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Supporting Information
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Multiphyletic origins of methylotrophy in Alphaproteobacteria, exemplified by
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comparative genomics of Lake Washington isolates
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David A.C. Becka,c, Tami L. McTaggarta, Usanisa Setboonsarnga*, Alexey Vorobeva**,
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Lynne Goodwine, Nicole Shapirod, Tanja Woyked, Marina G. Kalyuzhnayab,f, Mary E.
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Lidstroma,b, Ludmila Chistoserdova a#
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Departments of Chemical Engineeringa, Microbiologyb and eScience Institutec, University
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of Washington, Seattle, USA; DOE Joint Genome Institute, Walnut Creek, USAd, Los
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Alamos National Laboratory, Los Alamos, USAe, Biology Department, San Diego State
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University, San Diego, USAf
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#Address correspondence to: Ludmila Chistoserdova, milachis@u.washington.edu
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Contents
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Supplementary Text
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
Strain isolation and cultivation
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
DNA isolation, whole genome sequencing, assembly and genome annotation
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
Phylogenetic analysis
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
Reconstruction of methylotrophy metabolism pathways
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Supplementary References
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Supplementary Figures
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Supplementary Tables
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Strain isolation and cultivation. Strains Methylocsinus sp. LW3, Methylosinus sp.
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LW4, Methylosinus sp. LW5 and Labrys methylaminiphilus JLW10, isolated from Lake
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Washington, Seattle have been previously described (Auman et al., 2000; Miller et al.,
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2005). The remaining strains described here were isolated between 2004 and 2011.
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Sampling was carried out as previously described (Kalyuzhnaya et al., 2004). Samples
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were transferred to the laboratory on ice, where sub-samples originating from multiple
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sampling cores were mixed with each other and diluted with Lake Washington water
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collected as part of the sampling, to produce liquid slurries. These were incubated with
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one of the C1 substrates as listed in Supplementary Table 1. After a few transfers with
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dilutions 1:50, colony formation was observed on plates. The specific enrichment
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conditions for each strain are listed in Supplementary Table 1. Colonies were re-
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streaked for a few times onto the same medium, and then the identity of the strains was
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determined by polymerase chain reaction (PCR) amplification of 16S rRNA gene
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fragment using universal primers (27F and 1492R) followed by Sanger sequencing, as
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previously described (Kalyuzhnaya et al., 2009).
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All axenic cultures were routinely maintained on solid media supplemented by
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either methanol or methylamine. The purity was monitored via microscopy, 16S rRNA
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gene fragment amplification and sequencing, and ultimately via whole genome
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sequencing. Culture stocks were frozen at -800 with 10% DMSO.
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As methylotrophic representatives of most of these genera have been previously
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described (Doronina et al., 1998; 2002; Doronina and Trotsenkol 2003; McDonald et al.,
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2001; 2005; Firsova et al., 2009), the new strains from Lake Washington were only
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briefly phenotypically characterized. They all were facultative methylotrophs, being able
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to utilize a variety of multicarbon substrates such as glucose, fructose, succinate and
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pyruvate. They all also grew on rich media such as Lysogeny Broth or Nutrient medium
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(Difco). Growth temperature optima were around 30 0C. None of the strains revealed a
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propensity for lower temperature. However, in our enrichment cultures, the
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Methylobacterium types were clearly outcompeted by the Hyphomicrobium,
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Xanthobacter and Paracoccus types at 30 0C during initial incubations, as practically no
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pink colonies were detected after plating these enrichments. Conversely, the
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Methylobacterium types outcompeted all other alphaproteobacterial types in enrichments
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incubated at 10 0C, producing a significant crop of pink colonies on plates.
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DNA isolation, whole genome sequencing, assembly and genome annotation.
