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Heavy water and ¹⁵N labeling with NanoSIMS analysis reveals growth-rate dependent metabolic heterogeneity in chemostats

Kopf, Sebastian H. and McGlynn, Shawn E. and Green-Saxena, Abigail and Guan, Yunbin and Newman, Dianne K. and Orphan, Victoria J. (2015) Heavy water and ¹⁵N labeling with NanoSIMS analysis reveals growth-rate dependent metabolic heterogeneity in chemostats. Environmental Microbiology, 17 (7). pp. 2542-2556. ISSN 1462-2912. PMCID PMC4587896. http://resolver.caltech.edu/CaltechAUTHORS:20150220-131055817

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Abstract

To measure single cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labeling of microbial populations with heavy water (a passive tracer) and ¹⁵N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (~0.1 atom % ²H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (~6 hours, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium vs. amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1111/1462-2920.12752DOIArticle
http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12752/abstractPublisherArticle
http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12752/suppinfoPublisherSupporting Information
ORCID:
AuthorORCID
Newman, Dianne K.0000-0003-1647-1918
Orphan, Victoria J.0000-0002-5374-6178
Additional Information:© 2015 John Wiley & Sons, Inc. Accepted manuscript online: 5 FEB 2015; Manuscript Accepted: 12 DEC 2014. We thank Nathan Dalleska and the Caltech Environmental Analysis Center for instrumentation that benefited this project, Grayson Chadwick and Kat Dawson for helpful discussions, and members of the Newman and Orphan labs, as well as John Cliff and two anonymous reviewers for constructive criticism that improved the manuscript. This work was supported by grants from the Howard Hughes Medical Institute (HHMI) and the National Institutes of Health (Grant No. 5R01HL117328-03, to D.K.N.), and from the Gordon and Betty Moore Foundation (Grant No. GBMF3780 to V.J.O.). D.K.N. is an HHMI Investigator. S.H.K. is an HHMI International Student Research Fellow.
Funders:
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIH5R01HL117328-03
Gordon and Betty Moore FoundationGBMF3780
Subject Keywords:stable isotope labeling; NanoSIMS; single cell analysis; multi isotope imaging; metabolic heterogeneity
PubMed Central ID:PMC4587896
Record Number:CaltechAUTHORS:20150220-131055817
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20150220-131055817
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:55062
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:20 Feb 2015 21:23
Last Modified:24 Oct 2017 16:36

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