Supplementary material
A metagenome of a full-scale microbial community carrying out Enhanced Biological
Phosphorus Removal
Mads Albertsen, Lea Benedicte Skov Hansen, Aaron Marc Saunders, Per Halkjær Nielsen,
5
and Kåre Lehmann Nielsen
Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg
University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark
Supplementary Text 1 of 1.
10
An investigation of the reads mapping to the ppk1 gene of Accumulibacter was conducted to
evaluate the sensitivity and specificity of the reference assembly against the Accumulibacter
genome. 87 ppk1 sequences were obtained from NCBI and five ppk1 genes of closely related
species were included.
All ppk1 sequences were trimmed to the length of the smallest ppk1 fragments (1073 bp) and
15
clustered using cdhit-est v.4.2.1 (Li and Godzik, 2006) with the following parameters; -c 0.99
–r 1. A BLAST database was created from the resulting 68 non-redundant sequences. The
ppk1 sequences were assigned to different nodes in the phylogenetic tree using MEGAN. As
MEGAN assigns reads to nodes based on the species information in the BLAST hits, the
header of the individual ppk1 sequences were changed to reflect the topology of the
20
phylogenetic tree.
The metagenomic reads that matched the extracted region of the ppk1 gene in the
Accumulibacter genome in the original reference assembly, were extracted to investigate the
specificity of the reference mapping (inclusion of other bacteria in the mapping). These
sequences were matched to the ppk1 database using BLASTn with default parameters except
25
–word_size = 7, –outfmt 5 and –evalue 1e-5. The output was analysed in MEGAN.
In order to investigate the sensitivity (inclusion of most Accumulibacter clades in the
mapping) a reference assembly was conducted against the 68 Accumulibacter ppk1 genes
using CLCs reference mapping function requiring min. 85% identity over 70% of the read
length. Only reads with a minimum length of 60 bp were used. Otherwise the analysis was
30
conducted as the specificity analysis.
1 of 14
The high resolution of the diversity within the genus using the ppk1 gene was used as a test
case to validate the specificity (false positive matches) and the sensitivity (ability to recruit
reads from other Accumulibacter species) of the reference mapping. A total of 138 ppk1 genes
were used to construct a phylogenetic tree (Figure S5) and the phylogenetic position of each
35
sequence was mimicked in MEGAN for the assignment of individual reads to different nodes
on the tree. The specificity analysis showed that only 10 of the 268 ppk1 reads assigned to the
Accumulibacter IIA str. UW-1 ppk1 gene had a better match to non-Accumulibacter ppk1
sequences (Figure S7A). However, the sensitivity analysis showed that although we were able
to recruit most clade IIA ppk1 reads using the clade IIA str. UW-1 ppk1 gene, we were not
40
able to recruit more than approximately 30-50% of the reads from other Accumulibacter
species (Figure S7C).
2 of 14
Supplementary Figure 1 of 7.
45
Supplementary Figure 1. Histogram of length distribution of the de novo assembled contigs.
Contigs ≥ 300 bp were used for further analysis (blue bars).
3 of 14
50
Supplementary Figure 2 of 7.
Supplementary Figure 2. Histogram of the length distribution of ORFs with a significant
BLAST hit (e-value ≤ 1e-5) compared to ORFs where no significant hit could be found. The
55
“double curved” plots are due to the minimum contig size of 300 bp (100 amino acids).
4 of 14
Supplementary Figure 3 of 7.
60
Supplementary Figure 3. A) Species abundance curve. “Best hit” represent species
assignment based on best BLASTP hit. “10% Bitscore filter” represent species assignment if
the best BLASTP hit had a bitscore that is >10% higher than the second best BLASTP hit.
The graph only shows species with more than 100 ORFs assigned (100 ORFs ≈ 0.05% of all
65
ORFs). B) Species abundance chart. The 20 most abundant species are shown in the legend in
decreasing abundance. ORFs were assigned based on best BLASTP hit. C) Rarefaction
curves. The rarefaction function of MEGAN was used to create rarefaction curves at different
phylogenetic levels. The assignment is based on a 10% bitscore filter and minimum 5 ORFs
assigned.
70
5 of 14
Supplementary Figure 4 of 7.
Supplementary Figure 4. Annotation of ORFs in the largest contig (32884 bp). A yellow
75
ORF denote a significant blast hit (e-value ≤ 1e-5) whereas brown denotes no significant hit.
6 of 14
Supplementary Figure 5 of 7.
