Niches of Two Polysaccharide-Degrading Polaribacter Isolates from the North Sea During a Spring Diatom Bloom

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The ISME Journal (2015) 9, 1410–1422 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom

Peng Xing1,2,8, Richard L Hahnke1,8,9,FrankUnfried1,3,StephanieMarkert3, Sixing Huang1,9, Tristan Barbeyron4,JensHarder1,Do¨rte Becher5,ThomasSchweder3,6, Frank Oliver Glo¨ckner1,7, Rudolf I Amann1 and Hanno Teeling1 1Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen, Germany; 2State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, People’s Republic of China; 3Institute of Marine Biotechnology e.V., Greifswald, Germany; 4UPMC University Paris 6 and CNRS, UMR 7139 Marine Plants and Biomolecules, Station Biologique de Roscoff, Roscoff, Bretagne, France; 5Institute of Microbiology, Ernst-Moritz-Arndt-University, Greifswald, Germany; 6Pharmaceutical Biotechnology, Ernst-Moritz-Arndt-University, Greifswald, Germany and 7Jacobs University Bremen gGmbH, Bremen, Germany

Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplankton blooms. We analyzed two respective Polaribacter species by whole genome sequencing, comparative genomics, substrate tests and proteomics. Both can degrade algal polysaccharides but occupy distinct niches. The liquid culture isolate Polaribacter sp. strain Hel1_33_49 has a 3.0-Mbp genome with an overall peptidase:CAZyme ratio of 1.37, four putative polysaccharide utilization loci (PULs) and features proteorhodopsin, whereas the agar plate isolate Polaribacter sp. strain Hel1_85 has a 3.9-Mbp genome with an even peptidase:CAZyme ratio, eight PULs, a mannitol dehydrogenase for decomposing algal mannitol-capped polysaccharides but no proteorhodopsin. Unlike other sequenced Polaribacter species, both isolates have larger sulfatase- rich PULs, supporting earlier assumptions that Polaribacter take part in the decomposition of sulfated polysaccharides. Both strains grow on algal laminarin and the sulfated polysaccharide chondroitin sulfate. For strain Hel1_33_49, we identified by proteomics (i) a laminarin-induced PUL, (ii) chondroitin sulfate-induced CAZymes and (iii) a chondroitin-induced operon that likely enables chondroitin sulfate recognition. These and other data suggest that strain Hel1_33_49 is a planktonic flavobacterium feeding on proteins and a small subset of algal polysaccharides, while the more versatile strain Hel1_85 can decompose a broader spectrum of polysaccharides and likely associates with algae. The ISME Journal (2015) 9, 1410–1422; doi:10.1038/ismej.2014.225; published online 5 December 2014

