Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization

W.E. Huang, A. Ferguson, A.C. Singer, K.A. Lawson, I.P. Thompson, R.M. Kalin, M.J. Larkin, M.J. Bailey, A.S. Whiteley

Research output: Contribution to journalArticle

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Abstract

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (13C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 µM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.
LanguageEnglish
Pages234-241
Number of pages7
JournalApplied and Environmental Microbiology
Volume75
Issue number1
DOIs
Publication statusPublished - Jan 2009

Fingerprint

naphthalene
fluorescence in situ hybridization
Fluorescence In Situ Hybridization
Isotopes
stable isotopes
stable isotope
fluorescence
ribosomal RNA
Messenger RNA
Groundwater
Comamonadaceae
Acidovorax
biodegradation
Population
groundwater
prokaryote
cells
prokaryotic cells
Pseudomonas
RNA

Keywords

  • genetic functions
  • microbial populations
  • in situ
  • rRNA
  • mRNA
  • single-cell raman-fluorescence

Cite this

Huang, W.E. ; Ferguson, A. ; Singer, A.C. ; Lawson, K.A. ; Thompson, I.P. ; Kalin, R.M. ; Larkin, M.J. ; Bailey, M.J. ; Whiteley, A.S. / Resolving genetic functions within microbial populations : in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization. In: Applied and Environmental Microbiology. 2009 ; Vol. 75, No. 1. pp. 234-241.
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Resolving genetic functions within microbial populations : in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization. / Huang, W.E.; Ferguson, A.; Singer, A.C.; Lawson, K.A.; Thompson, I.P.; Kalin, R.M.; Larkin, M.J.; Bailey, M.J.; Whiteley, A.S.

In: Applied and Environmental Microbiology, Vol. 75, No. 1, 01.2009, p. 234-241.

Research output: Contribution to journalArticle

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