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Expanded description of relationship between isotope mass, element composition of DNA, and observed density shifts using stable isotope probing. Included newly developed variant, quantitative stable isotope probing, and provided examples of its use in microbial ecology. |
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'''Stable-isotope probing''' ('''SIP''') is a technique in [[microbial ecology]] for tracing uptake of [[nutrient]]s in [[biogeochemical cycling]] by microorganisms. A substrate is enriched with a heavier [[stable isotope]] that is consumed by the organisms to be studied.<ref name="Dumont_2005">{{cite journal | vauthors = Dumont MG, Murrell JC | title = Stable isotope probing - linking microbial identity to function | journal = Nature Reviews. Microbiology | volume = 3 | issue = 6 | pages = 499–504 | date = June 2005 | pmid = 15886694 | doi = 10.1038/nrmicro1162 | s2cid = 24051877 }}</ref><ref name="Neufeld_2007">{{cite journal | vauthors = Neufeld JD, Dumont MG, Vohra J, Murrell JC | title = Methodological considerations for the use of stable isotope probing in microbial ecology | journal = Microbial Ecology | volume = 53 | issue = 3 | pages = 435–42 | date = April 2007 | pmid = 17072677 | doi = 10.1007/s00248-006-9125-x | s2cid = 9417066 }}</ref> [[Biomarkers]] with the heavier isotopes incorporated into them can be separated from biomarkers containing the more naturally abundant lighter isotope by [[isopycnic centrifugation]]. For example, [[isotopes of carbon|<sup>13</sup>]][[carbon dioxide|CO<sub>2</sub>]] can be used to find out which organisms are actively [[photosynthesizing]] or consuming new photosynthate. As the biomarker, [[DNA]] with <sup>13</sup>C is then separated from DNA with <sup>12</sup>C by centrifugation. [[DNA sequencing|Sequencing]] the DNA identifies which organisms were consuming existing [[carbohydrates]] and which were using carbohydrates more recently produced from photosynthesis.<ref name=radajeweksi>{{cite journal | vauthors = Radajewski S, Ineson P, Parekh NR, Murrell JC | title = Stable-isotope probing as a tool in microbial ecology | journal = Nature | volume = 403 | issue = 6770 | pages = 646–9 | date = February 2000 | pmid = 10688198 | doi = 10.1038/35001054 | bibcode = 2000Natur.403..646R | s2cid = 4395764 }}</ref> SIP with <sup>18</sup>O-labeled water can be used to find out which organisms are actively growing, because oxygen from water is incorporated into DNA (and RNA) during synthesis.<ref name=schwartz>{{cite journal | vauthors = Schwartz E | title = Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H<sub>2</sub><sup>18</sup>O | journal = Applied and Environmental Microbiology | volume = 73 | issue = 8 | pages = 2541–2546 | date = February 2007 | pmid = 17322324 | doi = 10.1128/AEM.02021-06 | pmc = 1855593 | s2cid = 18653420 | doi-access = free }}</ref> |
'''Stable-isotope probing''' ('''SIP''') is a technique in [[microbial ecology]] for tracing uptake of [[nutrient]]s in [[biogeochemical cycling]] by microorganisms. A substrate is enriched with a heavier [[stable isotope]] that is consumed by the organisms to be studied.<ref name="Dumont_2005">{{cite journal | vauthors = Dumont MG, Murrell JC | title = Stable isotope probing - linking microbial identity to function | journal = Nature Reviews. Microbiology | volume = 3 | issue = 6 | pages = 499–504 | date = June 2005 | pmid = 15886694 | doi = 10.1038/nrmicro1162 | s2cid = 24051877 }}</ref><ref name="Neufeld_2007">{{cite journal | vauthors = Neufeld JD, Dumont MG, Vohra J, Murrell JC | title = Methodological considerations for the use of stable isotope probing in microbial ecology | journal = Microbial Ecology | volume = 53 | issue = 3 | pages = 435–42 | date = April 2007 | pmid = 17072677 | doi = 10.1007/s00248-006-9125-x | s2cid = 9417066 }}</ref> [[Biomarkers]] with the heavier isotopes incorporated into them can be separated from biomarkers containing the more naturally abundant lighter isotope by [[isopycnic centrifugation]]. For example, [[isotopes of carbon|<sup>13</sup>]][[carbon dioxide|CO<sub>2</sub>]] can be used to find out which organisms are actively [[photosynthesizing]] or consuming new photosynthate. As the biomarker, [[DNA]] with <sup>13</sup>C is then separated from DNA with <sup>12</sup>C by centrifugation. [[DNA sequencing|Sequencing]] the DNA identifies which organisms were consuming existing [[carbohydrates]] and which were using carbohydrates more recently produced from photosynthesis.