Introduction P. irgensii, P. gangjinensis, P. porphyrae, P. reichenbachii, P. sejongensis and P. huanghezhanensis (Bernardet, The genus Polaribacter belongs to the family 2010; Parte, 2014). The first described Polaribacter Flavobacteriaceae of the phylum Bacteroidetes. species were psychrophilic isolates from Arctic and To date, 12 Polaribacter species have been Antarctic sea ice (Gosink et al., 1998), but ever since, described with validly published names, P. atrinae Polaribacter have also been identified in temperate (Hyun et al., 2014), P. butkevichii, P. dokdonensis, marine habitats, both in seawater (Nedashkovskaya P. filamentus, P. franzmannii, P. glomeratus, et al., 2005; Yoon et al., 2006; Fukui et al., 2013) and sediments (Li et al., 2014). Based on in situ detection and 16S rRNA gene sequence analysis, it has Correspondence: Dr H Teeling, Department of Molecular Ecology, been suggested that the Polaribacter genus consists Max-Planck-Institute for Marine Microbiology, Celsiusstrae1, Bremen 28359, Germany. of cold- and warm-water-adapted phylotypes E-mail: [email protected] (Go´mez-Pereira et al., 2010; Dı´ez-Vives et al., 8These authors contributed equally to this work. 2013). Polaribacter species have also been isolated 9Current address: Leibniz Institute DSMZ-German Collection of from red (Fukui et al., 2013) and green macroalgae Microorganisms and Cell Cultures, Inhoffenstrae 7B, Braunsch- weig 38124, Germany. (Nedashkovskaya et al., 2013) and were found to Received 27 June 2014; revised 6 October 2014; accepted 23 thrive during diatom-dominated phytoplankton October 2014; published online 5 December 2014 blooms in the North Sea (Teeling et al., 2012) and Niches of two North Sea Polaribacter isolates P Xing et al 1411 the Antarctic Southern Ocean (Williams et al., phytoplankton and metagenome data support this 2013). Association of Polaribacter spp. with phyto- capability (Go´mez-Pereira et al., 2012; Teeling et al., plankton was also observed in the open North 2012). Atlantic (Go´mez-Pereira et al., 2012) and the coastal So far, limited genomic data are available for North Sea (Bennke et al., 2013) and has been members of the Polaribacter genus. A complete inferred in a recent study on an Antarctic polynya genome is present only for Polaribacter sp. MED152 (Kim et al., 2013). Because of their presence in cold (3.0 Mbp; Gonza´lez et al., 2008), an isolate from and temperate waters, in oligotrophic and copio- northwestern Mediterranean surface water. Further- trophic nutrient regimes within open ocean and more, draft genomes are available for two isolates coastal zones, members of the genus Polaribacter from Antarctic sea ice, P. irgensii 23-PT (B2.8 Mbp; have recently been recognized as ‘widely distributed two scaffolds, seven contigs) and P. franzmannii in marine habitats’ (Nedashkovskaya et al., 2013). 301T (B6.9 Mbp; 56 scaffolds, 69 contigs). Marine Bacteroidetes, in general, and marine In 2010, two novel Polaribacter species were isolated Flavobacteriia, in particular, have been ascribed to from surface seawater nearshore the North Sea island act as degraders of biopolymers, such as proteins Helgoland during a diatom-dominated spring phyto- and polysaccharides (for example, Thomas et al., plankton bloom (Hahnke and Harder, 2013; Hahnke 2011). This view is supported by high Flavobacteriia et al., 2014). Strain Hel1_85 (formerly Hel_I_85) was abundances in nutrient-rich habitats, such as aggre- isolated from agar plates and strain Hel1_33_49 in gates of particulate organic matter (Williams et al., liquid culture. Strain Hel1_33_49 did not grow on agar 2013) or in the microbiota of marine algae and plates, indicating distinct lifestyles of both strains. invertebrates (Di Camillo et al., 2012; Dong et al., In this study, we report almost complete genomes 2012). Also, so far sequenced genomes of marine of these Polaribacter strains. Annotations were Flavobacteriia have revealed clear adaptations combined with growth experiments on various towards biopolymer degradation (for example, substrates, including a suite of different carbohy- Bauer et al., 2006; Gonza´lez et al., 2008; Woyke drates and polysaccharides, to elucidate the meta- et al., 2009; Mann et al., 2013), as they feature high bolic potential and associated niches of both strains. proportions of peptidase and glycoside hydrolase These experiments were complemented by com- (GH) genes. Ferna´ndez-Go´mez et al. (2013) have parative genomics with 25 additional Flavobacter- shown that marine Bacteroidetes tend to have higher iaceae genomes and by proteomics of Hel1_33_49 numbers of peptidase than GH genes, whereas the cultures grown on the non-substituted algal poly- number in non-marine Bacteroidetes is about even. saccharide laminarin as well as the sulfated poly- However, depending on lifestyle, there is consider- saccharide chondroitin sulfate. able variation in the gene numbers for peptidases and carbohydrate-active enzymes (CAZymes; Lombard et al., 2014). Planktonic Flavobacteriia Material and methods such as Polaribacter sp. MED152 and Dokdonia donghaensis MED134 are subjected to rare feast and Substrate tests À 1 extended famine conditions. Both strains feature Various substrates (2.0gl ; Supplementary Table S1) relatively small genomes of around 3.0 Mbp, with were tested at 12 1C in marine mineral medium almost twice as many peptidases than CAZymes, (10 ml) as described previously (Mann et al., 2013), and can gain additional energy via the light-dependent including kappa- and iota-carrageenan, amorphous proton-pumping membrane protein proteorhodopsin, cellulose, glycogen, laminarin and xanthan. Volumes as has been inferred for Polaribacter sp. MED152 of 100 mlofeitherPolaribacter sp. Hel1_33_49 or (Gonza´lez et al., 2008) and verified for Dokdonia Hel1_85 stock culture were added as inocula. Optical donghaensis MED134 (Gonza´lez et al., 2011). density measurements (660 nm) were taken for 2 weeks In contrast, macroalgae-colonizing Flavobacteriia for monosaccharides and disaccharides and 8 weeks such as Zobellia galactanivorans DsijT (Barbeyron for polysaccharides and compared with inoculated et al., 2001) and Formosa agariphila KMM 3901T control cultures without saccharide supplements and (Nedashkovskaya et al., 2006) live under more stable non-inoculated, saccharide-supplemented media. conditions with ample nutrient supply. Both feature much larger genomes of 5.5 and 4.2 Mbp, respectively, and feature more CAZymes than peptidases DNA extraction, genome sequencing and annotation but are notably devoid of proteorhodopsin. Cells were grown on HaHa_100 medium (Hahnke Pure culture studies on Polaribacter strains have et al., 2014) at 12 1C in the dark until the late demonstrated growth on biopolymers, such as exponential phase and subsequently pelleted by aesculin, casein, starch and gelatin (Gosink et al., centrifugation (2500 g, 60 min). Wet cell pellets of 1998; Nedashkovskaya et al., 2005; Yoon et al., 2006; 0.5 g were dissolved in 10 ml sodium phosphate Lee et al., 2011; Fukui et al., 2013; Kim et al., 2013; buffer (pH 7.8) and incubated with 30 U a-amylase Nedashkovskaya et al., 2013). Growth on algal (10065, Sigma-Aldrich, Hamburg, Germany) at 37 1C polysaccharides has not yet been investigated for 60 min to remove glycoconjugates. Cells were in detail, but Polaribacter co-occurrences with subsequently centrifuged (30 000 g, 20 min), and

