Marc G. Dumont

Stable isotope probing - linking microbial identity to function.

Stable isotope probing (SIP) is a technique that is used to identify the microorganisms in environmental samples that use a particular growth substrate. The method relies on the incorporation of a substrate that is highly enriched in a stable isotope, such as 13C, and the identification of active microorganisms by the selective recovery and analysis of isotope-enriched cellular components. DNA and rRNA are the most informative taxonomic biomarkers and 13C-labelled molecules can be purified from unlabelled nucleic acid by density-gradient centrifugation. The future holds great promise for SIP, particularly when combined with other emerging technologies such as microarrays and metagenomics.

Methodological Considerations for the Use of Stable Isotope Probing in Microbial Ecology

Stable isotope probing (SIP) is a method used for labeling uncultivated microorganisms in environmental samples or directly in field studies using substrate enriched with stable isotope (e.g., 13C). After consumption of the substrate, the cells of microorganisms that consumed the substrate become enriched in the isotope. Labeled biomarkers, such as phospholipid-derived fatty acid (PLFA), ribosomal RNA, and DNA can be analyzed with a range of molecular and analytical techniques, and used to identify and characterize the organisms that incorporated the substrate. The advantages and disadvantages of PLFA-SIP, RNA-SIP, and DNA-SIP are presented. Using examples from our laboratory and from the literature, we discuss important methodological considerations for a successful SIP experiment.

Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil

Ammonia oxidation is an essential part of the global nitrogen cycling and was long thought to be driven only by bacteria. Recent findings expanded this pathway also to the archaea. However, most questions concerning the metabolism of ammonia-oxidizing archaea, such as ammonia oxidation and potential CO2 fixation, remain open, especially for terrestrial environments. Here, we investigated the activity of ammonia-oxidizing archaea and bacteria in an agricultural soil by comparison of RNA- and DNA-stable isotope probing (SIP). RNA-SIP demonstrated a highly dynamic and diverse community involved in CO2 fixation and carbon assimilation coupled to ammonia oxidation. DNA-SIP showed growth of the ammonia-oxidizing bacteria but not of archaea. Furthermore, the analysis of labeled RNA found transcripts of the archaeal acetyl-CoA/propionyl-CoA carboxylase (accA/pccB) to be expressed and labeled. These findings strongly suggest that ammonia-oxidizing archaeal groups in soil autotrophically fix CO2 using the 3-hydroxypropionate–4-hydroxybutyrate cycle, one of the two pathways recently identified for CO2 fixation in Crenarchaeota. Catalyzed reporter deposition (CARD)-FISH targeting the gene encoding subunit A of ammonia monooxygenase (amoA) mRNA and 16S rRNA of archaea also revealed ammonia-oxidizing archaea to be numerically relevant among the archaea in this soil. Our results demonstrate a diverse and dynamic contribution of ammonia-oxidizing archaea in soil to nitrification and CO2 assimilation and that their importance to the overall archaeal community might be larger than previously thought.

Regulation of methane oxidation in the facultative methanotroph Methylocella silvestris BL2

The molecular regulation of methane oxidation in the first fully authenticated facultative methanotroph Methylocella silvestris BL2 was assessed during growth on methane and acetate. Problems of poor growth of Methylocella spp. in small‐scale batch culture were overcome by growth in fermentor culture. The genes encoding soluble methane monooxygenase were cloned and sequenced, which revealed that the structural genes for soluble methane monooxygenase, mmoXYBZDC, were adjacent to two genes, mmoR and mmoG, encoding a σ54 transcriptional activator and a putative GroEL‐like chaperone, located downstream (3′) of mmoC. Transcriptional analysis revealed that the genes were all cotranscribed from a σ54‐dependent promoter located upstream (5′) of mmo X. The transcriptional start site was mapped. Transcriptional analysis of soluble methane monooxygenase genes and expression studies on fermentor grown cultures showed that acetate repressed transcription of sMMO in M. silvestris BL2. The possibility of the presence of a particulate, membrane‐bound methane monooxygenase enzyme in M. silvestris BL2 and the copper‐mediated regulation of soluble methane monooxygenase was investigated. Both were shown to be absent. A promoter probe vector was constructed and used to assay transcription of the promoter of the soluble methane monoxygenase genes of M. silvestris BL2 grown under various conditions and with different substrates. These data represent the first insights into the molecular physiology of a facultative methanotroph.

Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled (13)C-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA (<or= 40 kb) and cloned to produce a fosmid library of > 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.

DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment

A stable isotope probing (SIP) approach was used to study aerobic methane-oxidizing bacteria (methanotrophs) in lake sediment. Oligotrophic Lake Stechlin was chosen because it has a permanently oxic sediment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane monooxygenase enzyme, were analysed following the incubation of sediment with (13) CH(4) and the separation of (13) C-labelled DNA and RNA from unlabelled nucleic acids. The incubation with (13) CH(4) was performed over a 4-day time-course and the pmoA genes and transcripts became progressively labelled such that approximately 70% of the pmoA genes and 80% of the transcripts were labelled at 96 h. The labelling of pmoA mRNA was quicker than pmoA genes, demonstrating that mRNA-SIP is more sensitive than DNA-SIP; however, the general rate of pmoA transcript labelling was comparable to that of the pmoA genes, indicating that the incorporation of (13) C into ribonucleic acids of methanotrophs was a gradual process. Labelling of Betaproteobacteria was clearly seen in analyses of 16S rRNA by DNA-SIP and not by RNA-SIP, suggesting that cross-feeding of the (13) C was primarily detected by DNA-SIP. In general, we show that the combination of SIP approaches provided valuable information about the activity and growth of the methanotrophic populations and the cross-feeding of methanotroph metabolites by other microorganisms.

Anaerobic Consumers of Monosaccharides in a Moderately Acidic Fen

ABSTRACT 16S rRNA-based stable isotope probing identified active xylose- and glucose-fermenting Bacteria and active Archaea, including methanogens, in anoxic slurries of material obtained from a moderately acidic, CH4-emitting fen. Xylose and glucose were converted to fatty acids, CO2, H2, and CH4 under moderately acidic, anoxic conditions, indicating that the fen harbors moderately acid-tolerant xylose- and glucose-using fermenters, as well as moderately acid-tolerant methanogens. Organisms of the families Acidaminococcaceae, Aeromonadaceae, Clostridiaceae, Enterobacteriaceae, and Pseudomonadaceae and the order Actinomycetales, including hitherto unknown organisms, utilized xylose- or glucose-derived carbon, suggesting that highly diverse facultative aerobes and obligate anaerobes contribute to the flow of carbon in the fen under anoxic conditions. Uncultured Euryarchaeota (i.e., Methanosarcinaceae and Methanobacteriaceae) and Crenarchaeota species were identified by 16S rRNA analysis of anoxic slurries, demonstrating that the acidic fen harbors novel methanogens and Crenarchaeota organisms capable of anaerobiosis. Fermentation-derived molecules are conceived to be the primary drivers of methanogenesis when electron acceptors other than CO2 are absent, and the collective findings of this study indicate that fen soils harbor diverse, acid-tolerant, and novel xylose-utilizing as well as glucose-utilizing facultative aerobes and obligate anaerobes that form trophic links to novel moderately acid-tolerant methanogens.

Different bacterial populations associated with the roots and rhizosphere of rice incorporate plant-derived carbon

ABSTRACT Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH4, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with 13CO2 for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with 13C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the “Spartobacteria” and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (∼20%) than did those in the rhizosphere (∼4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment.

Autotrophic Growth of Bacterial and Archaeal Ammonia Oxidizers in Freshwater Sediment Microcosms Incubated at Different Temperatures

ABSTRACT Both bacteria and archaea potentially contribute to ammonia oxidation, but their roles in freshwater sediments are still poorly understood. Seasonal differences in the relative activities of these groups might exist, since cultivated archaeal ammonia oxidizers have higher temperature optima than their bacterial counterparts. In this study, sediment collected from eutrophic freshwater Lake Taihu (China) was incubated at different temperatures (4°C, 15°C, 25°C, and 37°C) for up to 8 weeks. We examined the active bacterial and archaeal ammonia oxidizers in these sediment microcosms by using combined stable isotope probing (SIP) and molecular community analysis. The results showed that accumulation of nitrate in microcosms correlated negatively with temperature, although ammonium depletion was the same, which might have been related to enhanced activity of other nitrogen transformation processes. Incubation at different temperatures significantly changed the microbial community composition, as revealed by 454 pyrosequencing targeting bacterial 16S rRNA genes. After 8 weeks of incubation, [13C]bicarbonate labeling of bacterial amoA genes, which encode the ammonia monooxygenase subunit A, and an observed increase in copy numbers indicated the activity of ammonia-oxidizing bacteria in all microcosms. Nitrosomonas sp. strain Is79A3 and Nitrosomonas communis lineages dominated the heavy fraction of CsCl gradients at low and high temperatures, respectively, indicating a niche differentiation of active bacterial ammonia oxidizers along the temperature gradient. The 13C labeling of ammonia-oxidizing archaea in microcosms incubated at 4 to 25°C was minor. In contrast, significant 13C labeling of Nitrososphaera-like archaea and changes in the abundance and composition of archaeal amoA genes were observed at 37°C, implicating autotrophic growth of ammonia-oxidizing archaea under warmer conditions.

Microbial Diversity in Hummock and Hollow Soils of Three Wetlands on the Qinghai-Tibetan Plateau Revealed by 16S rRNA Pyrosequencing

The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo) on the Qinghai-Tibetan Plateau, the world’s highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance), Actinobacteria (17.3%) and Bacteroidetes (11%) had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1–10%). In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales). Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta) and methanotrophs (mainly Methylocystis) compared with the other two wetlands. Principal coordinate analysis (PCoA) indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of the Qinghai-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem.