HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi.
AMF
Ammonia oxidation
Arbuscular mycorrhizal fungi
Archaea
Bacteria
Metagenomics
Microbial community
SIP
Soil
Stable-isotope probing
Journal
Microbiome
ISSN: 2049-2618
Titre abrégé: Microbiome
Pays: England
ID NLM: 101615147
Informations de publication
Date de publication:
25 11 2022
25 11 2022
Historique:
received:
29
07
2022
accepted:
04
10
2022
entrez:
26
11
2022
pubmed:
27
11
2022
medline:
30
11
2022
Statut:
epublish
Résumé
Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing-SIP-remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance-the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP-fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat-generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs' phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling. Video Abstract.
Sections du résumé
BACKGROUND
Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing-SIP-remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance-the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling.
RESULTS
Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to
CONCLUSIONS
Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP-fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat-generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs' phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling. Video Abstract.
Identifiants
pubmed: 36434737
doi: 10.1186/s40168-022-01391-z
pii: 10.1186/s40168-022-01391-z
pmc: PMC9700909
doi:
Substances chimiques
Ammonia
7664-41-7
Soil
0
Isotopes
0
DNA
9007-49-2
Types de publication
Video-Audio Media
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
199Informations de copyright
© 2022. The Author(s).
Références
Science. 2003 May 16;300(5622):1138-40
pubmed: 12750519
mBio. 2021 Apr 27;12(2):
pubmed: 33906922
Science. 2012 Aug 31;337(6098):1084-7
pubmed: 22936776
Nucleic Acids Res. 2017 Jan 4;45(D1):D535-D542
pubmed: 27899627
PLoS One. 2016 Jul 20;11(7):e0159415
pubmed: 27438604
Appl Environ Microbiol. 1997 Feb;63(2):408-13
pubmed: 16535505
New Phytol. 2018 Dec;220(4):1108-1115
pubmed: 29355963
ISME J. 2020 Jun;14(6):1520-1532
pubmed: 32203117
Int J Syst Evol Microbiol. 2016 Aug;66(8):3034-3040
pubmed: 27154284
ISME J. 2019 Sep;13(9):2162-2172
pubmed: 31053828
PeerJ. 2019 Jul 26;7:e7359
pubmed: 31388474
Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17989-94
pubmed: 23027926
Nature. 2000 Feb 10;403(6770):646-9
pubmed: 10688198
ISME J. 2016 May;10(5):1051-63
pubmed: 26528837
New Phytol. 2018 Dec;220(4):1161-1171
pubmed: 29355972
Ecology. 2014 May;95(5):1162-72
pubmed: 25000748
Proc Natl Acad Sci U S A. 2007 Oct 23;104(43):16970-5
pubmed: 17939995
Mycorrhiza. 2018 Apr;28(3):269-283
pubmed: 29455336
Nature. 2001 Sep 20;413(6853):297-9
pubmed: 11565029
Sci Rep. 2020 Jul 1;10(1):10689
pubmed: 32612216
Proc Natl Acad Sci U S A. 2014 Aug 26;111(34):12504-9
pubmed: 25114236
ISME J. 2021 Aug;15(8):2276-2288
pubmed: 33649552
New Phytol. 2009;181(1):199-207
pubmed: 18811615
Ecology. 2009 Jan;90(1):100-8
pubmed: 19294917
ISME J. 2015 Nov;9(11):2336-48
pubmed: 25822481
Front Microbiol. 2017 Aug 22;8:1593
pubmed: 28878752
Environ Microbiol. 2013 Jun;15(6):1795-809
pubmed: 23298189
Glob Chang Biol. 2022 Jan;28(1):128-139
pubmed: 34587352
Curr Opin Microbiol. 2009 Oct;12(5):547-52
pubmed: 19695948
Proc Natl Acad Sci U S A. 2010 Oct 5;107(40):17240-5
pubmed: 20855593
mSphere. 2021 Oct 27;6(5):e0008521
pubmed: 34468166
Commun Biol. 2019 Jun 21;2:233
pubmed: 31263777
Methods Mol Biol. 2012;881:375-408
pubmed: 22639220
New Phytol. 2018 Dec;220(4):1285-1295
pubmed: 29206293
ISME J. 2012 Oct;6(10):1978-84
pubmed: 22592820
Appl Microbiol Biotechnol. 1997 Sep;48(3):281-8