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1412 DNA was extracted according to the protocol of (Stamatakis and Ott, 2008) with the GTR substitu- Zhou et al. (1996). tion model and 1000 repetitions. Average nucleotide Sequencing was performed at LGC Genomics identity and digital DNA-DNA hybridization (Berlin, Germany) using the 454 GS FLX Ti platform were calculated using JSpecies (Richter and (454 Life Sciences, Branfort, CT, USA). The Rossello´-Mo´ra, 2009) and the Genome-to-Genome Hel1_33_49 genome was assembled from 733 179 Distance Calculator (Auch et al., 2010), respectively. reads from a standard shotgun library and 139 390 reads from a 3-kbp mate pair library (451.7 Mbp; 148 Â coverage) using Newbler v2.6. The initial Proteomics assembly comprised 38 contigs, 31 of which could Polaribacter sp. Hel1_33_49 was grown at 12 1Cin be joined into 10 scaffolds. The remaining gaps were modified HaHa_100 medium with 0.1 g l À 1 peptone, À 1 closed by PCR and Sanger sequencing yielding 0.1 g l casamino acids, 200 mM NH4Cl and 16 mM circular assemblies of 2 894 172 bp and 118 580 bp KH2PO4. Laminarin (L9634, Sigma-Aldrich Chemie (total: 3 012 752 bp). The latter does not carry any GmbH, Taufkirchen, Germany) or chondroitin known plasmid genes and likely constitutes a 4-sulfate (27042, Sigma-Aldrich) were used as repeat-bound integral piece of the chromosome. carbon source, and D-mannose served as control The Hel1_85 draft genome was created the same (concentrations: 2.0 g l À 1; Supplementary Figure S1). way with 646 110 reads from a standard shotgun The strain was adapted by three passages into the library and 135 989 reads of a 3-kbp mate pair library respective medium (1% inoculum), followed by (394.9 Mbp; 100 Â coverage). The initial assembly cultivation in 100 ml medium. All growth experiments comprised 83 contigs, 63 of which could be joined were carried out in triplicates. Cells were harvested by into 46 scaffolds. Subsequent gap closure yielded centrifugation (15 min; 13 000 g;41C), and the result- four larger contigs of 1 642 720, 1 466 007, 752 210 ing pellets were stored at À 80 1C until use. and 53 131 bp (total: 3 914 068 bp). The remaining Cell pellets were washed in TE buffer (10 mM gaps consist of four almost identical 16S-23S-5S Tris-HCl pH 7.5, 10 mM EDTA pH 8.0) containing rRNA operons that could not be traversed in several ‘cOmplete Protease Inhibitor’ (Roche, Berlin, targeted gap closure approaches. Germany). Soluble proteins were extracted by Gene prediction and annotation (including the sonication (2 Â 25 s), and the protein concentration phylogeny-guided CAZyme annotations provided was determined as described by Bradford (1976). in Supplementary Table S2) were performed as Fifteen micrograms of protein were loaded onto 10% described previously (Mann et al., 2013). Annotated polyacrylamide sodium dodecyl sulfate mini gels, genome sequences were submitted to NCBI’s Gen- separated (60 min, 150 V) and stained with Roti-Blue bank with the accession numbers JPDI00000000 quick (Carl Roth, Karlsruhe, Germany). Subse- (Hel1_33_49) and JPDS00000000 (Hel1_85). quently, entire gel lanes were excised, and proteins were digested as described by Heinz et al. (2012). Peptides were subjected to a reversed-phase C18 Comparative analysis column chromatography on a nano ACQUITY-UPLC We selected a total of 27 Flavobacteriaceae genomes (Waters Corporation, Milford, MA, USA) and sepa- from marine or aquatic habitats for comparative rated as described by Otto et al. (2010). Mass analysis (Supplementary Table S3). CAZymes were spectrometry (MS) and MS/MS data were deter- predicted using searches against the CAZy mined using an online-coupled LTQ-Orbitrap mass (Lombard et al., 2014), Pfam (Finn et al., 2014) and spectrometer (Thermo Fisher Scientific Inc., Wal- dbCAN (Yin et al., 2012) databases, and peptidases tham, MA, USA). MS spectra were searched against were predicted using similarity searches against the a target-decoy protein sequence database, including MEROPS database (Rawlings et al., 2014). The Pfam sequences of Polaribacter sp. Hel1_33_49 and of database was also used for the prediction of common laboratory contaminants. Validation of sulfatase (pE-13), bacteriorhodopsin (pE-5) and MS/MS-based peptide and protein identifications mannitol dehydrogenase (pE-5) frequencies. was performed with Scaffold (http://www.proteo- mesoftware.com) using the SEQUEST filter settings described by Heinz et al. (2012). Normalized spectral Phylogenetic analysis abundance factors (NSAFs) were calculated (Zybailov 16S rRNA gene sequences of Polaribacter strains et al., 2006) using Scaffold’s ‘exclusive spectrum count’ Hel1_33_49, Hel1_85 and all Flavobacteriaceae type for each protein, which allows to compare relative strains of the ‘The All-Species Living Tree’ project protein abundances between individual samples. (LTPs_111_SSU; Yarza et al., 2010) were aligned in ARB (Ludwig et al., 2004) using the SINA aligner (Pruesse et al., 2012) and manually corrected Results and Discussion according to secondary structure information. Bootstrapped maximum likelihood trees were Phylogeny constructed from sequences 41300 bp with and With three rRNA operons in Hel1_33_49 and four in without a 40% conservation filter using RAxML Hel1_85, both genomes feature about one rRNA