80
Supplementary Figure 5. Phylogenetic tree of ppk1 sequences. Sequences from Aalborg
East have been marked in red and the ppk1 sequence from “Candidatus Accumulibacter
phosphatis” clade IIA str. UW-1 has been marked in blue. In addition clade assignments have
been added. A putative new clade has been marked as IIx. The tree was first created on the
basis of 87 general ppk1 genes and only selected representative sequences are shown in the
85
final tree. The outgroup sequences (not shown on the tree) were Ralstonia eutropha
YP_300029, Ralstonia eutropha YP_729175 and Stenotrophomonas maltophilia K279a
CAQ44540.
7 of 14
Supplementary Figure 6 of 7.
90
Supplementary Figure 6. Comparison of genes prevalent in the different read pools based on
a reference mapping to the Accumulibacter genome. The percent read length covered was
used to compare presence or absence of genes. High identity reads (>95% identical at nt level,
x-axis) was compared with the rest of the read pool (≤95% identical at nt level, y-axis). Each
95
dot represents one gene. In order to compare which genes that differed between the high
(>95%) and low (≤95%) identity read pools, the read pool size of the low-identity group was
normalized (by subsampling) to the same size (179 741 reads) as the high-identity read pool,
thereby effectively comparing the prevalent genes in both read pools.
8 of 14
100
Supplementary Figure 7 of 7.
Supplementary Figure 7. Investigation of the specificity and sensitivity of the mapping of
metagenome reads to the genome of Accumulibacter clade IIA (NC_013194). MEGAN was
used to visualize the BLASTn results. A 10% bitscore difference was used to assign reads to
105
nodes. A) Investigation of the specificity of the mapping of the metagenome reads to the
Accumulibacter clade IIA ppk1 gene. The metagenome reads mapping to the clade IIA ppk1
gene were extracted and mapped to 68 non-redundant accumulibacter ppk1 genes and 5 ppk1
genes from closely related species. Few reads had best match to other species than
Accumulibacter. B) Investigation of the ability to include other Accumulibacter clades by the
110
use of the Accumulibacter clade IIA genome. The metagenome reads were mapped to 68 nonredundant Accumulibacter ppk1 genes and the extracted read pool was searched (BLASTn)
against all 68+5 ppk1 genes and visualised using MEGAN. C) The combination of panel A
and B reveals that most clade IIA reads are extractable using the clade IIA genome, however
only approximately 30% of reads matching other clades are extracted.
9 of 14
115
Supplementary Table 1 of 2.
10 of 14
Supplementary references for Table S1.
120
Crocetti GR., Hugenholtz P, Bond PL, Schuler A, Keller J, Jenkins D, Blackall LL (2000).
Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed
probes for their detection and quantitation. Appl Environ Microbiol 66:1175-1182.
Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M (2001). In situ characterization of
Nitrospira-like nitrite- oxidizing bacteria active in wastewater treatment plants. Appl Environ
Microbiol 67:5273-5284.
125
Daims H, Bruhl A, Amann R, Schleifer K-H,Wagner M (1999). The domain-specific probe
EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more
comprehensive probe set. Syst Appl Microbiol 22: 434–444.
Erhart R, Bradford D, Seviour RJ, Amann R, Blackall LL (1997). Development and use of
fluorescent in situ hybridization probes for the detection and identification of ‘Microthrix
parvicella’ in activated sludge. Systematic Appl Microbiol 20:310-318.
130
135
Flowers J, He S, Carvalho G, Peterson SB, Lopez C, Yilmaz S, Zilles JL, Morgenroth E,
Lemos PC, Reis MAM, Crespo MTB, Noguera DR, McMahon KD (2008). Ecological
differentiation of Accumulibacter in EBPR reactors. In: Proceedings of the Water
Environment Federation, WEFTEC 2008 (12):31-42.
Gieseke A, Purkhold U, Wagner M, Amann R, Schramm A (2001). Community structure and
activity dynamics of nitrifying bacteria in a phosphate-removing biofilm. Appl Environ
Microbiol 67:1351-1362.
Giuliano L, De Domenico M, De Domenico E, Hofle MG, Yakimov MM (1999).
Identification of culturable oligotrophic bacteria within naturally occurring bacterioplankton
communities of the Ligurian sea by 16S rRNA sequencing and probing. Micro Ecol 37:77-85.
