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. 2005 Feb;71(2):646-55.
doi: 10.1128/AEM.71.2.646-655.2005.

"V体育安卓版" Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2)

Martin Hesselsoe  1 , Jeppe Lund Nielsen, Peter Roslev, Per Halkjaer Nielsen
Affiliations

Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2)

Martin Hesselsoe et al. Appl Environ Microbiol. 2005 Feb.

V体育官网 - Abstract

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples VSports手机版. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (14)CO(2) assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO(2)-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO(2)-MAR results were supported by stable isotope analysis of (13)C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of (13)CO(2). In conclusion, the novel HetCO(2)-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms. .

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Figures (VSports app下载)

FIG. 1.
FIG. 1.
Isotope labeling of P. putida and E. coli with 14CO2 in the presence of different substrates and oxygen regimes. P. putida after 5 h with glucose is defined as index 1.
FIG. 2.
FIG. 2.
FISH, MAR, and superimposed MAR-FISH of P. putida after incubation with 14CO2. Glucose (A) and yeast extract (B) were used as substrates. (C) Negative control without any organic substrate addition. All samples were incubated at aerobic conditions. Scale bar, 10 μm.
FIG. 3.
FIG. 3.
MAR and FISH and superimposed MAR-FISH of E. coli after incubation with 14CO2. Glucose was used as energy substrate in the presence (A) or absence (B) of oxygen. (C) Negative control without any organic substrates added. Scale bars, 10 μm.
FIG. 4.
FIG. 4.
MAR of filamentous “Ca. Microthrix parvicella” in activated sludge after incubation with 14CO2 in the presence of unlabeled oleic acid under aerobic (A) and anaerobic (B) conditions. Scale bars, 10 μm.
FIG. 5.
FIG. 5.
MAR of filamentous “Ca. Microthrix parvicella” in activated sludge labeled with 14CO2 in the presence of oleic acid under anaerobic conditions (3 h), followed by the addition of oxygen (A), nitrate (B), or nitrite (C). Scale bar, 10 μm.
FIG. 6.
FIG. 6.
[13C]PLFA profiles from activated sludge with “Ca. Microthrix parvicella” incubated with oleic acid. (A) Comparison of 13C-PLFA profiles after incubation with oleic acid and 13CO2 in the presence or absence of oxygen. Enrichment in 13C is expressed as changes in δ13C compared to control samples incubated without 13CO2 (Δδ13C). (B) Comparison of 13C-PLFA profiles after incubation with either 13CO2 and nonlabeled oleic acid or nonlabeled CO2 and [13C]oleic acid. The relative distribution of 13C incorporated into PLFAs is expressed as a percentage.
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References

    1. Adamczyk, J., M. Hesselsoe, N. Iversen, A. Lehner, P. H. Nielsen, M. Schloter, P. Roslev, and M. Wagner. 2003. The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function. Appl. Environ. Microbiol. 69:6875-6887. - PMC - PubMed
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