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1413 operon per megabase pair (Supplementary Table Hel1_33_49 and Hel1_85 contain 5 and 11 SusD/ S4). The Hel1_33_49 16S rRNA gene sequence was TBDR gene pairs, respectively, 4 and 7 of which are nearly identical to previously obtained 16S rRNA part of putative PULs (Supplementary Figures S4 gene sequences from Helgoland bacterioplankton and S5). A single PUL in Hel1_33_49 and three in (99.8% identity), whereas the Hel1_85 16S rRNA Hel1_85 contain sulfatase genes (Supplementary sequence was distinct and exhibited a weaker Figures S4C and S5E, G and H). These PULs similarity to a sequence from the North Atlantic constitute the largest in both strains with up to 49 (97.8% identity) (Figure 1). Both strains have a 16S genes. rRNA identity of 98.3% and an average nucleotide Both strains contain genes for b-galactosidases identity of 80% (Supplementary Table S5). They (Hel1_33_49: 1; Hel1_85: 3) and a-glucosidases constitute distinct species that are closer related to (Hel1_33_49: 1; Hel1_85: 4). Such genes are typi- each other than to the sequenced Polaribacter cally induced by dissolved polymeric glucose. In strains MED152 and 23-PT. addition, strain Hel1_85 can likely degrade xylan as it does grow on xylose and features four b-xylosidase Degradation of biopolymers genes, three D-xylose proton-symporters (xylE) and one hexuronate transporter. In contrast, strain GH and polysaccharide utilization loci (PUL) gene Hel1_33_49 is notably devoid of these genes and repertoires. CAZymes and, in particular, GHs are does not grow on xylose. Furthermore, five putative centrally important for polysaccharide decomposi- PL7 alginate lyase genes were identified in strain tion. A comparison of the GH repertoires of strains Hel1_85, three in a single PUL (Supplementary Hel1_33_49 and Hel1_85 with 25 other sequenced Figure S5C). The polysaccharide alginate is found in marine Flavobacteriaceae comprising 133 GH brown alga cell walls, and alginate lyase excreting families (Supplementary Table S3) revealed GH13, Polaribacter have been described before as abundant GH2, GH16, GH3, GH43 and GH29 as the families members of epiphytic bacterial communities on with the highest mean abundances (Supplementary Laminaria sp. brown alga (Dong et al., 2012). Figure S2). No significant differences in GH abun- The storage polysaccharide of brown algae is dances were detected between Polaribacter and laminarin, a b-glucan of b-1,3-linked glucose resi- other Flavobacteriaceae members (Supplementary dues with occasional b-1,6-linked glucose side Figure S3). chains. These relatively short polysaccharides of Polysaccharide degradation and uptake genes in B25 residues are terminated by 1-linked D-mannitol Bacteroidetes are often co-located in operon-like at the reducing end (for example, Read et al., 1996). PULs (Sonnenburg et al., 2010). PULs comprise a Strain Hel1_85 contains a mannitol 2-dehydrogen- characteristic gene tandem coding for a SusD-like ase gene, which is absent from strain Hel1_33_49. substrate-binding protein and a TonB-dependent Mannitol 2-dehydrogenase allows conversion receptor (TBDR) together with CAZyme genes and of D-mannitol from mannitol-capped laminarin to often contain genes for transport, transcriptional D-fructose 6-phosphate that can subsequently be regulation, peptidases and sulfatases. Strains funneled into the Embden–Meyerhof–Parnas

Figure 1 Maximum likelihood tree based on Polaribacter spp. 16S rRNA gene sequences. Strains Hel1_33_49 and Hel1_85 are depicted in bold. Bootstrap values are based on 1000 sub-samplings. Polaribacter species with sequenced genomes are marked by an asterisk (*). Outgroup: Tenacibaculum spp. Bar: 0.05 substitutions per nucleotide position.