140
145
Hess A, Zarda B, Hahn D, Haner A, Stax D, Hohener P, Zeyer J (1997). In situ analysis of
denitrifying toluene- and m-xylene-degrading bacteria in a diesel fuel-contaminated
laboratory aquifer column. Appl Environ Microbiol 63:2136-2141.
Hugenholtz P, Tyson GW, Webb RI, Wagner AM, Blackall LL (2001). Investigation of
Candidate division TM7, a recently recognizedmajor lineage of the domain bacteriawith no
known pure-culture representatives. Appl Environ Microbiol 67:411-419.
Kanagawa T, Kamagata Y, Aruga S, Kohno T, Horn M, Wagner M (2000). Phylogenetic
analysis of and oligonucleotide probe development for Eikelboom type 021N filamentous
bacteria isolated from bulking activated sludge. Appl Environ Microbiol 66:5043-5052.
150
Kong YH, Beer M, Rees GN, Seviour RJ (2002). Functional analysis of microbial
communities in aerobiceanaerobic sequencing batch reactors fed with different phosphorus/
carbon (P/C) ratios. Microbiology-Sgm 148:2299-2307.
11 of 14
Kong Y, Nielsen JL, Nielsen PH (2005). Identity and ecophysiology of uncultured
actinobacterial polyphosphate- accumulating organisms in full-scale enhanced biological
phosphorus removal plants. Appl Environ Microbiol 71:4076-4085.
155
160
165
Kragelund C, Levantesi C, Borger A, Thelen K, Eikelboom D, Tandoi V, Kong Y,
Krooneman J, Larsen P, Thomsen TR, Nielsen PH (2008). Identity, abundance and
ecophysiology of filamentous bacteria belonging to the Bacteroidetes present in activated
sludge plants. Microbiology 154:886-894.
Lajoie CA, Layton AC, Gregory IR, Sayler GS, Taylor DE, Meyers AJ (2000). Zoogloeal
clusters and sludge dewatering potential in an industrial activated-sludge wastewater
treatment plant. Water Environ Research 72:56-64.
Levantesi C, Rossetti S, Thelen K, Kragelund C, Krooneman J, Eikelboom D, Nielsen PH,
Tandoi V (2006). Phylogeny, physiology and distribution of ‘Candidatus Microthrix calida’,
anew Microthrix species isolated from industrial activated sludge wastewater treatment
plants. Environ Microbiol 8:1552-1563.
Maixner F, Noguera DR, Anneser B, Stoecker K, Wegl G, Wagner M, Daims H (2006).
Nitrite concentration influences the population structure of Nitrospira-like bacteria. Environ
Microbiol 8:1487-1495.
170
Mobarry BK, Wagner M, Urbain V, Rittmann BE, Stahl DA (1996). Phylogenetic probes for
analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ Microbiol
62:2156-2162.
Nguyen HTT, Le VQ, Hansen AA, Nielsen JL, Nielsen PH (2011). High diversity and
abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated
sludge systems. FEMS Microbiol Ecol 76:256-267.
175
Rossello-Mora RA, Wagner M, Amann R, Schleifer KH (1995). The abundance of Zoogloea
ramigera in sewage treatment plants. Appl Environ Microbiol 61:702-707.
Schauer M, Hahn MW (2005). Diversity and phylogenetic affiliations of morphologically
conspicuous large filamentous bacteria occurring in the pelagic zones of a broad spectrumof
freshwater habitats. Appl Environ Microbiol 71:1931-1940.
180
185
Thomsen TR, Nielsen JL, Ramsing NB, Nielsen PH (2004). Micromanipulation and further
identification of FISH-labelled microcolonies of a dominant denitrifying bacterium in
activated sludge. Environ Microbiol 6:470-479.
Trebesius K, Leitritz L, Adler K, Schubert S, Autenrieth IB, Heesemann J (2000). Culture
independent and rapid identification of bacterial pathogens in necrotising fasciitis and
streptococcal toxic shock syndrome by fluorescence in situ hybridisation. Medical Microbiol
Immun 188:169-175.
12 of 14
Supplementary Table 2 of 2
190
Table S2 Selected reference genomes from Dinsdale et al., (2008b) used for comparison with
the metagenome obtained in the current study. In addition a metagenome from a non-EPBR
wastewater treatment plant was included (Sanapareddy et al., 2009).
