Environmental Microbiology - Supporting Information Text
Halorhabdus
tiamatea:
Proteogenomics
and
glycosidase
activity
measurements identify the first cultivated euryarchaeon from a deep-sea
5
anoxic brine lake as potential polysaccharide degrader
Johannes Werner1,2,*, Manuel Ferrer3,*, Gurvan Michel4, Alexander J. Mann1,2, Sixing
Huang1, Silvia Juarez5, Sergio Ciordia5, Juan P. Albar5, María Alcaide3, Violetta La
Cono6, Michail M. Yakimov6, André Antunes7, Marco Taborda8, Milton S. da Costa9,
10
Tran Hai10, Frank Oliver Glöckner1,2, Olga V. Golyshina10, Peter N. Golyshin10,†,‡,
Hanno Teeling1,‡: The MAMBA consortium
1
15
20
25
30
Max Planck Institute for Marine Microbiology, Celsiusstraße 1, 28359 Bremen,
Germany
2 Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany
3 CSIC, Institute of Catalysis, Marie Curie, 2, 28049 Madrid, Spain
4 UPMC University Paris 6 and CNRS, UMR 7139 Marine Plants and Biomolecules,
Station Biologique de Roscoff, 29682 Roscoff, Bretagne, France
5 Proteomic Facility, CNB-National Centre for Biotechnology, CSIC, Darwin 3,
28049 Madrid, Spain
6 Institute for Coastal Marine Environment (IAMC), Laboratory of Marine Molecular
Microbiology, CNR, Spianata S. Raineri, 86, 98122 Messina, Italy
7 Institute for Biotechnology and Bioengineering (IBB), Centre of Biological
Engineering, Micoteca da Universidade do Minho, University of Minho, Braga,
Portugal
8 Microbiology Unit, BIOCANT Biotechnological Park, 3060-197 Cantanhede,
Portugal
9 Department of Life Sciences, University of Coimbra, 3001-401 Coimbra, Portugal
10 School of Biological Sciences, Bangor University, Deiniol Road, LL57 2UW
Gwynedd, UK
‡
*, These authors contributed equally to this work
† Corresponding author: Peter N. Golyshin; phone: +44 1248 38 3629, fax: Fax +44
1248 37 0731
35
Running title: Proteogenomics of the halophile H. tiamatea
1
Table of Contents
Supporting Text
Transporters in H. tiamatea
40
.................................................................... 3
Motility and chemotaxis in H. tiamatea
....................................................... 3
Phosphonate utilization in H. tiamatea
....................................................... 4
Fermentations in H. tiamatea
.................................................................... 4
Biotechnological aspects of polysaccharide-degrading halophiles
............... 5
Presence of H tiamatea-like species in the Mediterranean DHAL Medee
45
Biogeographical aspects
...... 6
............................................................................... 7
Supporting experimental procedures
Sampling and sequencing of the Medee ANR26 enrichment
....................... 8
Gene prediction and annotation of the ANR26 metagenome
....................... 9
Taxonomic analysis of the ANR26 metagenome
50
...................................... 10
CARD-FISH probe design and CARD-FISH on Medee samples
References
............... 10
....................................................................................................... 11
Supporting figure and table legends
.................................................................. 18
2
Supporting text
55
Transporters in H. tiamatea
H. tiamatea lacks tripartite ATP-independent periplasmic (TRAP) transporters, TonBdependent transporters (TBDT) and phosphotransferase transport systems (PTS).
Instead, it has mostly ABC-type transport systems and permeases. Surprisingly, one
transporter of the tripartite tricarboxylate transporter family (TctA) was found, which
60
were believed to only occur in Bacteria, not in Archaea or Eukaryota (Winnen et al.,
2003). Ion transporters could be annotated for the transport of sodium, potassium,
magnesium, ferrous and ferric iron, copper, zinc, various toxic heavy metal ions
(cobalt, cadmium, mercury, lead), phosphate and divalent anions. The H. tiamatea
genome encodes three sodium/proton antiporters and eleven potassium uptake
65
systems. Annotated transporters for organic compounds comprised phosphonates,
lipids, glutamine, amino acids, di- and oligopeptides, malonate, putrescine,
xanthine/uracil/thiamine/ascorbate, vitamin B12, thiamine, ribose, xylulose and
maltose/maltodextrin.