<ref name=radajeweksi>{{cite journal | vauthors = Radajewski S, Ineson P, Parekh NR, Murrell JC | title = Stable-isotope probing as a tool in microbial ecology | journal = Nature | volume = 403 | issue = 6770 | pages = 646–9 | date = February 2000 | pmid = 10688198 | doi = 10.1038/35001054 | bibcode = 2000Natur.403..646R | s2cid = 4395764 }}</ref> SIP with <sup>18</sup>O-labeled water can be used to find out which organisms are actively growing, because oxygen from water is incorporated into DNA (and RNA) during synthesis.<ref name=schwartz>{{cite journal | vauthors = Schwartz E | title = Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H<sub>2</sub><sup>18</sup>O | journal = Applied and Environmental Microbiology | volume = 73 | issue = 8 | pages = 2541–2546 | date = February 2007 | pmid = 17322324 | doi = 10.1128/AEM.02021-06 | pmc = 1855593 | s2cid = 18653420 | doi-access = free }}</ref> |
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When DNA is the biomarker, SIP can be performed using isotopically labeled C, H, O, or N, though <sup>13</sup>C is used most often. The density shift is proportional to the change in density in the DNA, which depends on the difference in mass between the rare and common isotopes for a given element, and on the abundance of elements in the DNA. For example, the difference in mass between <sup>18</sup>O and <sup>16</sup>O (two atomic mass units) is twice that between <sup>13</sup>C and <sup>12</sup>C (one atomic mass unit), so incorporation of <sup>18</sup>O into DNA will cause a larger per atom density shift than will incorporation of <sup>13</sup>C. Conversely, DNA contains nearly twice as many carbon atoms (11.25 per base, on average) as oxygen atoms (6 per base), so at equivalent labeling (e.g., 50 atom percent <sup>13</sup>C or <sup>18</sup>O), DNA labeled with <sup>18</sup>O will be only slightly more dense than DNA fully labeled with <sup>13</sup>C. Similarly, nitrogen is less abundant in DNA (3.75 atoms per base, on average), so a weaker DNA buoyant density shift is observed with <sup>15</sup>N- versus <sup>13</sup>C-labeled or <sup>18</sup>O-labeled substrates. Larger buoyant density shifts are observed when multiple isotope tracers are used.<ref name="Cupples_2007">{{cite journal | vauthors = Cupples AM, Shaffer EA, Chee-Sanford JC, Sims GK | title = DNA buoyant density shifts during 15N-DNA stable isotope probing | journal = Microbiological Research | volume = 162 | issue = 4 | pages = 328–34 | date = 2007 | pmid = 16563712 | doi = 10.1016/j.micres.2006.01.016 | doi-access = free }}</ref> Because density shifts as a predictable function of the change in mass caused by isotope assimilation, stable isotope probing can be modeled to estimate the amount of isotope incorporation, an approach called quantitative stable isotope probing (qSIP)<ref>{{Cite journal|last=Hungate|first=Bruce A.|last2=Mau|first2=Rebecca L.|last3=Schwartz|first3=Egbert|last4=Caporaso|first4=J. Gregory|last5=Dijkstra|first5=Paul|last6=van Gestel|first6=Natasja|last7=Koch|first7=Benjamin J.|last8=Liu|first8=Cindy M.|last9=McHugh|first9=Theresa A.|last10=Marks|first10=Jane C.|last11=Morrissey|first11=Ember M.|date=2015|editor-last=Schloss|editor-first=P. D.|title=Quantitative Microbial Ecology through Stable Isotope Probing|url=https://journals.asm.org/doi/10.1128/AEM.02280-15|journal=Applied and Environmental Microbiology|language=en|volume=81|issue=21|pages=7570–7581|doi=10.1128/AEM.02280-15|issn=0099-2240|pmc=PMC4592864|pmid=26296731}}</ref>, which has been applied to microbial communities in soils<ref>{{Cite web|last=Starr|first=Evan P.|last2=Shi|first2=Shengjing|last3=Blazewicz|first3=Steven J.|last4=Koch|first4=Benjamin J.|last5=Probst|first5=Alexander J.|last6=Hungate|first6=Bruce A.|last7=Pett-Ridge|first7=Jennifer|last8=Firestone|first8=Mary K.|last9=Banfield|first9=Jillian F.|title=Stable-Isotope-Informed, Genome-Resolved Metagenomics Uncovers Potential Cross-Kingdom Interactions in Rhizosphere Soil|url=https://journals.asm.org/doi/abs/10.1128/mSphere.00085-21|url-status=live|access-date=2021-09-20|website=mSphere|language=en|doi=10.1128/msphere.00085-21}}</ref>, marine sediments<ref>{{Cite journal|last=Coskun|first=Ömer K.|last2=Özen|first2=Volkan|last3=Wankel|first3=Scott D.|last4=Orsi|first4=William D.|date=2019|title=Quantifying population-specific growth in benthic bacterial communities under low oxygen using H218O|url=https://www.nature.com/articles/s41396-019-0373-4|journal=The ISME Journal|language=en|volume=13|issue=6|pages=1546–1559|doi=10.1038/s41396-019-0373-4|issn=1751-7370|pmc=PMC6776007|pmid=30783213}}</ref>, and decomposing leaves<ref>{{Cite journal|last=Hayer|first=Michaela|last2=Schwartz|first2=Egbert|last3=Marks|first3=Jane C.