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1414 pathway. Presence of this gene in strain Hel1_85 Polaribacter genomes contain only two to three hence supports possible association with brown sulfatase genes (Supplementary Table S4). In con- algae. Diatoms in contrast feature chrysolaminarin trast, strains Hel1_33_49 and Hel1_85 harbor 12 and as main storage polysaccharides (Beattie et al., 33 sulfatases, respectively, with as many as 9 1961). Chrysolaminarin has a similar structure than (Supplementary Figure S4C) and 8 (Supplementary laminarin, but reducing ends are not capped by Figure S5G) sulfatases in a single PUL. Strain mannitol residues. Hel1_85 furthermore features a PUL-like gene cluster without a susD-like gene in which 14 Carbohydrate and polysaccharide utilization. sulfatases are co-located with GHs (Supplementary Growth studies using a suite of different carbohy- Figure S5D). Large numbers of diverse GH genes in drate and polysaccharide substrates revealed that these sulfatase-rich PULs indicate that they target strains Hel1_33_49 and Hel1_85 both grew on more than one type or rather heterogeneous sulfated D-mannose and D-maltose, but differed in the polysaccharides. This is supported by growth of utilization of other tested monosaccharides, disac- both Helgoland strains on sulfated polysaccharides, charides and trisaccharides. As predicted, strain such as kappa- and iota-carrageenan, agar and Hel1_85 grew on mannitol, and strain Hel1_33_49 xanthan (Supplementary Table S1). did not. Neither strain could use D-trehalose, The sole sulfatase-containing PUL of the North L-rhamnose and N-acetyl-D-glucosamine as is also Sea isolate Hel1_33_49 (Supplementary Figure 3C) true for their closest type strains (Supplementary exhibits a strong synteny with the partial PUL from Table S1). Both strains were able to grow on the the Atlantic fosmid S3_860 (Figure 3). In both PULs, polysaccharides cellulose, carboxymethyl cellulose, three GH92 a-1,2-mannosidases are clustered with laminarin, agar, xanthan, kappa- and iota-carrageenan, five sulfatases. A recent study on the human gut glycogen and chondroitin sulfate. A surprising obser- microbe Bacteroides thetaiotaomicron indicated vation was that cells of both Polaribacter isolates that its arsenal of family GH92 exo-acting enzymes increased in lengths from approximately 0.8 to 3–4 mm decomposes a-mannoside-containing N-glycans and during growth on laminarin or chondroitin sulfate. glycoproteins from land plant cell walls (Zhu et al., Laminarin and chondroitin sulfate are readily meta- 2010). It is possible that Polaribacter sp. Hel1_33_49 bolized by marine microbial communities (Arnosti, can utilize similarly structured but sulfated poly- 2008; Arnosti et al., 2011) and were thus selected for saccharides from marine algae. Furthermore, the further tests with strain Hel1_33_49. presence of such highly similar PULs in distinct Polaribacter species from the North Sea and Atlantic Decomposition of laminarin. Both strains Hel1_33_49 Ocean (97.6% 16S rRNA sequence identity) indi- and Hel1_85 grow on laminarin and contain a cates that this PUL is widespread and functionally putative laminarin-specific PUL (Supplementary important in marine systems. Figures S4D and S5B) with GH16 (Labourel et al., Chondroitin sulfate is a heterogeneous polysac- 2014), GH17 and one putative GH30 family b-1,6- charide composed of alternating N-acetylgalactosa- glucanase but lack GH55 family b-1,3-glucanases mine and glucuronic acid that carries sulfate (Ishida et al., 2009). moieties at various positions. It is not a common Proteomic analysis of strain Hel1_33_49 on lami- substrate in surface seawater, but it is readily narin supplemented medium revealed a significant metabolized by microbial communities and might upregulation of the respective PUL vs D-mannose- mimic polysaccharides of similar composition, such supplied controls (Figure 2a, Supplementary as transparent exopolymers of diatoms that are Table S6). Syntenic PULs were also found in other known to contain high proportions of glucuronic Flavobacteriaceae (Figure 2b). Induction of this PUL acids and sulfated monosaccharides (for example, by laminarin was recently demonstrated in a Zhang et al., 2008). It has been shown that detailed proteomics approach for ‘Gramella forsetii’ Polaribacter abundance of up to 7% of the bacter- KT0803 (Kabisch et al., 2014), which has been ioplankton (Go´mez-Pereira et al., 2010) coincided isolated earlier from the same North Sea sampling with peaking potential for chondroitin sulfate site as strain Hel1_33_49 (Eilers et al., 2001). decomposition in the North Atlantic Ocean (Arnosti et al., 2012). However, the so far sequenced Decomposition of chondroitin sulfate. Previous Polaribacter genomes do not contain any known work indicates that members of Polaribacter are chondroitinase genes. Therefore, chondroitin sulfate able to degrade sulfated algal polysaccharides. First, decomposition in Polaribacter might not be Go´mez-Pereira et al. (2012) obtained a fosmid mediated by a specific PUL but rather initiated by (S3_860) from the North Atlantic Ocean that distinct sulfatases that desulfate chondroitin sulfate harbored a 16S rRNA gene affiliating with Polar- to a glycosaminoglycan composed of N-acetylgalac- ibacter together with a partial PUL with five tosamine and glucuronic acid, which is subse- sulfatase genes. Additionally, Polaribacter sulfatase quently decomposed. expression was found to increase during a diatom- Proteomic analyses (Supplementary Table S6) of dominated phytoplankton bloom in the North Sea chondroitin sulfate-grown Hel1_33_49 cultures (Teeling et al., 2012). However, to date sequenced revealed differential expression of one sulfotransferase,

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1415 a 300 laminarin 250 mannose

200 1,000 × 150

100 %NSAF 50

2,315,000 2,310,000 2,305,000 2,300,000 2,295,000 2,290,000 [bp] b

P. sp. Hel1_33_49 # nc 16 3 17 30

P. sp. Hel1_85 16 nc 3 17 30

P. sp. MED152 16 16 17 30

P. irgensii 23-PT 16 16 16 3

F. agariphila KMM3901T 16 17 2 30

L. blandensis MED217T 16 3 16 16 # 'G. forsetii' KT0803 16 16 3 16

F. johnsoniae UW101T 16 16 16

TBDR SusD-like protein transcriptional regulator other transporter glycoside hydrolase other Figure 2 Laminarin-induced locus of Polaribacter sp. Hel1_33_49: (a) Differential expression of the genes in laminarin- vs mannose- supplied cells. Ordinate: NSAFs; abscissa: position in the Hel1_33_49 genome (large contig; Supplementary Figure S4D). (b) Gene arrangement of the locus as well as similar loci in other Flavobacteriaceae, including Polaribacter sp. Hel1_85 (see Supplementary Figure S5B); numbers indicate GH families (nc ¼ not classified); species that have been shown to use laminarin in cultivation experiments are marked by an asterisk (*); species where laminarin usage is supported by proteomics are marked by a hash (#).

