Niches of two polysaccharide-degrading Polaribacter isolates from the North
Sea during a spring diatom bloom
Peng Xing1,2,$, Richard L. Hahnke1,$,†, Frank Unfried1,3, Stephanie Markert3, Sixing
5
Huang1,†, Tristan Barbeyron4, Jens Harder1, Dörte Becher5, Thomas Schweder3,6,
Frank Oliver Glöckner1,7, Rudolf I. Amann1, Hanno Teeling1*
1
10
2
†
15
3
4
5
20
6
7
25
30
Max Planck Institute for Marine Microbiology, Celsiusstraße 1, 28359 Bremen,
Germany
State Key Laboratory of Lake Science and Environment, Nanjing Institute of
Geography and Limnology, Chinese Academy of Sciences, 210008 Nanjing,
People’s Republic of China
Current address: Leibniz Institute DSMZ-German Collection of Microorganisms and
Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany
Institute of Marine Biotechnology e.V., Walther-Rathenau-Straße 49A, 17489
Greifswald, Germany
UPMC University Paris 6 and CNRS, UMR 7139 Marine Plants and Biomolecules,
Station Biologique de Roscoff, 29682 Roscoff, Bretagne, France
Institute
of
Microbiology,
Ernst-Moritz-Arndt-University,
Friedrich-Ludwig-Jahn-Straße 15, 17487 Greifswald, Germany
Pharmaceutical
Biotechnology,
Ernst-Moritz-Arndt-University,
Felix-Hausdorff-Straße 3, 17487 Greifswald, Germany
Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany
$
These authors contributed equally to this work.
* Corresponding author: Hanno Teeling, Max-Planck-Institute
Microbiology, Celsiusstraße 1, 28359 Bremen
Tel.: +49 421 2028 976; Fax: +49 421 2028 870
Running title: Niches of two North Sea Polaribacter isolates
for
Marine
Supplementary Information Contents
Supplementary
Text.
Estimation
of
in
situ
abundances
by
metagenome
read-recruitment / Degradation of monosaccharides / Adaptations to micro-oxic and
35
anoxic conditions / Assimilation of nitrogen and sulfur / Gliding motility;
Supplementary Figure S1 Growth curves of Polaribacter sp. Hel1_33_49.
Supplementary Figure S2 Variation of abundant GH families in 27 marine
Flavobacteriaceae genomes.
Supplementary Figure S3 CAZyme category comparison between Polaribacter spp.
40
and other Flavobacteriaceae.
Supplementary Figure S4 Putative PULs of Polaribacter sp. Hel1_33_49.
Supplementary Figure S5 Putative PULs of Polaribacter sp. Hel1_85.
Supplementary
Figure S6
MEROPS
peptidase
families
in
27
marine
Flavobacteriaceae.
45
Supplementary Figure S7 MEROPS category comparison between Polaribacter spp.
and other Flavobacteriaceae.
Supplementary Figure S8 Tree of bacteriorhodopsin sequences for Polaribacter sp.
Hel1_33_49 and relatives.
Supplementary Figure S9 Conserved DNA photolyase/cryptochrome gene clusters
50
among three proteorhodopsin-containing Polaribacter strains.
Supplementary Figure S10 Relationships between mannitol dehydrogenase,
bacteriorhodopsin and sulfatase gene copy numbers and genome size in 27
Flavobacteriaceae.
Supplementary Figure S11 Rnf operon of Polaribacter sp. Hel1_85.
55
Supplementary Figure S12 Clustered assimilatory nitrate reduction genes in
Polaribacter sp. Hel1_33_49 and sp. Hel_I_85.
Supplementary Table S1 Substrate utilization of Polaribacter strains.
Supplementary Table S2 Manually annotated CAZymes and sulfatases in
Polaribacter sp. Hel1_33_49 and sp. Hel1_85.
60
Supplementary Table S3 Reference Flavobacteriaceae and their peptidase and
CAZyme gene frequencies according to automated annotation.
Supplementary Table S4 Features of currently available Polaribacter genomes.
Supplementary Table S5 ANI- and dDDH-based comparison of sequenced
Polaribacter strains.
65
Supplementary Table S6 Proteome data for Polaribacter sp. Hel1_33_49.
Supplementary Table S7 Recruitment of metagenomic reads by the genomes of
Polaribacter sp. Hel1_33_4 and s. Hel1_85.
Supplementary Table S8 Gliding motility genes in sequenced Polaribacter strains.
70
Supplementary Text
Estimation of in situ abundances by metagenome read-recruitment
We used the genomes of Polaribacter sp. Hel1_33_49 and Hel1_85 for the
recruitment of reads from 26 metagenomes of sea surface bacterioplankton from the
English Channel and the North Sea (Table S7). The English Channel metagenomes
75
were obtained from eight samples taken in 2008 at the long-term sample station L4 in
the framework of the Western Channel Observatory Microbial Metagenomics Study
(Gilbert et al., 2010). The North Sea metagenomes were obtained from 18 samples
taken in 2008 - 2012 at the isolation site of Polaribacter sp. Hel1_33_49 and Hel1_85
- the long-term sample station "Kabeltonne" near the island Helgoland. These
80
publically available metagenomes were produced in the framework of the Microbial
Interactions in Marine Systems (MIMAS) project (Teeling et al., 2012) and the Coastal
Microbe Taxonomic & Genomic Observatory (COGITO) community sequencing
project of the DOE Joint Genome Institute.