70
Motility and chemotaxis in H. tiamatea
In the original H. tiamatea description no flagella could be observed (Antunes et al.,
2008), but the genome encodes archaeal flagella genes, such as flagellin
components (flaBEFGHIJ), a flagellin-related protein and a flagellin cluster protein.
Also, chemotaxis and chemotaxis transduction genes were found. This indicates that
75
either H. tiamatea can produce a flagellum like H. utahensis (Wainø et al., 2000) or
that it has recently lost this capability.
3
Phosphonate utilization in H. tiamatea
H. tiamatea is known to harbor genes for methylphosphonate degradation (Antunes
et al., 2011). The complete genome revealed all genes for methylphosphonate
80
degradation that with the exception of phnP (Podzelinska et al., 2009) are all
arranged in a single cluster (phnGHIJKLM) (Kamat et al., 2011). This pathway is
thought to scavenge additional phosphate when inorganic phosphate is limiting and
releases the methylphosphonate methyl moiety in the form of methane.
85
Fermentations in H. tiamatea
H. tiamatea relies on a fermentative lifestyle under anoxic conditions with hydrogen,
L-lactate, D-lactate, acetate and possibly succinate as fermentation products. L- and
D-lactate are not only produced from the reduction of pyruvate, but also from
methylglyoxal, a toxic byproduct of the EMP pathway that results from
90
dephosphorylation
of
D-glyceraldehyde-3-phosphate
and
dihydroxyacetone-
phosphate. The H. tiamatea genome harbors the genes required for methylglyoxal
detoxification via L-lactaldehyde and (R)-S-lactoylglutathione to L- and D-lactate,
respectively.
H. tiamatea does have an acetoacetate decarboxylase that would enable it to
95
decarboxylate acetoacetate to acetone. However, it does not seem to have a suitable
2-propanol dehydrogenase for the subsequent reduction of acetone to 2-propanol,
and without this terminal reduction step there would be no disposal of reducing
equivalents and thus no benefit. Since the specificities of alcohol dehydrogenases
are difficult to predict, there might be an alternate alcohol dehydrogenase that
4
100
catalyzes this reaction. It is also not clear, whether H. tiamatea can co-ferment amino
acids.
Biotechnological aspects of polysaccharide-degrading halophiles
In halophiles, two major strategies have evolved to cope with high salinities and
105
prevent enzymes from denaturing and precipitation (Galinski, 1995). The first is to
counter high osmolarities by intracytoplasmic accumulation of compatible solutes like
quaternary amines or sugars such as trehalose (organic osmolyte strategy), and the
second is to accumulate high levels of internal potassium (and to lesser extents
sodium) chloride (salt-in strategy). The salt-in strategy is energetically favorable
110
(Oren, 1999), but requires cytoplasmic enzymes with amino acid compositions that
ensure acidic protein surfaces. As a consequence, these enzymes operate over a
wide pH range under high ionic strengths in aqueous solutions (Unsworth et al.,
2007), and some even preserve their function in certain organic solvents and ionic
liquids. Such enzymes are particularly promising targets for industrial applications,
115
even more so since many halophiles are also moderate thermophiles. However, in
spite of this undisputed potential (Litchfield, 2011), so far only β-carotene from the
algae Dunaliella, bacteriorhodopsin from Halobacterium and ectoine from Halomonas
are used and besides ectoine production no large-scale industrial application has
emerged (Ma et al., 2010; Oren, 2010).
