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Supplementary information
Haloplanus rubicundus sp. nov., an extremely halophilic archaeon isolated from solar
salt
Yeon Bee Kima,b, Joon Yong Kimc, Hye Seon Songa,b, Se Hee Leea, Na-Ri Shind, Jin-Woo
Baec, Jinjong Myounge, Ki-Eun Leef, In-Tae Chaf, Jin-Kyu Rheeb*, and Seong Woon Roha*
aMicrobiology and Functionality Research Group, World Institute of Kimchi, Gwangju
61755, Republic of Korea
bDepartment of Food Science and Engineering, Ewha Womans University, Seoul 03760,
Republic of Korea
cDepartment of Biology, Kyung Hee University, Seoul 02447, Republic of Korea
dBiological Resource Center, Korea Research Institute of Bioscience and Biotechnology,
Jeollabuk-do 56212, Republic of Korea
eKorea Zoonosis Research Institute, Chonbuk National University, Jeollabuk-do 54531,
Republic of Korea.
fMicroorganism Resources Division, National Institute of Biological Resources, Incheon
22689, Republic of Korea
*Corresponding author.
E mail addresses: seong18@gmail.com (S. W. Roh), jkrhee@ewha.ac.kr (J. Rhee)
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Supplementary Table S1. 16S rRNA gene sequence similarities between the two novel
strains and ten related type strains in the order of high similiarity value.
Taxon Strain AccessionSimilarity (%)
1 2 3 4
Haloplanus natans DSM 17983 KE386573 98.78 98.64 98.57 98.57
Haloplanus aerogenes CGMCC 1.10124 jgi.1058094 98.44 98.44 98.50 98.50
Haloplanus vescus CGMCC 1.8712 jgi.1058054 98.30 98.16 98.10 98.10
Haloplanus salinus YGH66 JQ237126 97.77 97.91 97.84 97.84
Haloplanus litoreus GX21 HM063950 96.93 96.79 96.72 96.72
Haloplanus salinarum SP28 KX151776 96.52 96.38 96.31 96.31
Haloplanus rallus MBLA0036 KU933931 96.46 96.33 96.26 96.26
Haloplanus ruber R35 HM159597 96.02 96.16 96.09 96.09
Haloprofundus marisrubri SB9 KJ999759 91.65 91.65 91.58 91.58
Salinigranum salinum YJ-50-S2 KC918822 91.32 91.32 91.39 91.39
16S rRNA genes: 1, Haloplanus rubicundus CBA1112T rrnA; 2, Hpn. rubicundus CBA1112T
rrnB; 3, Hpn. rubicundus CBA1113 rrnA; 4, Hpn. rubicundus CBA1113 rrnB.
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Supplementary Table S2. in silico DDH (isDDH) values between CBA1112T, CBA1113,
and closely related Haloplanus strains.
Query genome Reference genome isDDH value Model confidence interval (%)
Hpn. rubicundus CBA1112T Hpn. rubicundus CBA1113 89.5 87.1‒91.5
Hpn. natans JCM 14081T 31.0 28.6‒33.5
Hpn. aerogenes JCM 16430T 28.9 26.5‒31.4
Hpn. vescus JCM 16055T 25.4 23.1‒27.9
Hpn. salinus JCM 18368T 38.6 36.1‒41.1
Hpn. rubicundus CBA1113 Hpn. natans JCM 14081T 30.9 28.5‒33.4
Hpn. aerogenes JCM 16430T 28.9 26.5‒31.4
Hpn. vescus JCM 16055T 25.5 23.2‒28.0
Hpn. salinus JCM 18368T 38.5 36.0‒41.0
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Supplementary Table S3. Strain-specific POGs annotated and classified using the KEGG database.