|last4=Koch|first4=Benjamin J.|last5=Morrissey|first5=Ember M.|last6=Schuettenberg|first6=Alexa A.|last7=Hungate|first7=Bruce A.|date=2016|title=Identification of growing bacteria during litter decomposition in freshwater through quantitative stable isotope probing|url=https://onlinelibrary.wiley.com/doi/10.1111/1758-2229.12475|journal=Environmental Microbiology Reports|language=en|volume=8|issue=6|pages=975–982|doi=10.1111/1758-2229.12475|issn=1758-2229}}</ref> to compare rates of growth and substrate assimilation among different microbial taxa. |
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When DNA is the biomarker, SIP can be performed using isotopically labeled C, H, O, or N, though <sup>13</sup>C is used most often. A weaker DNA buoyant density shift is observed when <sup>15</sup>N- versus <sup>13</sup>C-labeled substrates were used in pure culture. Conversely, a very strong buoyant density shift was observed when both labels were used.<ref name="Cupples_2007">{{cite journal | vauthors = Cupples AM, Shaffer EA, Chee-Sanford JC, Sims GK | title = DNA buoyant density shifts during 15N-DNA stable isotope probing | journal = Microbiological Research | volume = 162 | issue = 4 | pages = 328–34 | date = 2007 | pmid = 16563712 | doi = 10.1016/j.micres.2006.01.016 | doi-access = free }}</ref> |
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== See also == |
== See also == |
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== References == |
== References == |
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<references /> |
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{{reflist}} |
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== Further reading == |
== Further reading == |
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Revision as of 14:19, 20 September 2021
Stable-isotope probing (SIP) is a technique in microbial ecology for tracing uptake of nutrients in biogeochemical cycling by microorganisms. A substrate is enriched with a heavier stable isotope that is consumed by the organisms to be studied.[1][2] Biomarkers with the heavier isotopes incorporated into them can be separated from biomarkers containing the more naturally abundant lighter isotope by isopycnic centrifugation. For example, 13CO2 can be used to find out which organisms are actively photosynthesizing or consuming new photosynthate. As the biomarker, DNA with 13C is then separated from DNA with 12C by centrifugation. Sequencing the DNA identifies which organisms were consuming existing carbohydrates and which were using carbohydrates more recently produced from photosynthesis.[3] SIP with 18O-labeled water can be used to find out which organisms are actively growing, because oxygen from water is incorporated into DNA (and RNA) during synthesis.[4]
When DNA is the biomarker, SIP can be performed using isotopically labeled C, H, O, or N, though 13C is used most often. The density shift is proportional to the change in density in the DNA, which depends on the difference in mass between the rare and common isotopes for a given element, and on the abundance of elements in the DNA. For example, the difference in mass between 18O and 16O (two atomic mass units) is twice that between 13C and 12C (one atomic mass unit), so incorporation of 18O into DNA will cause a larger per atom density shift than will incorporation of 13C. Conversely, DNA contains nearly twice as many carbon atoms (11.25 per base, on average) as oxygen atoms (6 per base), so at equivalent labeling (e.g., 50 atom percent 13C or 18O), DNA labeled with 18O will be only slightly more dense than DNA fully labeled with 13C. Similarly, nitrogen is less abundant in DNA (3.75 atoms per base, on average), so a weaker DNA buoyant density shift is observed with 15N- versus 13C-labeled or 18O-labeled substrates. Larger buoyant density shifts are observed when multiple isotope tracers are used.[5] Because density shifts as a predictable function of the change in mass caused by isotope assimilation, stable isotope probing can be modeled to estimate the amount of isotope incorporation, an approach called quantitative stable isotope probing (qSIP)[6], which has been applied to microbial communities in soils[7], marine sediments[8], and decomposing leaves[9] to compare rates of growth and substrate assimilation among different microbial taxa.
