802071 99.5 Coccinistipes vermicola EF108212 92.6 62 FR714981 L,G5m R Bacteroidetes uncultured bacterium FJ352171 98.1 Gilvibacter sediminis AB255368 92.6 63 FR714982 L R Bacteroidetes uncultured flavobacterium FJ744887 95.4 Owenweeksia hongkongensis AB125062 92.5 65 FR714984 L D/R Bacteroidetes Chryseobacterium sp. 18C EU057839 100.0 Chryseobacterium joostei EF204455 99.3 67 FR714986 L,G5m D/R Bacteroidetes uncultured bacterium EU802159 98.5 Cellulophaga fucicola EU939693 88.1 69 FR714988 L,G5m D/R Bacteroidetes uncultured Bacteroidetes GQ348552 99.5 Aureimarina marisflavi EF108215 96.0 71 FR714990 G80-110 R Bacteroidetes flavobacterium GMD102 DQ660394 98.0 Flavobacterium kamogawaensis AB275999 95.6 76 FR714994 G80m D Bacteroidetes bacterium DG890 AY258122 96.8 Owenweeksia hongkongensis AB125062 95.3 78 FR714995 G80m D Bacteroidetes uncultured Bacteroidetes GQ350619 95.8 Kordia algicida FJ015036 85.0 79 FR714996 G80m D Bacteroidetes uncultured Bacteroidetes GQ350621 100.0 Marinoscillum pacificum DQ660388 93.6 80 FR714997 G5m R Bacteroidetes uncultured bacterium FJ350314 97.0 Owenweeksia hongkongensis AB125062 87.9 82 FR714998 G5m R Bacteroidetes uncultured flexibacterium EU703449 99.7 Prolixibacter bellariivorans AY918928 86.8 84 FR715000 G5m R Bacteroidetes uncultured bacterium FJ973584 98.5 Arenibacter latericius AF052742 93.7 85 FR715001 G5m R Bacteroidetes uncultured flexibacterium EU641216 98.5 Algoriphagus aquatilis EU313811 97.0 86 FR715002 G5m R Bacteroidetes uncultured bacterium FJ354886 95.9 Owenweeksia hongkongensis AB125062 91.1 98 FR715004 G5m D Bacteroidetes uncultured bacterium EU800828 95.8 Gracilimonas tropica EF988655 86.6 99 FR715005 G5m D Bacteroidetes uncultured bacterium FJ350767 98.4 Formosa crassostrea EU328155 93.9 113 FR715017 G138m R Bacteroidetes uncultured Bacteroidetes FJ497473 93.0 NA

10 10 FR714949 T,L,G5-120m D Alphaproteobacteria uncult. alphaproteobacterium GU061257 98.8 Pelagibacter ubique CP000084 98.8 16 FR714952 T,L,G5-120m D Alphaproteobacteria uncultured bacterium EU802244 99.3 Pelagibacter ubique EU410957 92.4 27 FR714960 G120-138m R Alphaproteobacteria uncult. alphaproteobacterium FJ497399 98.0 Marinosulfonomonas U62894 96.6 methylotropha 42 FR714970 G110-120m D/R Alphaproteobacteria uncult. alphaproteobacterium U70679 100.0 Caedibacter caryophilus X71837 87.5 45 FR714973 T,G5-110m R Alphaproteobacteria Roseobacter sp. WM2 EF044234 98.1 Jannaschia helgolandensis DQ915603 96.8 48 FR714976 T,L R Alphaproteobacteria uncult. alphaproteobacterium AM936707 98.8 Thalassobaculum litoreum EF203900 94.4 49 FR714977 T,L,G5-120m D Alphaproteobacteria uncultured bacterium EU802205 99.0 Pelagibacter ubique EU410957 85.8 60 FR714980 L R Alphaproteobacteria uncultured bacterium GQ396964 100.0 Nitrobacteria novellus DQ205093 99.3 72 FR714991 G30-80m R Alphaproteobacteria uncultured marine bacterium DQ071108 94.7 Aquamicrobium defluvii Y15403 80.9 87 FR715003 G5m R Alphaproteobacteria uncultured bacterium GQ109020 99.7 Methylobacterium brachiatum EU730910 99.7 103 FR715007 G150-175m R Alphaproteobacteria uncult. deltaproteobacterium FJ205325 89.5 Pelobacter seleniigenes DQ991964 87.4 43 FR714971 T,L,G5m D/R Betaproteobacteria uncultured bacterium EU443090 99.3 Burkholderia sordidicola DQ256491 93.9 64 FR714983 L,G5m D/R Betaproteobacteria uncultured bacterium GQ240201 99.5 Aquamonas fontana AB120967 98.8 66 FR714985 L D Betaproteobacteria uncultured bacterium EU287085 98.5 Methylophilus methylotrophus GQ175365 96.6 83 FR714999 G5m R Betaproteobacteria uncultured bacterium FJ352945 95.5 Massilia lutea AY966001 90.5 112 FR715016 G150m R Betaproteobacteria uncultured bacterium DQ836754 98.3 Candidatus Nitrotoga arctica GQ420877 96.2 123 FR715023 G110m D Betaproteobacteria uncult. betaproteobacterium GQ347909 98.3 Azoarcus communis AF011343 92.0 4 FR714944 G138-225m D/R Deltaproteobacteria uncul. deltaproteobacterium GQ348213 99.0 Desulfobacterium anilini EU020016 93.4 7 FR714947 G30-80m D/R Deltaproteobacteria uncult. verrucomicrobium EU350909 93.8 Desulfobulbus mediterraneus AF354663 85.9 25 FR714958 G138m R Deltaproteobacteria uncultured bacterium EU362299 93.9 Desulforhopalus singaporensis AF118453 92.4 30 FR714963 G120m R Deltaproteobacteria uncult. deltaproteobacterium GQ346922 93.0 Anaeromyxobacter FJ190060 89.3 dehalogenans 39 FR714968 G120-175m D/R Deltaproteobacteria uncultured bacterium AY133461 98.5 Desulfobacula toluolica X70953 96.8 105 FR715009 G175m R Deltaproteobacteria uncultured bacterium FJ462011 91.7 Desulfobotulus sapovorans FJ789839 89.7 107 FR715011 G175m R Deltaproteobacteria uncult. deltaproteobacterium GQ349156 95.7 Desulfobacula toluolica X70953 94.8 109 FR715013 G150m R Deltaproteobacteria uncultured bacterium EU362299 92.8 Desulfocapsa sulfexigens Y13672 90.5 111 FR715015 G138-150m D/R Deltaproteobacteria uncultured bacterium EU362299 98.8 Desulfocapsa sulfexigens Y13672 96.4 114 FR715018 G138m R Deltaproteobacteria Uncult. deltaproteobacterium GQ349156 95.9 Desulfobacula toluolica X70953 94.2 26 FR714959 G120-225m D/R Epsilonproteobacteria uncul. epsilonproteobacteri. EF405798 99.5 Sulfurimonas autotrophica AB088431 95.8 70 FR714989 G5-175m D/R Epsilonproteobacteria uncultured Arcobacter sp. DQ234112 99.3 Arcobacter butzleri U34388 96.8 1 FR714941 G175m R Gammaproteobacteria uncultured proteobacterium FJ497298 98.0 Thiomicrospira frisia AF013974 88.6 33 FR714965 G120m D Gammaproteobacteria uncultured bacterium GQ259224 97.5 Marinobacter flavimaris FJ161304 91.4 34 FR714966 L D Gammaproteobacteria uncultured bacterium DQ337061 99.0 Marinobacterium EU573965 86.3 nitratireducens 37 FR714967 G80-225m D/R Gammaproteobacteria uncultured bacterium EU134767 99.8 Vesicomyosocius okutanii AP009247 96.1 53 FR714979 G5&175m D/R Gammaproteobacteria Rheinheimera sp. 3006 AM110966 100.0 Rheinheimera baltica AJ441082 100.0