Strain K numberNo. ofmatched gene
Orthology description Pathway Subcategory Category
Haloplanus rubicundusCBA1112
K07213 1 ATOX1, ATX1, copZ, golB; copper chaperone Mineral absorption Digestive system Organismal Systems
K00694 1 bcsA; cellulose synthase (UDP-forming) Starch and sucrose metabolism
Carbohydrate metabolism Metabolism
Biofilm formation - Escherichia coli
Cellular community - prokaryotes
Cellular Processes
K10742 1 DNA2; DNA replication ATP-dependent helicase Dna2 DNA replication Replication and repair Genetic Information Processing
K02316 1 dnaG; DNA primase DNA replication Replication and repair Genetic Information Processing
K04043 1 dnaK, HSPA9; molecular chaperone DnaK RNA degradation Folding, sorting and degradation
Genetic Information Processing
Longevity regulating pathway - worm
Aging Organismal Systems
Tuberculosis Infectious disease: bacterial Human Diseases
K01470 1 E3.5.2.10; creatinine amidohydrolase Arginine and proline metabolism
Amino acid metabolism Metabolism
K04042 1 glmU; bifunctional UDP-N-acetylglucosamine pyrophosphorylase /Glucosamine-1-phosphate N-acetyltransferase
Amino sugar and nucleotide sugar
metabolism
Carbohydrate metabolism Metabolism
K10726 1 mcm, cdc21; replicative DNA helicase Mcm DNA replication Replication and repair Genetic Information Processing
K01893 1 NARS, asnS; asparaginyl-tRNA synthetase Aminoacyl-tRNA biosynthesis
Translation Genetic Information Processing
K11936 1 pgaC, icaA; poly-beta-1,6-N-acetyl-D-glucosamine synthase Biofilm formation - Escherichia coli
Cellular community - prokaryotes
Cellular Processes
K01007 1 pps, ppsA; pyruvate, water dikinase Pyruvate metabolism Carbohydrate metabolism Metabolism
Carbon fixation pathways in prokaryotes
Energy metabolism Metabolism
Methane metabolism Energy metabolism Metabolism
K01318 1 sspA; glutamyl endopeptidase Quorum sensing Cellular community - Cellular Processes
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prokaryotesK06013 1 STE24; STE24 endopeptidase Terpenoid backbone
biosynthesisMetabolism of terpenoids
and polyketidesMetabolism
K00788 1 thiE; thiamine-phosphate pyrophosphorylase Thiamine metabolism Metabolism of cofactors and vitamins
Metabolism
K03657 2 uvrD, pcrA; DNA helicase II / ATP-dependent DNA helicase PcrA Nucleotide excision repair
Replication and repair Genetic Information Processing
Mismatch repair Replication and repair Genetic Information Processing
K13525 1 VCP, CDC48; transitional endoplasmic reticulum ATPase Protein processing in endoplasmic reticulum
Folding, sorting and degradation
Genetic Information Processing
Legionellosis Infectious disease: bacterial Human Diseases
K01537 1 ATP2C; P-type Ca2+ transporter type 2C - - -
K06930 1 bat; HTH-type transcriptional regulator, bacterioopsin transcriptional activator and related proteins
- - -
K07282 1 capA, pgsA; gamma-polyglutamate biosynthesis protein CapA - - -
K10725 3 cdc6A; archaeal cell division control protein 6 - - -
K01449 1 cwlJ, sleB; N-acetylmuramoyl-L-alanine amidase - - -
K19175 1 dptH; DNA phosphorothioation-dependent restriction protein DptH - - -
K05521 1 draG; ADP-ribosyl-[dinitrogen reductase] hydrolase - - -
K00590 4 E2.1.1.113; site-specific DNA-methyltransferase (cytosine-N4-specific)
- - -
K00571 2 E2.1.1.72; site-specific DNA-methyltransferase (adenine-specific) - - -
K03687 1 GRPE; molecular chaperone GrpE - - -
K02486 1 K02486; two-component system, sensor kinase - - -
K07058 1 K07058; membrane protein - - -
K07454 1 K07454; putative restriction endonuclease - - -
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K07465 1 K07465; putative RecB family exonuclease - - -
K07496 1 K07496; putative transposase - - -
K07498 1 K07498; putative transposase - - -
K07499 2 K07499; putative transposase - - -
K10716 1 kch, trkA, mthK, pch; voltage-gated potassium channel - - -
K07448 1 mrr; restriction system protein - - -
K12287 1 mshQ; MSHA biogenesis protein MshQ - - -
K03496 1 parA, soj; chromosome partitioning protein - - -