120
Polysaccharide-degrading
halophiles
such
as
H. tiamatea
are
particular
promising sources to screen for enzymes with biotechnological applicability. For
example, H. tiamatea contains halotolerant xylanases. Xylans are hemicelluloses that
act as glue between cellulose and lignin, and constitute the second most abundant
5
polysaccharide in nature. Thus, halotolerant xylanases might be used to separate
125
unwanted lignin from cellulose in biofuel production (Ma et al., 2010; Anderson et al.,
2011). Likewise, halotolerant cellulases are of interest for biofuel production, such as
for example the potential modular GH9 family cellulase of H. tiamatea. This is also
the reason why the GH5 family cellulase of H. utahensis has recently been studied in
detail (Zhang et al., 2011).
130
Presence of H tiamatea-like species in the Mediterranean DHAL Medee
We enriched microbes from brine of the Eastern Mediterranean DHAL Medee
(south-west off the Western coast of Crete) and subsequently applied shotgun
metagenomics. Metagenome analyses revealed that the enrichment culture termed
135
ANR26 consisted of ~80-95% of Halorhabdus species. The longest assembled contig
(183,988 bp) featured a complete 16S rRNA gene with 99.7% identity to H. tiamatea
and 99.4% to H. utahensis (Fig. 1), but average nucleotide identities (ANI) between
all strains confirmed that they constitute distinct species. ANI values of the four
longest Halorhabdus-related contigs indicated a closer relatedness of the ANR26
140
Halorhabdus species to H. tiamatea (ANI: 86.95) than to H. utahensis (ANI: 82.95).
Presence of Halorhabus species in Medee was verified by catalyzed reporter
deposition fluorescence in situ hybridization (CARD-FISH) of Medee brine (Fig. S2)
with a novel Halorhabdus-specific probe (Halo178; see Supporting Experimental
Procedures). However, the abundances were too low to quantify in situ cell numbers.
145
CAZyme frequencies of the ANR26 enrichment were similar to those of the
completely sequenced Halorhabdus species, and correlated better with those of
H. tiamatea than of H. utahensis (Table S4). For instance, GH5 numbers were low
6
(two as in H. tiamatea instead of seven as in H. utahensis) and the GH13 numbers
were high. However, the profiles of the ANR26 enrichment and H. tiamatea were not
150
identical, as for example the ANR26 enrichment lacked GH51 genes that in
H. tiamatea except for one are encoded by the putative plasmid. This indicates that
the enriched H. tiamatea-like species from the Medee DHAL lacked this plasmid.
Biogeographical aspects
155
The similarity of H. tiamatea and the Halorhabdus species identified in the Medee
ANR26 enrichment might be explained by the common geological history of the
Mediterranean and Red Sea basins. The desiccation-driven formation of lateMiocene evaporites and their submergence in the early Pliocene occurred
simultaneously in both basins at a time, when the Red Sea had no link with the
160
Indian Ocean (the Bab el Mandeb Strait opening emerged more recently due to the
separation of Nubian, Somalian and Arabian plates). This suggests that the
Mediterranean and Red Seas most probably were part of the same hydrological
system in the Mio-Pliocene, when the evaporites developed (Hsü et al., 1978).
However, the Mediterranean and the Red Sea do not share such a common
165
geological history with the Great Salt Lake of Utah, the isolation source of
H. utahensis. Thus, the strong similarities in genome organization as well as
ecological niches as polysaccharide degraders of H. tiamatea and H. utahensis
indicates exchange between their seemingly isolated habitats. This corroborates a
previous study on seemingly isolated Haloquadratum walsbyi strains from salterns in
170
Spain and Australia (Dyall-Smith et al., 2011). These H. walsbyi isolates featured
highly similar genomes, supporting that these species are effectively dispersed
7
around the globe (Dyall-Smith et al., 2011), as is implied by Lourens Baas Becking's
statement that "everything is everywhere, but, the environment selects" (Baas
Becking, 1934).
175
In recent years, members of genus Halorhabdus have been identified in more
and more hypersaline habitats (Fan et al., 2003; van der Wielen et al., 2005; Pan et
al., 2006; Jiang et al., 2007; Bodaker et al., 2009; Makhdoumi-Kakhki et al., 2012;
Sangwan et al., 2012). This might fortify the view that Halorhabdus species can
occupy a distinct niche as polysaccharide degraders in hypersaline environments
180
worldwide.