Figure 3 Synteny between a PUL in Polaribacter sp. strain Hel1_33_49 (Supplementary Figure S4C) and a partial PUL on fosmid S3_860 from the Atlantic Ocean identified by Go´mez-Pereira et al. (2012). Cho, Man and Lam indicate proteins that were only detected in chondroitin sulfate-, mannose- and laminarin-supplied cells, respectively. Numbers above genes represent gene locus tags; numbers in genes represent CAZyme family affiliations. The asterisk (*) indicates a possible sequencing error mimicking a gene fusion.

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1416 three sulfatases and one gene with sulfatase and the assimilatory nitrate/nitrite reductases were only GH99 domains. The latter resides in the sulfatase- expressed in laminarin-supplied cultures. This suggests containing PUL with high synteny to fosmid S3_860 that strain Hel1_33_49 can effectively scavenge (Figure 3) and likely represents two distinct nitrogen from the chondroitin sulfate glycosamino- genes that are artificially fused due to a sequencing glycan backbone, whereas it needs additional error. This is supported by an amino-acid identity nitrogen when grown on nitrogen-free laminarin. of ca. 95% of the N-terminal sulfatase and of ca. One of the two mannose-6-phosphate isomerase 90% of the C-terminal GH (PHEL49_1323) with and a putative mannan endo-1,4-b-mannosidase corresponding genes on the fosmid sequence. (GH26) were downregulated with chondroitin Expression of this sulfatase/GH99 locus was only sulfate vs mannose-supplied controls. Surprisingly, detected with chondroitin sulfate (1.3%NSAF Â 1000) these enzymes were also expressed to similar and not with laminarin or mannose. A second extents in laminarin-grown cultures as in the sulfatase (PHEL49_1477) was detected only with controls (%NSAF Â 1000: 31.2 vs 38.8 and 43.6 vs chondroitin sulfate (7.0%NSAF Â 1000) or lami- 41.7), which indicates co-regulation of the laminarin narin (2.3%NSAF Â 1000) but not with mannose. and mannose metabolism. Such co-regulations for The remaining two sulfatases (PHEL49_1332/1334) naturally co-occurring saccharides have been were present only in mannose-supplied cultures, described before, for example, for the b-1,3-glucan whereas the sulfotransferase (PHEL49_498) was laminarin and a-1,4-glucans in ‘Gramella forsetii’ higher expressed with chondroitin sulfate (12 Â ) KT0803 (Kabisch et al., 2014). and mannose (7 Â ) than with laminarin. Ten GHs Hel1_33_49 cultures grown on chondroitin (including families GH13, GH16, GH65, GH74 sulfate showed an unexpected upregulation of a and GH99), eight GTs (including families GT2, GT4 PUL-like operon without known CAZymes and GT19) and two non-GH carbohydrate-binding (Figure 4) composed of genes for a TBDR (2.8 Â ), a proteins (CBM4 and CBM50) were more than SusD-like protein (3.5 Â ), an integrin-like protein twofold upregulated with chondroitin sulfate, (only detected with chondroitin sulfate), an arylsul- whereas five GHs (including families GH13, GH16, fate sulfotransferase (1.8 Â ), a methyltransferase GH31 and GH81) were more than twofold down- (1.5 Â ) and a conserved hypothetical protein regulated vs mannose-supplied controls. Further- (4.6 Â ). The N-terminal half of the integrin-like more, glutamate synthase and glutamine synthetase protein resembles the human cartilage acidic protein were downregulated with chondroitin sulfate, and CRTAC1 (Steck et al., 2007), which might specifically

Figure 4 Chondroitin sulfate-induced locus in Polaribacter sp. Hel1_33_49. (a) Differential expression with chondroitin sulfate and laminarin vs mannose. Ordinate: NSAFs; abscissa: position in the Hel1_33_49 genome (large contig). (b) Gene arrangement of the locus as well as similar loci in other Flavobacteriaceae.