K00344 1 qor, CRYZ; NADPH:quinone reductase - - -
K02485 1 rssB, hnr; two-component system, response regulator - - -
K03294 1 TC.APA; basic amino acid/polyamine antiporter, APA family - - -
K06957 1 tmcA; tRNA(Met) cytidine acetyltransferase - - -
K04763 1 xerD; integrase/recombinase XerD - - -
K07114 1 yfbK; Ca-activated chloride channel homolog - - -
K16263 1 yjeH; amino acid efflux transporter - - -
K16267 1 zipB; zinc and cadmium transporter - - -
K06915 2 K06915; uncharacterized protein - - -
K06921 1 K06921; uncharacterized protein - - -
K06987 1 K06987; uncharacterized protein - - -
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K07052 4 K07052; uncharacterized protein - - -
K07064 2 K07064; uncharacterized protein - - -
K07076 1 K07076; uncharacterized protein - - -
K09807 1 K09807; uncharacterized protein - - -
Haloplanus rubicundusCBA1113
K00128 1 ALDH; aldehyde dehydrogenase (NAD+) Glycolysis / Gluconeogenesis
Carbohydrate metabolism Metabolism
Ascorbate and aldarate metabolism
Carbohydrate metabolism Metabolism
Pyruvate metabolism Carbohydrate metabolism Metabolism
Fatty acid degradation Lipid metabolism Metabolism
Glycerolipid metabolism Lipid metabolism Metabolism
Valine, leucine and isoleucine degradation
Amino acid metabolism Metabolism
Lysine degradation Amino acid metabolism Metabolism
Arginine and proline metabolism
Amino acid metabolism Metabolism
Histidine metabolism Amino acid metabolism Metabolism
Tryptophan metabolism Amino acid metabolism Metabolism
beta-Alanine metabolism Metabolism of other amino acids
Metabolism
Insect hormone biosynthesis
Metabolism of terpenoids and polyketides
Metabolism
Limonene and pinene degradation
Metabolism of terpenoids and polyketides
Metabolism
Chloroalkane and chloroalkene degradation
Xenobiotics biodegradation and metabolism
Metabolism
K01438 1 argE; acetylornithine deacetylase Arginine biosynthesis Amino acid metabolism Metabolism
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K03407 1 cheA; two-component system, chemotaxis family, sensor kinase CheA
Two-component system Signal transduction Environmental Information Processing
Bacterial chemotaxis Cell motility Cellular Processes
K00390 1 cysH; phosphoadenosine phosphosulfate reductase Sulfur metabolism Energy metabolism Metabolism
K06223 2 dam; DNA adenine methylase Mismatch repair Replication and repair Genetic Information Processing
K01439 1 dapE; succinyl-diaminopimelate desuccinylase Lysine biosynthesis Amino acid metabolism Metabolism
K00558 1 DNMT1, dcm; DNA (cytosine-5)-methyltransferase 1 Cysteine and methionine metabolism
Amino acid metabolism Metabolism
MicroRNAs in cancer Cancer: overview Human Diseases
K16937 1 doxD; thiosulfate dehydrogenase [quinone] large subunit Sulfur metabolism Energy metabolism Metabolism
K01051 1 E3.1.1.11; pectinesterase Pentose and glucuronate interconversions
Carbohydrate metabolism Metabolism
Two-component system Signal transduction Environmental Information Processing
K08688 1 E3.5.3.3; creatinase Arginine and proline metabolism
Amino acid metabolism Metabolism
K18128 1 gadh; D-glyceraldehyde dehydrogenase (NADP+) Pentose phosphate pathway
Carbohydrate metabolism Metabolism
K05831 1 lysK, argE; [amino group carrier protein]-lysine/ornithine hydrolase
Lysine biosynthesis Amino acid metabolism Metabolism
Arginine biosynthesis Amino acid metabolism Metabolism
K00971 1 manC, cpsB; mannose-1-phosphate guanylyltransferase Fructose and mannose metabolism
Carbohydrate metabolism Metabolism
Amino sugar and nucleotide sugar
metabolism
Carbohydrate metabolism Metabolism
K10726 1 mcm, cdc21; replicative DNA helicase Mcm DNA replication Replication and repair Genetic Information Processing
K00140 1 mmsA, iolA, ALDH6A1; malonate-semialdehyde dehydrogenase (acetylating)/ methylmalonate-semialdehyde dehydrogenase
Propanoate metabolism Carbohydrate metabolism Metabolism
Inositol phosphate metabolism
Carbohydrate metabolism Metabolism