11 75 FR714993 G80-175m D Gammaproteobacteria Acinetobacter johnsonii Z93440 99.5 Acinetobacter johnsonii Z93440 99.5 102 FR715006 G30-110m D Gammaproteobacteria uncultured proteobacterium GQ348678 98.3 Pseudomonas tolaasii EU169190 90.2 106 FR715010 G175m R Gammaproteobacteria uncultured proteobacterium FJ497298 95.7 Dechloromarinus chlorophilus AF170359 90.4 108 FR715012 G175m R Gammaproteobacteria uncultured bacterium GQ903305 89.0 Thioprofundum lithotrophica AB468957 89.0 110 FR715014 G150m R Gammaproteobacteria uncult. alphaproteobacterium GQ350982 99.3 Pseudomonas marincola FJ869035 88.7 122 FR715022 G120m D Gammaproteobacteria uncultured bacterium EU438597 97.0 Thiohalophilus DQ469584 91.0 thiocyanatoxydans 2 FR714942 G138-175m R Chloroflexi uncultured chloroflexi GQ351076 96.0 Levilinea saccharolytica AB109439 87.9 29 FR714962 G110-120m D/R Chloroflexi uncultured chloroflexi GQ349484 99.8 Dehalobium chlorocoercia AF393781 83.6 NP FR715024 G110-225m D/R Crenachaeota uncultured crenarchaeote EF591452 100.0 Nitrosopumilus maritimus DQ085097 99.8 14 FR714950 T,L,G5-30m D/R Cyanobacteria uncultured synechococcus EU703476 99.0 Synechococcus rubescens AM709629 99.0 17 FR714953 L,G5m R Cyanobacteria Cyanobium sp. NS01 AY172837 99.8 Synechococcus rubescens AM709629 97.6 19 FR714954 L,G5-80m R Cyanobacteria uncultured cyanobacterium AM259255 99.0 Snowella litoralis AJ781041 99.0 3 FR714943 G120-175m D/R Deferribacteres uncultured marine bacterium GQ351079 97.3 Ruminococcus callidus X85100 82.4 24 FR714957 G138-175m D/R Deferribacteres uncultured marine bacterium GQ350776 98.8 Clostridium halophilum X77837 82.0 104 FR715008 G175m R Firmicutes uncultured bacterium AB433161 90.9 Carboxydocella ferrireducens EF092457 84.4 115 FR715019 G120m R Planctomycetes uncultured bacterium FJ930400 86.0 Planctomyces maris AJ231184 83.0 121 FR715021 G110-120m D Planctomycetes uncultured bacterium EU491044 96.6 Cand. Kuenenia stuttgartiensis CT573071 85.1 50 FR714978 T D Verrucomicrobia unidentified bacterium LD29 AF009975 99.5 Chthoniobacter flavus AY388649 91.7 73 FR714992 G80m R Verrucomicrobia uncult. gammaproteobacteri. EU050837 97.5 Pelagicoccus mobilis AB286015 91.8 a G= Station G (depth m), T= T1, L=LL12 b Type of fingerprint where phylotype was detected. D=DNA-based, RNA-based, D/R= DNA- and RNA-based c 16S rRNA gene similarity

NA = not applicable, i.e. the closest described species has a similarity of <80% as indicated in the Material and Methods.

12