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Biomass for genomic DNA isolation was collected from plates. DNA was isolated in
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accordance with recommendations of the Department of Energy's Joint Genome Institute
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(DOE-JGI; Walnut Creek, CA). The genomes were sequenced using the Illumina (HiSeq
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2000) sequencing platform, at the Joint Genome Institute (JGI) production facility
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(http://www.jgi.doe.gov/). Sequencing reads were assembled using one or a combination
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of the following assemblers: ALLPATHS versions 39750 and r42328; Velvet version
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1.1.05; Phrap version 4.24; IDBA-UD version 1.0.9, as part of the JGI/Los Alamos
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National Laboratory assembly pipeline (Mavromatis et al., 2012). The genomes were
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annotated using the JGI annotation pipeline and uploaded as part of the IMG interface
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(http://img.jgi.doe.gov/cgi-bin/w/main.cgi; Markowitz et al., 2012).
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Phylogenetic analysis. Sequences were aligned using MUSCLE v3.8.31 (Edgar,
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2004). The set of protein orthologs was computed from the translated gene sequences
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for the genomes discussed herein using OrthoMCL v2.0.8 (Li et al., 2003). Single copy
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per genome orthologs (SCGO) were identified from the OrthoMCL results. Each SCGO
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member was aligned with MUSCLE v3.8.31 (Edgar, 2004) and the full set of alignments
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concatenated into a single file with FASconCAT v1.0 (Kück and Meusemann, 2010).
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RAxML v7.7.2 was used to compute the best-scoring maximum likelihood tree using the
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PROTGAMMAJTTF mode with partitions and 100 bootstrap replicates. 16S rRNA gene
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sequences were aligned and trees constructed similarly, with the addition of sequences
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of related organisms. Trees were rendered using the Interactive Tree Of Life software
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(iTOL; Letunic and Bork, 2011). Average amino acid identity (AAI) values were
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computed via reciprocal BLAST best hits between pairs of proteomes similarly with
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previously described methods (Konstantinidis and Tiedje, 2005).
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Reconstruction of methylotrophy metabolism pathways. Automated gene
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annotations created using the IMG pipeline were curated manually for genes involved in
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key metabolic pathways. Reconstruction of methylotrophy pathways was modeled after
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prior analysis of the genomes of Methylobacterium extorquens (Chistoserdova et al.,
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2003; Vuilleumier et al., 2009; Guffaz et al., 2014), Methylocella silvestris BL2 (Chen et
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al., 2010) and Methylosinus trichosporium OB3b (Matsen et al., 2013; Yang et al., 2013),
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as well as after activity-based analysis of specific genes/enzymes (Yoshida et al., 1994;
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Hagishita et al., 1996; Chistoserdova et al., 2003). Homologs of the previously described
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genes were identified in the newly sequenced genomes using comparative genomics
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tools that are parts of the IMG system. Genes without homologs in previously described
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genomes were searched using word searches (for example, ‘phosphoenolpyruvate
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carboxylase’ etc.), and their annotations were validated via BLAST against the non-
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redundant NCBI database.
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characterization of methanotrophic isolates from freshwater lake sediment. Appl Environ
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al. (2011) An integrated proteomics/ transcriptomics approach points to oxygen as the
main electron sink for methanol metabolism in Methylotenera mobilis. J Bacteriol 193:
4758-4765.
Chistoserdova, L., Chen, S.W., Lapidus, A., and Lidstrom, M.E. (2003) Methylotrophy in
Methylobacterium extorquens AM1 from a genomic point of view. J Bactriol 185: 29802987.
Hagishita, T., Yoshida, T., Izumi, Y., and Mitsunaga, T. (1996) Cloning and expression of
the gene for serine-glyoxylate aminotransferase from an obligate methylotroph
Hyphomicrobium methylovorum GM2. Eur J Biochem 241: 1-5.
Doronina, N.V., and Trotsenko, Y.A. (2003) Reclassification of 'Blastobacter viscosus' 7d
and 'Blastobacter aminooxidans' 14a as Xanthobacter viscosus sp. nov. and
Xanthobacter aminoxidans sp. nov. Int J Syst Evol Microbiol 53: 179-182.