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Valine, leucine and isoleucine degradation
Amino acid metabolism Metabolism
beta-Alanine metabolism Metabolism of other amino acids
Metabolism
K01893 1 NARS, asnS; asparaginyl-tRNA synthetase Aminoacyl-tRNA biosynthesis
Translation Genetic Information Processing
K01480 1 speB; agmatinase Arginine and proline metabolism
Amino acid metabolism Metabolism
K01791 1 wecB; UDP-N-acetylglucosamine 2-epimerase (non-hydrolysing) Amino sugar and nucleotide sugar
metabolism
Carbohydrate metabolism Metabolism
Two-component system Signal transduction Environmental Information Processing
Biofilm formation - Vibrio cholerae
Cellular community - prokaryotes
Cellular Processes
K02472 1 wecC; UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase Amino sugar and nucleotide sugar
metabolism
Carbohydrate metabolism Metabolism
Two-component system Signal transduction Environmental Information Processing
Biofilm formation - Vibrio cholerae
Cellular community - prokaryotes
Cellular Processes
K16898 2 addA; ATP-dependent helicase/nuclease subunit A - - -
K16899 2 addB; ATP-dependent helicase/nuclease subunit B - - -
K06930 2 bat; HTH-type transcriptional regulator, bacterioopsin transcriptional activator and related proteins
- - -
K10725 2 cdc6A; archaeal cell division control protein 6 - - -
K19174 1 dptG; DNA phosphorothioation-dependent restriction protein DptG - - -
K00786 1 E2.4.-.-; glycosyltransferase - - -
K01274 1 E3.4.13.-; D-alanyl-D-alanine dipeptidase - - -
K07062 1 fitB; toxin FitB [EC:3.1.-.-] - - -
K07332 1 flaI-A, flaI; archaeal flagellar protein FlaI - - -
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K03466 1 ftsK, spoIIIE; DNA segregation ATPase FtsK/SpoIIIE, S-DNA-T family
- - -
K01138 1 K01138; uncharacterized sulfatase - - -
K07228 1 K07228; TrkA domain protein - - -
K07468 1 K07468; putative ATP-dependent DNA ligase - - -
K07498 2 K07498; putative transposase - - -
K07499 2 K07499; putative transposase - - -
K09967 1 K09967; uncharacterized protein - - -
K10716 2 kch, trkA, mthK, pch; voltage-gated potassium channel - - -
K07452 1 mcrB; 5-methylcytosine-specific restriction enzyme B - - -
K18828 1 mvpA, vapC; tRNA(fMet)-specific endonuclease VapC - - -
K01262 1 pepP; Xaa-Pro aminopeptidase - - -
K01271 1 pepQ; Xaa-Pro dipeptidase - - -
K11928 1 putP; sodium/proline symporter - - -
K06218 1 relE, stbE; mRNA interferase RelE/StbE - - -
K03546 1 sbcC, rad50; DNA repair protein SbcC/Rad50 - - -
K03529 1 smc; chromosome segregation protein - - -
K03294 1 TC.APA; basic amino acid/polyamine antiporter, APA family - - -
K03307 1 TC.SSS; solute:Na+ symporter, SSS family - - -
K03733 1 xerC; integrase/recombinase XerC - - -
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K04763 2 xerD; integrase/recombinase XerD - - -
K07459 1 ybjD; putative ATP-dependent endonuclease of the OLD family - - -
K06915 2 K06915; uncharacterized protein - - -
K06922 1 K06922; uncharacterized protein - - -
K07052 1 K07052; uncharacterized protein - - -
K07095 1 K07095; uncharacterized protein - - -
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Supplementary Fig. S1. Genes of strains CBA1112T (red bar) and CBA1113 (blue bar)
functionally annotated using the COG database. One-letter abbreviations for the COG
categories: D, cell cycle control, cell division, chromosome partitioning; M, cell
wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification,
protein turnover, and chaperones; T, signal transduction mechanisms; U, intracellular
trafficking, secretion, and vesicular transport; V, defense mechanisms; B, chromatin structure
and dynamics; J, translation, ribosomal structure and biogenesis; K, transcription; L,
replication, recombination, and repair; C, energy production and conversion; E, amino acid
transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport
and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P,
inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport, and
catabolism; S, function unknown.