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1417 recognize chondroitin. Such integrin-like proteins that cannot degrade peptides 430 amino acids and are also present in strain Hel1_85 and Dokdonia are likely used to decompose external oligopeptides donghaensis MED134 where they are also co-localized prior to uptake. with TBDRs/SusD-like proteins. Conversely, the arylsulfate sulfotransferase of Hel1_33_49 has not been found in any other Polaribacter strain so far. Light-dependent proteins Bacterial rhodopsins are light sensors or light- Protein utilization. Peptidases of 90 different dependent ion pumps. The latter can either translo- MEROPS families (Rawlings et al., 2014) were cate protons or sodium ions across the cytoplasmic detected among the 27 investigated Flavobacteriaceae membrane, which contributes to the chemiosmotic genomes (Supplementary Figure S6, Supplementary potential that can be used, for example, for ATP Table S3) dominated by cysteine (C), metallo (M) and production, or function as chloride importers serine (S) families (Supplementary Figure S7). as has been shown for the marine flavobacterium Twenty-seven MEROPS families were common to Nonlabens marinus S1–08T (Yoshizawa et al., 2014). all Flavobacteriaceae, with the highest average Rhodopsin genes have been estimated to be present gene numbers in families S09 (7.0 Mbp À 1), S33 in about 50% of the North Sea bacterioplankton, and M38 (both 2.8 Mbp À 1). Mesoflavibacter most of which absorbs green light (Riedel et al., zeaxanthinifaciens S86 (Oh et al., 2011a) and 2010). Gonza´lez et al. (2008) hypothesized that Flavobacteriaceae bacterium S85 (Oh et al., during oligotrophic conditions Polaribacter sp. 2011b) had the highest (68.0 Mbp À 1) and lowest MED152 uses surplus ATP from its proton-pumping (28.7 Mbp À 1) peptidase density, respectively. Both rhodopsin for anaplerotic carbon dioxide fixation were isolated from seawater in Micronesia but seem via pyruvate and phosphoenolpyruvate carboxylase. to exhibit a pronounced niche separation. This is supported by recent stable isotope experi- Sequenced Polaribacter strains (excluding the ments that demonstrated active assimilation of fragmentary P. franzmannii 301T genome) have dissolved inorganic carbon in Polaribacter in the 161–167 peptidases. In comparison to the other northeast Pacific (DeLorenzo et al., 2012). investigated Flavobacteriaceae, Polaribacter have The rhodopsin light-absorbing cofactor retinal is elevated proportions of serine (S) peptidases, and produced from oxidative cleavage of b-carotene strain Hel1_33_49 also has an elevated proportion of by 15,15’-monooxygenases. Both rhodopsin and metallo (M) peptidases. Members of these families 15,15’-monooxygenase genes are present in mediate the degradation and uptake of extracellular Polaribacter strains MED152, 23-PT and proteins, suggesting that digestion of external Hel1_33_49 but notably absent from strain proteins and subsequent uptake of oligopeptides Hel1_85. The Leu105 of the Hel1_33_49 rhodopsin and amino acids constitutes an important carbon sequence indicates a 490-nm light absorption and nitrogen source for both Helgoland strains. maximum (Man et al., 2003). Its sequence resem- Kirchman (2000) estimated that B50% of bacterial bles that of the Polaribacter isolate SA4–10 (99.7% production in the oceans is supported by amino amino-acid identity; Supplementary Figure S8), acids as a key nitrogen source. This agrees with the for which rhodopsin functionality has been finding that strain Hel1_33_49 did not grow in demonstrated (Yoshizawa et al., 2012). medium without yeast extract, casamino acids or Strain Hel1_33_49 expressed its rhodopsin when peptone and due to a lack of detectable asparagine grown on chondroitin sulfate even in the dark synthetase is likely auxotrophic for this amino acid. (Supplementary Table S6), similar to Dokdonia sp. There are notable differences in the peptidase PRO95, which expresses rhodopsin independent of profiles of Polaribacter sp. strain Hel1_33_49 and light conditions and organic substrate supply Hel1_85. For example, strain Hel1_33_49 contains (Riedel et al., 2010). In contrast, no rhodopsin more M38 and S09 peptidases than strain Hel1_85, expression was observed when strain Hel1_33_49 which in turn has more S08 and S41 peptidases. was grown on laminarin or mannose. This suggests However, MEROPS family assignments are of that rhodopsin expression in Hel1_33_49 depends limited value in linking peptidase profiles to on the availability of specific substrates. The effect environmental processes. Distinct peptidases of light on rhodopsin expression has been demon- with varying specificities are known to act on strated for marine model bacteria (Go´mez- polypeptides (for example, M01), oligopeptides (for Consarnau et al., 2007; Riedel et al., 2010; Kimura example, S09, M20) and carboxy/aminopeptidases et al., 2011; Steindler et al., 2011) and in field (for example, M14/M42). Strains Hel1_33_49 and studies (Lami et al., 2009; Poretsky et al., 2009). Hel1_85 have six and eight exported family M01 Possible effects of nutrient availability on rhodopsin aminopeptidases, respectively. Elevated aminopepti- expression are still not well studied, but it was dase activities have been reported for marine bacteria recently shown by Akram et al. (2013) that nutrient that grow on high-molecular weight dissolved organic limitation and not light exposure regulates differ- nitrogen as sole nitrogen source (Korth et al., 2012; ential rhodopsin expression in Vibrio sp. AND4. Calleja et al., 2013). Polaribacter sp. Hel1_85 has 27 Photolyases and cryptochromes are further light- signal peptide-containing S09 prolyl oligopeptidases dependent proteins in Polaribacter strain Hel1_33_49.

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1418

GHs T 90 PLs Zobellia galactanivorans DsiJ CEs CBMs Flavobacterium johnsoniae UW101T GTs C family peptidases 80 S family peptidases M family peptidases other peptidases

70 Formosa agariphila KMM3901T

60 'Gramella forsetii' KT0803

CAZy families 50 Dokdonia Polaribacter sp. Hel1 85 donghaensis MED134 Polaribacter sp. MED152 40

Polaribacter sp. Hel1 33 49 Polaribacter irgensii 23-PT 30

2 3 4 5 6

20 genome size [Mbp]

0 50 100 150 200 (GH + PL + CE)

70 40 (GH+PL+CE) peptidases

60 30

50 Polaribacter sp. Hel1_33_49 20

40 peptidases / Mbp (GH + PL CE) / Mbp Polaribacter sp. Hel1_85 10 30

20 0 23456 7 genome size [Mbp] Figure 5 Comparison of 27 Flavobacteriaceae genomes based on automated annotations (Supplementary Table S3): (a) Interdependency of the number of degradative CAZymes and the number of CAZyme families (trend line: logarithmic regression). Genomes are represented by circles with radii proportional to genome sizes. Relative proportions of CAZymes vs peptidases are shown for each genome. (b) Densities of CAZymes with degradation functions and peptidases vs genome size (trend lines: second-order polynominal regressions).