Doronina, N.V., Trotsenko, Y.A., Kuznetzov, B.B., and Tourova, T.P. (2002) Emended
description of Paracoccus kondratievae. Int J Syst Evol Microbiol 52: 679-682.
Doronina, N.V., Trotsenko, Y.A., Krausova, V.I., Boulygina, E.S., and Tourova, T.P.
(1998) Methylopila capsulata gen. nov., sp. nov., a novel non-pigmented aerobic
facultatively methylotrophic bacterium. Int J Syst Bacteriol 48: 1313-1321.
Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res 32: 1792-1797.
Firsova, J., Doronina, N., Lang, E., Spröer, C., Vuilleumier, S., and Trotsenko, Y. (2009)
Ancylobacter dichloromethanicus sp. nov.--a new aerobic facultatively methylotrophic
bacterium utilizing dichloromethane. Syst Appl Microbiol 32: 227-232.
Gruffaz, C., Muller, E.E., Louhichi-Jelail, Y., Nelli, Y.R., Guichard, G., and Bringel, F.
(2014) Genes of the N-methylglutamate pathway are essential for growth of
Methylobacterium extorquens DM4 with monomethylamine. Appl Environ Microbiol 80:
3541-3550.
Kalyuhznaya, M.G., Martens-Habbena, W., Wang, T., Hackett, M. Stolyar, S.M. Stahl,
D.A, et al. (2009) Methylophilaceae link methanol oxidation to denitrification in
freshwater lake sediment as suggested by stable isotope probing and pure culture
analysis. Environ Microbiol Rep 1: 385-392.
Konstantinidis, K.T., and Tiedje, J.M. (2005) Towards a genome-based taxonomy for
prokaryotes. J Bacteriol 187: 6258-6264.
Kück, P., and Meusemann, K. (2010) FASconCAT: Convenient handling of data
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of phylogenetic trees made easy. Nucleic Acids Res 39: W475-478.
Li, L., Stoeckert, C.J., Jr, and Roos, D.S. (2003) OrthoMCL: identification of ortholog
groups for eukaryotic genomes. Genome Res 13: 2178-2189.
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(2012). IMG: the Integrated Microbial Genomes database and comparative analysis
system. Nucleic Acids Res 40: D115-122.
Matsen, J.B., Yang, S., Stein, L.Y., Beck, D., and Kalyuzhnaya, M.G. (2013) Global
molecular analyses of methane metabolism in methanotrophic Alphaproteobacterium,
Methylosinus trichosporium OB3b. Part I: Transcriptomic Study. Front Microbiol 4: 40.
Mavromatis, K., Land, M.L., Brettin, T.S., Quest, D.J., Copeland, A., Clum, A. et al.
(2012). The fast changing landscape of sequencing technologies and their impact on
microbial genome assemblies and annotation. PLoS One 7: e48837.
McDonald, I.R., Doronina, N.V., Trotsenko, Y.A., McAnulla, C., and Murrell, J.C. (2001)
Hyphomicrobium chloromethanicum sp. nov. and Methylobacterium chloromethanicum
sp. nov., chloromethane-utilizing bacteria isolated from a polluted environment. Int J Syst
Evol Microbiol 51: 119-122.
McDonald, I.R., Kämpfer, P., Topp, E., Warner, K.L., Cox, M.J., Hancock, T.L. et al.
(2005) Aminobacter ciceronei sp. nov. and Aminobacter lissarensis sp. nov., isolated
from various terrestrial environments. Int J Syst Evol Microbiol 55: 1827-1832.
Miller, J.A., Kalyuzhnaya, M.G., Noyes, E., Lara, J.C., Lidstrom, M.E., and
Chistoserdova, L. (2005) Labrys methylaminiphilus Sp. Nov., a new facultatively
methylotrophic bacterium from a freshwater lake sediment. Internat J Syst Evol Microbiol
55: 1247-1253.
Yang, S., Matsen, J.B., Konopka, M., Green-Saxena, A., Clubb J, Sadilek, M. et al.
