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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.

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 ((13)C)-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 muM. 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.

SUBMITTER: Huang WE

PROVIDER: S-EPMC2612214 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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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 Wei E WE Ferguson Andrew A Singer Andrew C AC Lawson Kathryn K Thompson Ian P IP Kalin Robert M RM Larkin Michael J MJ Bailey Mark J MJ Whiteley Andrew S AS

Applied and environmental microbiology 20081107 1

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 ((13)C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) rec ...[more]

PMID: 18997025

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Project description:A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-N mg of MLVSS(-1) h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [(12)C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [(14)C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

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Dataset Information

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.

Publications

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.

Similar Datasets

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