pubmed: 9352672
Glob Chang Biol. 2022 Apr;28(8):2527-2540
pubmed: 34989058
Appl Environ Microbiol. 2002 Nov;68(11):5367-73
pubmed: 12406726
Front Microbiol. 2021 Feb 19;12:574060
pubmed: 33679625
Nature. 2011 Jan 6;469(7328):58-63
pubmed: 21209659
Nat Commun. 2021 Jun 7;12(1):3381
pubmed: 34099669
ISME J. 2007 Oct;1(6):480-91
pubmed: 18043650
Int J Syst Evol Microbiol. 2017 Dec;67(12):5067-5079
pubmed: 29034851
Appl Environ Microbiol. 2007 May;73(10):3196-204
pubmed: 17369332
Appl Environ Microbiol. 2012 Mar;78(5):1544-55
pubmed: 22194293
mBio. 2021 Jan 5;12(1):
pubmed: 33402535
Biochim Biophys Acta. 2013 Oct;1830(10):4482-90
pubmed: 23688399
New Phytol. 2022 Oct;236(1):210-221
pubmed: 35633108
J Proteome Res. 2014 Mar 7;13(3):1200-10
pubmed: 24467184
Front Microbiol. 2016 May 12;7:711
pubmed: 27242732
Environ Microbiol. 2009 Jul;11(7):1658-71
pubmed: 19236445
Mycorrhiza. 2009 Apr;19(4):239-246
pubmed: 19101737
Trends Microbiol. 2012 Nov;20(11):523-31
pubmed: 22959489
Environ Microbiol. 2018 Mar;20(3):1112-1119
pubmed: 29411496
ISME J. 2022 Apr;16(4):1140-1152
pubmed: 34873295
Front Microbiol. 2016 Jan 07;6:1469
pubmed: 26779135
Curr Opin Biotechnol. 2016 Oct;41:14-18
pubmed: 27035052
Proc Natl Acad Sci U S A. 2021 Nov 23;118(47):
pubmed: 34799453
Mol Ecol. 2016 Oct;25(19):4818-35
pubmed: 27545292
mSystems. 2022 Oct 27;:e0041722
pubmed: 36300946
Biochemistry. 1966 Jan;5(1):236-44
pubmed: 4161040
Front Microbiol. 2016 May 12;7:703
pubmed: 27242725
mSystems. 2021 May 11;6(3):
pubmed: 33975968
Nat Commun. 2020 Aug 4;11(1):3897
pubmed: 32753587
ISME J. 2022 Dec;16(12):2752-2762
pubmed: 36085516
Environ Microbiol. 2005 Jun;7(6):828-38
pubmed: 15892702
J Forensic Sci. 2013 Jan;58 Suppl 1:S173-5
pubmed: 23082821
Genome Res. 2017 May;27(5):824-834
pubmed: 28298430
Appl Environ Microbiol. 2010 Oct;76(20):6920-7
pubmed: 20802074
Appl Environ Microbiol. 2015 Nov;81(21):7570-81
pubmed: 26296731
Nucleic Acids Res. 2022 Jan 7;50(D1):D785-D794
pubmed: 34520557
Nat Protoc. 2007;2(4):860-6
pubmed: 17446886
ISME J. 2021 Sep;15(9):2738-2747
pubmed: 33782569
Genome Res. 2015 Jul;25(7):1043-55
pubmed: 25977477
Front Microbiol. 2013 Jun 14;4:149
pubmed: 23785358
Nucleic Acids Res. 2012 Jul;40(Web Server issue):W445-51
pubmed: 22645317
Antonie Van Leeuwenhoek. 2002 Aug;81(1-4):343-51
pubmed: 12448732
J Bacteriol. 2011 May;193(9):2367-8
pubmed: 21398538
Proc Natl Acad Sci U S A. 2011 May 17;108(20):8420-5
pubmed: 21525411
Appl Environ Microbiol. 2021 Nov 10;87(23):e0170621
pubmed: 34524899
Microb Ecol. 2011 May;61(4):911-6
pubmed: 21365231
mSystems. 2020 Jul 21;5(4):
pubmed: 32694124
Front Microbiol. 2019 Nov 13;10:2650
pubmed: 31798566
Environ Microbiol. 2013 Jun;15(6):1870-81
pubmed: 23360621
Appl Environ Microbiol. 2015 Feb;81(4):1513-19
pubmed: 25527556
ISME J. 2020 Apr;14(4):999-1014
pubmed: 31953507
Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):E911-20
pubmed: 25605935
Bioinformatics. 2019 Nov 15;:
pubmed: 31730192
ISME J. 2019 Feb;13(2):413-429
pubmed: 30258172
Microbiome. 2021 Oct 18;9(1):208
pubmed: 34663463
New Phytol. 2015 Mar;205(4):1537-1551
pubmed: 25382456
Environ Microbiol. 2018 Jan;20(1):44-61
pubmed: 29027346
Nucleic Acids Res. 2018 Jan 4;46(D1):D624-D632
pubmed: 29145643
Microbiome. 2018 Sep 15;6(1):158
pubmed: 30219103
Nat Ecol Evol. 2019 Jul;3(7):1064-1069
pubmed: 31209289
J Biol Chem. 2018 Jan 26;293(4):1243-1258
pubmed: 29196602
Bioinformatics. 2020 Apr 1;36(7):2251-2252
pubmed: 31742321
Environ Microbiol. 2022 Jan;24(1):357-369
pubmed: 34811865
Methods Mol Biol. 2019;2046:109-128
pubmed: 31407300
FEMS Microbiol Ecol. 2012 Apr;80(1):236-47
pubmed: 22224699
Nat Biotechnol. 2017 Aug 8;35(8):725-731
pubmed: 28787424
ISME J. 2016 Aug;10(8):1836-45
pubmed: 26882267