Metagenome Name
Soudan Red Stuff
Soudan Black Stuff
Low Saltern microbes
Medium Saltern Microbes (MB1110)
Medium saltern microbes (MB1111)
Low saltern pond plasmids (TT)
High saltern microbial (HB1128)
Salton Sea Bacteria 1
Medium salinity microbial (MB1116)
Low salinity microbial (LB1128)
Line Islands Kingman Reef B2 bacteria
Line Islands Christmas Reef B3 bacteria
Line Islands Palmyra F8 Bacteria
DMSP 1 (MAM.1)
DMSP 2 (MAM.2)
VAN 2 (MAM 4)
Tilapia pond microbes
Healthy Tilapia pond microbes
Healthy Prebead tank microbes
Tpond microbe 3
Rios Mesquites Stromatolites bacteria
Pozas Azule II stromatolite microbes
Healthy slime bacteria
Morbid slime bacteria
Healthy gut bacteria
Morbid gut bacteria
Non-EBPR wastewater treatment plant
Environment
Subterranean
Subterranean
Hyper-Saline
Hyper-Saline
Hyper-Saline
Hyper-Saline
Hyper-Saline
Hyper-Saline
Hyper-Saline
Hyper-Saline
Marine
Marine
Marine
Marine
Marine
Marine
Freshwater
Freshwater
Freshwater
Freshwater
Microbialites
Microbialites
Fish
Fish
Fish
Fish
WWTP
MG-RAST ID
4440281
4440282
4440437
4440435
4440434
4440090
4440419
4440329
4440425
4440426
4440037
4440041
4440039
4440364
4440360
4440363
4440440
4440413
4440411
4440422
4440060
4440067
4440059
4440066
4440055
4440056
N/A
Reference
Edwards et al., 2006
Edwards et al., 2006
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Swan et al., 2010
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Dinsdale et al., 2008a
Dinsdale et al., 2008a
Dinsdale et al., 2008a
Mou et al., 2008
Mou et al., 2008
Mou et al., 2008
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Rodriguez-Brito et al 2009
Breitbart et al., 2009
Desnues et al., 2008
Angly et al., 2009
Angly et al., 2009
Angly et al., 2009
Angly et al., 2009
Sanapareddy et al., 2009
Supplementary references for Table S1.
195
Angly FE, Willner D, Prieto-Davó A, Edwards RA, Schmieder R, Vega-Thurber R, et al.
(2009). The GAAS metagenomic tool and its estimations of viral and microbial average
genome size in four major biomes. PLoS Computational Biology 5:e1000593.
Breitbart M, Hoare A, Nitti A, Siefert J, Haynes M, Dinsdale E, et al. (2009). Metagenomic
and stable isotopic analyses of modern freshwater microbialites in Cuatro Ciénegas, Mexico.
Environ Microbiol 11:16-34.
13 of 14
200
Desnues C, Rodriguez-Brito B, Rayhawk S, Kelley S, Tran T, Haynes M, et al. (2008).
Biodiversity and biogeography of phages in modern stromatolites and thrombolites. Nature
452:340-343.
Dinsdale EA, Pantos O, Smriga S, Edwards RA, Angly F, Wegley L, et al. (2008a). Microbial
ecology of four coral atolls in the Northern Line Islands. PloS one 3:e1584.
205
Dinsdale EA, Edwards RA, Hall D, Angly F, Breitbart M, Brulc JM, et al. (2008b).
Functional metagenomic profiling of nine biomes. Nature 452:629–632.
Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, Peterson DM, et al.
(2006). Using pyrosequencing to shed light on deep mine microbial ecology. BMC genomics
7:57.
210
Mou X, Sun S, Edwards RA, Hodson RE, Moran MA. (2008). Bacterial carbon processing by
generalist species in the coastal ocean. Nature 451:708-711.
Rodriguez-Brito B, Li L, Wegley L, Furlan M, Angly F, Breitbart M, et al. (2010). Viral and
microbial community dynamics in four aquatic environments. The ISME J 4:739-751.
215
Sanapareddy N, Hamp TJ, Gonzalez LC, Hilger HA, Fodor AA, Clinton SM. (2009).
Molecular diversity of a North Carolina wastewater treatment plant as revealed by
pyrosequencing. Appl Environ Microbiol 75:1688-1696.
Swan BK, Ehrhardt CJ, Reifel KM, Moreno LI, Valentine DL. (2010). Archaeal and bacterial
communities respond differently to environmental gradients in anoxic sediments of a
California hypersaline lake, the Salton Sea. Appl Environ Microbiol 76:757-768.
220
14 of 14