Reads were quality filtered prior to mapping. Illumina reads were filtered using a
85
script from Murat Eren (https://github.com/meren/illumina-utils/blob/master/scripts/ana
alyze-illumina-quality-minoche) that implements criteria suggested by Minoche et al.
(2010), and 454 reads were de-duplicated and filtered using prinseq v.0.20.4
(Schmieder & Edwards, 2011) (parameters: -derep 12345 -min_len 150 -max_len 450
-trim_qual_left 30 -trim_qual_right 30 -min_qual_score 10). Reads were subsequently
90
mapped onto both Polaribacter genomes using smalt v.0.7.6 (parameters for indexing:
-k 20 -s 10) and only reads were retained with a minimum of 95% nucleotide identity to
one of the genomes.
The proportion of recruited reads by either genome was below 1% for all 26
metagenomes (Supplementary Table S7). Abundances were highest in the 2010
95
North Sea metagenomes during a phytoplankton bloom 12 days before (2010/04/08)
and 14 days after (2010/05/04) the isolation date of both strains (2010/04/20). The
maximum abundance of Polaribacter sp. Hel1_33_49 was detected on 2010/05/04
with 0.5% recruited reads, which covered 99.1% of the genome with an average depth
of coverage of 59.6 x.
100
Degradation of monosaccharides
Both North Sea Polaribacter strains have complete genes for the Embden
Meyerhof-Parnas (EMP) pathway and Krebs cycle. Fructose can be converted to
fructose-6-phosphate, and galactose can be converted to glucose-1-phosphate and
105
subsequently funneled into glycolysis. Both strains also harbor a predicted
keto-deoxy-phosphogluconate aldolase, the key enzyme of the Entner-Doudoroff
pathway, but lack the oxidative part of the pentose phosphate pathway.
Adaptations to micro-oxic and anoxic conditions
110
All Polaribacter type strains are capable of fermentation (Bernardet, 2010), but
Polaribacter sp. MED152 is a strict aerobe (González et al., 2008). Based on gene
repertoires, strains Hel1_33_49 and Hel1_85 can perform mixed acid fermentations
and produce lactate, formate, acetate, acetaldehyde, ethanol, oxaloacetate and
succinate. Strain Hel1_85 has further adaptations to low-oxygen conditions as they
115
might occur on biofilms on algae. It features genes for high oxygen affinity cytochrome
bd- and cbb3-type terminal oxidases. The latter is also present in strain MED152
(González et al., 2008), but absent from the strain Hel1_33_49 and Polaribacter
irgensii 23-PT.
Polaribacter sp. Hel1_85 is the only known Polaribacter species that features the
120
rnfCDGEAB operon. Phylogenetic analysis of this operon suggested lateral transfer
from a gammaproteobacterial species (Supplementary Figure S11). The rnf operon
encodes a membrane-bound electron transport complex that couples the ferredoxin
and pyridine dinucleotide pools (Buckel & Thauer, 2013). The exergonic oxidation of
reduced ferredoxin with pyridine dinucleotides can be coupled to translocation of
125
either protons or sodium ions across the cytoplasmic membrane and might enable
strain
Hel1_85
to gain
additional energy from reduced ferredoxin
under
oxygen-limiting or anoxic conditions.
Assimilation of nitrogen and sulfur
130
Polaribacter strains Hel1_33_49 and Hel1_85 each grow on amino acids as a sole
nitrogen source, but also have genes for assimilatory nitrate reduction. These genes
are co-localized in a cluster similar to that in Flavobacterium johnsoniae UW101T
(Supplementary Figure S12) but are not present in other sequenced Polaribacter
genomes. In contrast, genes for assimilatory sulfate reduction are present in all
135
sequenced Polaribacter genomes.
Gliding motility
Gliding enables bacteria to (i) move on rather dry surfaces, (ii) penetrate into and
migrate within complex organic matrices to reach polymeric, non-diffusible substrates,
140
and (iii) move in fluidic environments without loosing contact to the substratum
(Reichenbach, 1981). Flavobacteria have been frequently found attached to
aggregates (Kirchman, 2002; Gómez-Pereira et al., 2010) and described either as
non-motile or to move by gliding (Bernardet, 2010).
Most of the essential genes for Bacteroidetes-type gliding motility were identified
145
in the pelagic Polaribacter spp. Hel1_33_49, Hel1_85 and MED152 and the sea ice
isolate P. irgensii 23-PT (McBride & Zhu, 2013), including genes for the Por secretion
system (gldKLMN, sprAET), the GldI peptidoprolyl isomerase, the GldAFG
ATP-binding cassette transporter, and the proteins GldBCDEFGH (Supplementary
Table S8). Despite its genomic potential for gliding, P. irgensii 23-PT has been
150
described as non-gliding (Gosink et al., 1998; Yoon et al., 2006). One possible
explanation is that all four investigated Polaribacter genomes contain adhesins
(Supplementary Table S8), which might have recognition functions to ensure that
gliding is only induced in the presence of specific substrates. Gliding motility was
observed on agar plates for strain Hel1_85 (Hahnke & Harder, 2013), gliding for strain
155
Hel1_33_49 could be demonstrated using the hanging-drop technique (Skerman,
1967).
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