The ISME Journal Niches of two North Sea Polaribacter isolates P Xing et al 1419 Gonza´lez et al. (2008) identified a set of three in 2008–2012 from the North Sea Polaribacter’s different DNA photolyase/cryptochrome genes in isolation site (Teeling et al., 2012) and the the Polaribacter sp. MED152 genome, which might English Channel (Gilbert et al., 2010). The propor- function as blue light photoreceptors and mediate tion of recruited reads at X95% sequence identity gene expression levels. We found that the respective by each genome was o1% for all metagenomes locushasastrongsyntenyamongthethreerhodopsin- (Supplementary Table S7 and Supplementary Text). containing Polaribacter strains Hel1_33_49, MED152 However, Polaribacter abundances in the North Sea and 23-PT, with amino-acid sequence identities are dynamic and peak during spring phytoplankton 486% among all DNA photolyase/cryptochrome blooms (Teeling et al., 2012). It is thus likely that genes (Supplementary Figure S9). higher abundances of either of the two Polaribacter strains were missed by any of the metagenome sampling dates. Read abundances were consistently Niche separation higher for Polaribacter sp. Hel1_33_49 than for Comparisons of the CAZyme and peptidase profiles Hel1_85, which supports genomic analyses indicat- of the 27 analyzed Flavobacteriaceae revealed ing that the former is better equipped for a pronounced differences between both North Sea planktonic lifestyle than the latter. Polaribacter strains (Figure 5a). Strain Hel1_33_49 has a smaller genome with less CAZymes representing less families and, based on manual CAZyme Concluding remarks annotations, a peptidase:CAZyme ratio of 1.37. In contrast, strain Hel1_85 has a larger genome with Polaribacter sp. Hel1_33_49 and Hel1_85 were more CAZymes representing more families an even isolated from the same sample but exhibit detectable peptidase:CAZyme ratio. In general, small Flavo- differences with respect to their genomes, physiol- bacteriaceae genomes tend to have a higher pepti- ogies and inferred lifestyles. Strain Hel1_33_49 dase density than larger genomes, while the constitutes what might be considered a typical opposite trend is true for the number of CAZymes pelagic representative with a smaller genome, with degradation functions (Figure 5b). Although rhodopsin, a limited number of PULs in conjunction Hel1_33_49 and Hel1_85 feature similar CAZyme with a higher peptidase:CAZyme ratio and no densities, they exhibit notably different peptidase mannitol dehydrogenase. Strain Hel1_85, in con- densities. trast, has a larger genome with more PULs in Flavobacteriaceae species with high peptidase:- conjunction with a lower peptidase:CAZyme ratio, CAZyme ratios include known planktonic species features a mannitol dehydrogenase but no rhodop- such as Polaribacter sp. MED152 and thus are likely sin and seems better equipped to cope with low adapted towards carbohydrate-depleted marine oxygen conditions as they might occur in biofilms habitats and preferentially feed on proteins. By (see Supplementary Text). This species possibly contrast, CAZyme-rich species with lower peptidase constitutes a Polaribacter representative that can proportions include known algae-associated associate with algae, which, however, has to be species such as Zobellia galactanivorans DsijT and confirmed in a future in situ localization study. Formosa agariphila KMM 3901T, reflecting that Strain Hel1_85 also has a higher density of histidine these species have an ample supply of algal kinases and response regulators than strain polysaccharides. Further commonalities among Hel1_3_49 and thus seems to be the more adaptive these species are (i) the tendency for larger genomes, and versatile of the two (Supplementary Table S4). (ii) the absence of rhodopsins, and (iii) often Both strains grow on laminarin and chondroitin presence of mannitol dehydrogenases (Supplementary sulfate, and for strain Hel1_33_49 we identified (i) a Figure S10). laminarin-dependent PUL, (ii) chondroitin sulfate- Although the characteristics of strain Hel1_33_49 induced CAZymes and (iii) a novel operon that match those of typical planktonic Flavobacteria- might enable recognition of chondroitin sulfate-like ceae, Hel1_85 lies in between those of planktonic substrates. In comparison to the genomic data from and algae-associated species (Figure 5) and might Polaribacter spp., it is evident that the genus thrive under both conditions. Algae-association of Polaribacter is not only broad in terms of phylogeny Polaribacter sp. Hel1_85 is supported by the fact but also in terms of genome sizes, physiological traits and associated ecological niches. that it can use laminarin, D-mannitol and alginate as carbon sources, which are typical constituents of brown algae, its high number of sulfatases and its lack of rhodopsin genes. Conflict of Interest The authors declare no conflict of interest.

In situ abundance We used the genomes of the two North Sea Acknowledgements Polaribacter strains for read-recruitment with 26 We thank Nils O¨ ster, Lennart Kappelmann and Karen bacterioplankton metagenomes that were obtained Kru¨ ger for bioinformatics and Meghan Chafee for critical

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