Supporting experimental procedures
Sampling and sequencing of the Medee ANR26 enrichment
Brine of the Mediterranean DHAL Medee was sampled on 17th of December 2007
185
during the SAMCA MedBio 2 cruise of the R/V Urania (34° 19.778’ N, 22° 31.341’ E,
-3,040 m depth). Aboard, 10 ml of the sample were immediately incubated with 25 ml
of DSM medium 141 in an anaerobic atmosphere (N2/CO2, 80/20) and later cultivated
in the laboratory at 15 °C for six months (ANR26 enrichment). Total DNA was
extracted using the G'NOME DNA extraction Kit (BIO 101/Qbiogene, Morgan Irvine,
190
CA, USA) according to the manufacturer’s instructions, and subsequently sequenced
on a 454 FLX Ti sequencer (454 Life Sciences). In total 677,220 reads were
generated. Assembly with Newbler v. 2.3 resulted into 1,647 contigs comprising
3,918,195 bp (mean length: 2,379 bp; max. length: 183,988 bp; 67 contigs >10 kbp).
8
The metagenome sequences have been deposited at the ENA with the accession
195
number ERP002033.
Gene prediction and annotation of the ANR26 metagenome
Genes were called with the metagenome gene finder MetaGene (Noguchi et al.,
2006) in combination with a subsequent calling of ORFs exceeding 150 bp in the
200
intergenic regions in order to ensure that almost no genes were overlooked.
Annotation was carried out using a modified GenDB v. 2.2.1 annotation system
(Meyer et al., 2003). For each predicted gene, similarity searches were performed
against the NCBI non-redundant protein database (Pruitt et al., 2004), SWISSPROT
(The UniProt Consortium, 2011), KEGG (Kanehisa and Goto, 2000) and against the
205
protein family databases Pfam (Finn et al., 2009), InterPro (Hunter et al., 2011), COG
(Tatusov et al., 2003) and CAZy (Cantarel et al., 2009). Signal peptides were
predicted with SignalP v. 3.0 (Nielsen et al., 1999; Emanuelsson et al., 2007) and
transmembrane helices with TMHMM v. 2.0 (Krogh et al., 2001). Ribosomal RNA
genes were identified via BLAST searches (Altschul et al., 1997) against public
210
nucleotide databases and transfer RNA genes using tRNAScan-SE v. 1.21 (Lowe
and Eddy, 1997). Based on these observations, annotations were derived in an
automated fashion using a fuzzy logic-based approach (MicHanThi software; Quast,
2006).
9
215
Taxonomic analysis of the ANR26 metagenome
A metagenome taxonomic classification pipeline described elsewhere (Ferrer et al.,
2012; Teeling et al., 2012) was used for taxonomic analysis of the metagenome. Of
all reads, 583,865 (93.1%) were classified on superkingdom level, 583,814 (93.1%)
on phylum level, 548,712 (87.4%) on class level and 551,419 (87.9%) on genus
220
level. Of the classified reads, 74.9% were assigned to the genus Halorhabdus.
Metagenome 16S rRNA gene fragment analysis with the SILVA NGS pipeline,
v. 4.1.7 (Klindworth et al., 2012) confirmed the strong dominance of Halorhabdus-like
species in the ANR26 enrichment. Of the 390 reads carrying partial 16S rRNA genes,
295 had BLAST hits and 257 (87.1%) were assigned to the Halorhabdus genus,
225
while 95 (24.4%) of all 16S rRNA gene fragments had no relative in the SILVA
database (SILVA release 108) (Quast et al., 2013).