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Supplementary Fig. S2. The functional gene annotations of strains CBA1112T (red bar) and
CBA1113 (blue bar) using the KEGG database.
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Supplementary Fig. S3. A circular phylogenetic tree based on 16S rRNA gene sequence
with environmental Haloplanus sequences. The tree was constructed using the neighbor-
joining (NJ) algorithm with bootstrap values of 1,000. Natrinema soli 3-5T was used as an
outgroup taxon.
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Supplementary Fig. S4. Functional POG annotation of six Haloplanus strains using COG
database. One-letter abbreviations for the COG categories: D, cell cycle control, cell division,
chromosome partitioning; M, cell wall/membrane/envelope biogenesis; N, cell motility; O,
post-translational modification, protein turnover, and chaperones; T, signal transduction
mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense
mechanisms; B, chromatin structure and dynamics; J, translation, ribosomal structure and
biogenesis; K, transcription; L, replication, recombination, and repair; C, energy production
and conversion; E, amino acid transport and metabolism; F, nucleotide transport and
metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and
metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q,
secondary metabolites biosynthesis, transport, and catabolism; S, function unknown. Core,
accessory, and unique genes represented as blue, green, or red bars, respectively.
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Supplementary Fig. S5. Functional POG annotation of six Haloplanus strains using KEGG
database. Core, accessory, and unique genes represented as blue, green, or red bars,
respectively.
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Supplementary Fig. S6. Histograms showing unique POG distributions of strains CBA1112T
(red bar) and CBA1113 (blue bar) based on (A) COG categories and (B) KEGG pathways.
One-letter abbreviations for the COG categories: D, cell cycle control, cell division,
chromosome partitioning; M, cell wall/membrane/envelope biogenesis; O, post-translational
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modification, protein turnover, and chaperones; T, signal transduction mechanisms; V,
defense mechanisms; J, translation, ribosomal structure and biogenesis; K, transcription; L,
replication, recombination, and repair; C, energy production and conversion; E, amino acid
transport and metabolism; G, carbohydrate transport and metabolism; Q, secondary
metabolites biosynthesis, transport, and catabolism; P, inorganic ion transport and
metabolism; S, function unknown.
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Supplementary Fig. S7. The phylogenetic tree using concatenated core genes of six
Haloplanus strains. The tree was constructed using the NJ method with 1,000 bootstrap
replications.
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Supplementary Fig. S8. Two-dimensional thin-layer chromatograms of total polar lipids
extracted from strains CBA1112T (A) and CBA1113 (B). PG, Phosphatidylglycerol; PGP-Me,
phosphatidylglycerol phosphate methyl ester; PGS, phosphatidylglycerol sulfate; S-DGD-1,
sulfated mannosyl glucosyl diether; GL, unidentified glycolipid; L1–L6, unidentified lipids.
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Supplementary Fig. S9. Fragment recruitment plot of Santa Pola saltern metagenomic reads
to genomes of Haloplanus rubicundus strain CBA1112T (A, C, E, G) and CBA1113 (B, D, F,
H). Santa Pola metagenomes of 13% (A, B), 19% (C, D), 33% (E, F), and 37% (G, H)
salinities were mapped to the genomes of strain CBA1112T and CBA1113 with percent
identity on the y-axis and the hit position along the genome on the x-axis.
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