See also
References
- ^ Dumont MG, Murrell JC (June 2005). "Stable isotope probing - linking microbial identity to function". Nature Reviews. Microbiology. 3 (6): 499–504. doi:10.1038/nrmicro1162. PMID 15886694. S2CID 24051877.
- ^ Neufeld JD, Dumont MG, Vohra J, Murrell JC (April 2007). "Methodological considerations for the use of stable isotope probing in microbial ecology". Microbial Ecology. 53 (3): 435–42. doi:10.1007/s00248-006-9125-x. PMID 17072677. S2CID 9417066.
- ^ Radajewski S, Ineson P, Parekh NR, Murrell JC (February 2000). "Stable-isotope probing as a tool in microbial ecology". Nature. 403 (6770): 646–9. Bibcode:2000Natur.403..646R. doi:10.1038/35001054. PMID 10688198. S2CID 4395764.
- ^ Schwartz E (February 2007). "Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H218O". Applied and Environmental Microbiology. 73 (8): 2541–2546. doi:10.1128/AEM.02021-06. PMC 1855593. PMID 17322324. S2CID 18653420.
- ^ Cupples AM, Shaffer EA, Chee-Sanford JC, Sims GK (2007). "DNA buoyant density shifts during 15N-DNA stable isotope probing". Microbiological Research. 162 (4): 328–34. doi:10.1016/j.micres.2006.01.016. PMID 16563712.
- ^ Hungate, Bruce A.; Mau, Rebecca L.; Schwartz, Egbert; Caporaso, J. Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J.; Liu, Cindy M.; McHugh, Theresa A.; Marks, Jane C.; Morrissey, Ember M. (2015). Schloss, P. D. (ed.). "Quantitative Microbial Ecology through Stable Isotope Probing". Applied and Environmental Microbiology. 81 (21): 7570–7581. doi:10.1128/AEM.02280-15. ISSN 0099-2240. PMC 4592864. PMID 26296731.
{{cite journal}}: CS1 maint: PMC format (link) - ^ Starr, Evan P.; Shi, Shengjing; Blazewicz, Steven J.; Koch, Benjamin J.; Probst, Alexander J.; Hungate, Bruce A.; Pett-Ridge, Jennifer; Firestone, Mary K.; Banfield, Jillian F. "Stable-Isotope-Informed, Genome-Resolved Metagenomics Uncovers Potential Cross-Kingdom Interactions in Rhizosphere Soil". mSphere. doi:10.1128/msphere.00085-21. Retrieved 2021-09-20.
{{cite web}}: CS1 maint: url-status (link) - ^ Coskun, Ömer K.; Özen, Volkan; Wankel, Scott D.; Orsi, William D. (2019). "Quantifying population-specific growth in benthic bacterial communities under low oxygen using H218O". The ISME Journal. 13 (6): 1546–1559. doi:10.1038/s41396-019-0373-4. ISSN 1751-7370. PMC 6776007. PMID 30783213.
{{cite journal}}: CS1 maint: PMC format (link) - ^ Hayer, Michaela; Schwartz, Egbert; Marks, Jane C.; Koch, Benjamin J.; Morrissey, Ember M.; Schuettenberg, Alexa A.; Hungate, Bruce A. (2016). "Identification of growing bacteria during litter decomposition in freshwater through quantitative stable isotope probing". Environmental Microbiology Reports. 8 (6): 975–982. doi:10.1111/1758-2229.12475. ISSN 1758-2229.
Further reading
- Berry D, Loy A (December 2018). "Stable-Isotope Probing of Human and Animal Microbiome Function". Trends in Microbiology. 26 (12): 999–1007. doi:10.1016/j.tim.2018.06.004. PMC 6249988. PMID 30001854.
- Egert M, Weis S, Schnell S (October 2018). "RNA-based stable isotope probing (RNA-SIP) to unravel intestinal host-microbe interactions". Methods. 149. San Diego, Calif.: 25–30. doi:10.1016/j.ymeth.2018.05.022. PMID 29857194.
- Jiang B, Jin N, Xing Y, Su Y, Zhang D (November 2018). "Unraveling uncultivable pesticide degraders via stable isotope probing (SIP)" (PDF). Critical Reviews in Biotechnology. 38 (7): 1025–1048. doi:10.1080/07388551.2018.1427697. PMID 29385846. S2CID 1218065.
- Wegener G, Kellermann MY, Elvert M (October 2016). "Tracking activity and function of microorganisms by stable isotope probing of membrane lipids". Current Opinion in Biotechnology. 41: 43–52. doi:10.1016/j.copbio.2016.04.022. PMID 27179643.