CARD-FISH probe design and CARD-FISH on Medee samples
Presence of Halorhabdus spp. in the DHAL Medee was analyzed via CARD-FISH
230
(Pernthaler et al., 2002, Pernthaler et al., 2004; Thiele et al., 2011). In brief, after
lysozyme and hydrochloric acid treatment for cell permeabilization, the hybridization
was carried out with a final probe concentration of 28 fmol/µl for 2 h at 35 °C. Signal
amplification was done using Alexa Fluor® 488 labeled tyramide (1 µg/ml final
concentration) for 45 minutes at room temperature. Both hybridization and
235
amplification were done on glass slides in humidity chambers. All filters were
counterstained with DAPI (4′,6-diamidino-2-phenylindole). The applied probes were
ARCH915 (Stahl and Amann, 1991), NON338 (Wallner et al., 1993) and a newly
10
developed Halorhabdus-specific probe Halo178 (5’ CCA GCT GGC GAG TCG TAT
3’).
240
Halo178 and two helper probes were designed using ARB (Ludwig et al., 2004)
and the Silva 108 database. The helper probes (Halo178-h1: 5’ CGG ACA TTA GCC
TCA GTT TC 3’ and Halo178-h2: 5’ TTT CCG ACT CGC CGC ACT 3’) were used to
increase the target site accessibility (Fuchs et al., 2000). The probe Halo178 was
used in an equimolar mixture with the helpers at a formamide concentration of 25%.
245
The Halo178 probe was evaluated with a high in silico specificity for Halorhabdus
spp., as no other taxon was found with less than 1.0 weighted mismatches.
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Supporting figure and table legends
Figure S1.
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Whole genome alignment between H. utahensis and H. tiamatea. The genome of
H. tiamatea was split into 50 bp fragments and subsequently mapped on the
H. utahensis with SSAHA2 v. 2.5 (Ning et al., 2001).
Figure S2.
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Numbers of GHs in archaeal genomes according to the CAZyme database (Cantarel
et al., 2009). From top to bottom: Halorhabdus tiamatea SARL4BT, Haloterrigena
turkmenica 4kT, Halorhabdus utahensis AX-2T, Halopiger xanaduensis SH-6T,
Caldivirga maquilingensis IC-167T, Ignisphaera aggregans AQ1.S1T, Sulfolobus
islandicus REY15AT, Sulfolobus solfataricus P2T, Sulfolobus islandicus Y.N.15.51T,
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Sulfolobus islandicus M.16.27T.
Figure S3.
Unrooted phylogenetic tree of characterized GH13 family proteins and GH13 family
proteins of H. tiamatea (see Table S3 for details). Sequences were aligned with
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MAFFT (Katoh et al., 2002) and the tree was computed with the program PhyML
(Guindon and Gascuel, 2003). Numbers indicate the bootstrap values (100
replicates). Proteins from H. tiamatea are marked by black diamonds.
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Figure S4.
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Identification of Hrd. species in a sample from the Eastern Mediterranean DHAL
Medee. A: DAPI staining; B: hybridization with the probe Halo178 and helper probes
Halo178-h1 and Halo178-h2, showing typical pleomorphic cells of Hrd. species.
Images were post-processed with Autoquant X (A and B) and Imaris 7.4.0 (only B).
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Table S1.
Annotations of all 50 glycoside hydrolases in H. tiamatea, including gene identifiers
(locus tags), closest characterized homologues, and EC numbers.
Table S2.
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Differentially expressed proteins of H. tiamatea grown on HBM liquid medium with
2% and 5% oxygen in the headspace compared to 0% oxygen. Protein annotations
and their associated biological processes are shown. Panel A shows 435 proteins
unambiguously identified with at least two peptides. Panel B shows 183 proteins
identified with on one peptide. Protein identification and quantitation was conducted
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with MASCOT v. 2.3.01 (panel A contains protein, and panel B peptide scores). Log2
ratios of the relative protein/peptide abundances at 2% versus 0% oxygen and 5%
versus 0% oxygen are shown.
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440
Table S3.
NCBI GenPept labels and accession numbers of the GH13 proteins that were used in
the phylogenetic analysis.
Table S4.
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Glycoside hydrolases in the genomes of H. utahensis (according to the CAZy
database as of 2013/12/10), H. tiamatea, and the ANR26 enrichment from the
Eastern Mediterranean DHAL Medee. The first number represents absolute counts,
the number in parentheses the relative number per Mbp, and the number in boldface
reflects GHs detected in the proteome.
450
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