The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13 CO 2 respired from [ 13 C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that 13 C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [ 13 C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [ 13 C]phenol, and (iii) 12 daily doses of [ 13 C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13 CO 2 evolution by a factor of 10. Furthermore, imaging of 13 C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13 C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13 C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of ␣-, -, and ␥-proteobacteria and high-G؉C-content grampositive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.
This study was designed to characterize naphthalene metabolism in Polaromonas naphthalenivorans CJ2. Comparisons were completed using two archetypal naphthalenedegrading bacteria: Pseudomonas putida NCIB 9816-4 and Ralstonia sp. strain U2, representative of the catechol and gentisate pathways, respectively. Strain CJ2 carries naphthalene catabolic genes that are homologous to those in Ralstonia sp. strain U2. Here we show that strain CJ2 metabolizes naphthalene via gentisate using respirometry, metabolite detection by GC-MS and cell-free enzyme assays. Unlike P. putida NCIB 9816-4 or Ralstonia sp. strain U2, strain CJ2 did not grow in minimal medium saturated with naphthalene. Growth assays revealed that strain CJ2 is inhibited by naphthalene concentrations of 78 mM (10 p.p.m.) and higher, and the inhibition of growth is accompanied by the accumulation of orange-coloured, putative naphthalene metabolites in the culture medium. Loss of cell viability coincided with the appearance of the coloured metabolites, and analysis by HPLC suggested that the accumulated metabolites were 1,2-naphthoquinone and its unstable auto-oxidation products. The naphthoquinone breakdown products accumulated in inhibited, but not uninhibited, cultures of strain CJ2. Furthermore, naphthalene itself was shown to directly inhibit growth of a regulatory mutant of strain CJ2 that is unable to metabolize naphthalene. These results suggest that, despite being able to use naphthalene as a carbon and energy source, strain CJ2 must balance naphthalene utilization against two types of toxicity.
We used a combination of stable isotope probing (SIP), gas chromatography-mass spectrometry-based respiration, isolation/cultivation, and quantitative PCR procedures to discover the identity and in situ growth of soil microorganisms that metabolize benzoic acid. We added [13 C]benzoic acid or [ 12 C]benzoic acid (100 g) once, four times, or five times at 2-day intervals to agricultural field plots. After monitoring 13 CO 2 evolution from the benzoic acid-dosed soil, field soils were harvested and used for nucleic acid extraction and for cultivation of benzoate-degrading bacteria. Exposure of soil to benzoate increased the number of culturable benzoate degraders compared to unamended soil, and exposure to benzoate shifted the dominant culturable benzoate degraders from Pseudomonas species to Burkholderia species. Isopycnic separation of heavy [13 C]DNA from the unlabeled fraction allowed terminal restriction fragment length polymorphism (T-RFLP) analyses to confirm that distinct 16S rRNA genes were localized in the heavy fraction. Phylogenetic analysis of sequenced 16S rRNA genes revealed a predominance (15 of 58 clones) of Burkholderia species in the heavy fraction. Burkholderia sp. strain EBA09 shared 99.5% 16S rRNA sequence similarity with a group of clones representing the dominant RFLP pattern, and the T-RFLP fragment for strain EBA09 and a clone from that cluster matched the fragment enriched in the [ 13 C]DNA fraction. Growth of the population represented by EBA09 during the field-dosing experiment was demonstrated by using most-probable-number-PCR and primers targeting EBA09 and the closely related species Burkholderia hospita. Thus, the target population identified by SIP not only actively metabolized benzoic acid but reproduced in the field upon the addition of the substrate.Soil environments are commonly carbon limited (1), and carbon input through decomposition, industrial spills, or other disturbances can lead to an increase in microbial activity (36). In order to understand the population dynamics of bacteria in soils, it is necessary to understand which organisms respond to increases in carbon availability and how the population changes. Investigations using stable isotope probing (SIP) are particularly suited to identifying bacteria that metabolize a specific carbon compound because cellular biomarkers used to identify organisms become 13 C labeled when organisms metabolize and incorporate 13
SummaryWe demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring 13 C assimilation into individual bacterial cells grown on 13 Clabeled organic compounds in the laboratory and in field soil. We grew pure cultures of Pseudomonas putida NCIB 9816-4 in minimal media with known mixtures of 12 C-and 13 Cglucose and analyzed individual cells via SIMS imaging. Individual cells yielded signals of masses 12, 13, 24, 25, 26, and 27 as negative secondary ions indicating the presence of 12 C − , 13 C − , 24 ( 12 C 2 ) − , 25 ( 12 C 13 C) − , 26 ( 12 C 14 N) − , and 27 ( 13 C 14 N) − ions, respectively. We verified that ratios of signals taken from the same cells only changed minimally during a ∼4.5-min period of primary O 2 + beam sputtering by the dynamic SIMS instrument in microscope detection mode. There was a clear relationship between mass 27 and 26 signals in Psuedomonas putida cells grown in media containing varying proportions of 12 C-to 13 C-glucose: a standard curve was generated to predict 13 C-enrichment in unknown samples. We then used two strains of Pseudomonas putida able to grow on either all or only a part of a mixture of 13 C-labeled and unlabeled carbon sources to verify that differential 13 C signals measured by SIMS were due to 13 C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving 12 C-or 13 C-phenol: (i) cells enriched in 13 C were heterogeneously distributed among soil populations; (ii) 13 C-labeled cells were detected in soil that was dosed a single time with 13 C-phenol; and (iii) in soil that received 12 doses of 13 C-phenol, 27% of the cells in the total community were more than 90% 13 C-labeled.
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