(2013) Global molecular analyses of methane metabolism in methanotrophic
Alphaproteobacterium, Methylosinus trichosporium OB3b. Part II. Metabolomics and
13C-labeling study. Front Microbiol 4: 70.
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Supplementary Figure 1. Maximum likelihood phylogenetic trees of crotonyl-CoA
carboxylase/reductase (a), ethylmalonyl-CoA mutase (b) and malate dehydrogenase (c).
Different colors denote different families within Alphaproteobacteria and correspond to
the colors in Figures 1 and 2.
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Supplementary Figure 2. Maximum likelihood phylogenetic trees of the MxaF subunit
of calcium-dependent methanol dehydrogenase (a) and the alpha subunit of Nmethylglutamate dehydrogenase (b). Different colors denote different families within
Alphaproteobacteria and correspond to the colors in Figures 1 and 2.
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Supplementary Table 1. Strain isolation details, genome statistics and accession numbers
Strain
Aminobacter sp. 108
Year
isolated
2011
Medium/
substrate3
2/MA
Temperature
0C
30
Total
nucleotides
6,112,094
Ancylobacter sp. 117
2011
2/Me
30
Ancylobacter sp. 501b
2011
3/Me
Ancylobacter sp. FA202
2006
Hyphomicrobium sp. 802
GC%
63.37
Predicted
proteins
5,904
With function
predictions
4,881
NCBI accession
ARCZ00000000.1
4,635,764
68.17
4,280
3,783
JNLC00000000.1
Room
5,108,628
67.37
4,635
3,965
ARJL00000000.1
1/Me
Room
5,354,552
67.31
4,888
4,079
KB904818.1
2004
1/Me
Room
4,333,578
59.47
4,205
3,150
JAFM01000001.1
Hyphomicrobium sp. 99
2011
1/MA
30
4,108,307
59.54
3,953
3,006
XXXX00000000.1
Labrys methylaminiphilus
JLW10
Methylobacterium sp. 10
20031
2/MA
Room
7,517,092
62.99
6,932
5,822
XXXX00000000.1
2011
1/Me
10
4,982,370
66.55
4,658
3,615
JAEV00000000.1
Methylobacterium sp. 77
2011
2/Me
10
4,664,957
66.67
4,307
3,291
ARCS00000000.1
Methylobacterium sp. 88A
2011
1/Me
10
4,889,301
67.14
4,568
3,437
AQVT00000000.1
Methylopila sp. 73B
2004
2/Me
Room
4,382,767
69.35
4,147
3,408
JAFV01000001.1
Methylopila sp. M107
2004
2/Me
Room
4,453,493
67.98
4,210
3,165
ARWB01000001.1
Methylosinus sp. LW3
19962
4/CH4
Room
5,090,474
64.66
4,676
3,483
AZUO00000000.1
Methylosinus sp. LW4
19962
4/CH4
Room
4,824,446
64.91
4,412
3,312
ARAB00000000.1
Methylosinus sp. LW5
19962
4/CH4
Room
4,760,983
64.84
4,375
3,383
JMKQ00000000.1
Methylosinus sp. PW1
19962
4/CH4
Room
5,128,790
64.68
4,826
3,534
JQNK00000000.1
Paracoccus sp. N5
2004
1/For
Room
4,354,541
67.70
4,210
3,395
AQUO00000000.1
Xanthobacter sp. 126
2011
1/MA
30
5,472,264
67.80
5,125
4,234
JAFO01000001.1
Xanthobacter sp. 91
2011
1/Me
30
5,339,523
68.09
4,918
4,231
JNIB00000000.1
1Miller
et al., 2005; 2Auman et al., 2000; 3The following media were used: 1, lake water, 2, MM2 (Beck et al., 2011), 3, Hypho (Beck et al., 2011), 4, NMS (Auman
et al., 2000). MA, 1 mM methylamine, with periodic additions; Me, 1 mM methanol, with periodic additions; For, 5 mM formate, with periodic additions.
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