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Transcript
Advanced Environmental Biotechnology II Week 09 - Stable-isotope probing Based on Chapter 7 Stable-isotope probing Stefan Radajewski and J.Colin Murrell in Molecular Microbial Ecology BIOS Advanced Methods. (2005) Osborn, A. Mark.; Smith, Cindy J. Eds. Taylor & Francis Routledge 7.1 Introduction Finding the metabolic function of groups in microbial communities is difficult. One way to do this has involved isolating, identifying and characterizing microorganisms which have a particular function. A functional group can sometimes be found by small subunit rRNA gene similarities, then molecular biological techniques are used to investigate these closely related populations in situ. Another method looks at the similarities of genes that encode key enzymes in metabolic pathways, ‘functional genes’. Many microorganisms will share metabolic functions, and therefore some new organisms will be found using these molecular approaches. However these methods will not work for all uncultivated taxa. A different way to link metabolic function with taxonomic identity is first to find the function of uncultivated microbial populations and then find their identity using molecular biological techniques. Several techniques involve the use of substrates labeled with radioisotopes or stable-isotopes. These methods can link identity, activity and function under conditions similar to in situ. Microautoradiography, developed for microscopic observation of microorganisms involved in uptake of radiolabeled substrates, has recently been combined with molecular identification using 16S rRNA probes and fluorescent in situ hybridization. Figure 1 | Cultivation-independent identification of microorganisms using radioisotopes. a FISH (fluorescence in situ hybridization)–microautoradiography. An environmental sample is incubated with labelled substrates such as 3H-acetate, 14Cpyruvate, 14C-butyrate or 14C-bicarbonate, and then fixed onto a glass slide. Fluorescently labelled oligonucleotide probes specific for various 16S rRNA sequences are hybridized to the sample. The slides are treated to show radioactive cells. FISH plus radioactivity can find microorganisms that are present and metabolizing the radiolabelled substrate. Figure 1 | Cultivation-independent identification of microorganisms using radioisotopes. b Isotope array. The environmental sample is incubated with a 14C-labelled substrate, then RNA is extracted from the sample. RNA is then labelled with a fluorescent dye and hybridized to an oligonucleotide array containing DNA probe sequences specific for the 16S rRNA genes of the bacteria of interest. Fluorescence can show which microorganisms are in the sample, and radioactivity shows which of the microbes metabolized the labelled substrate and incorporated the isotope into their RNA. Substrates labeled with 14C or 13C have also been added to environmental samples and found as labeled lipids that can be compared with the lipids of cultivated strains. The technique of stable-isotope probing (SIP) used substrates highly enriched with 13C to recover the DNA of functional groups of microorganisms, which were identified using molecular biological techniques. This lecture will introduce the basis of SIP, outline technical considerations for the use of stable-isotopes, and provide examples of its application. 7.2 Stable-isotope labeling of DNA There is a low natural abundance of certain stable-isotopes: the stable carbon isotopes are 12C (98.9%) and 13C (1.1%), and stable nitrogen isotopes are 14N (99.63%) and 15N (0.37%). Substrates that are highly enriched in the rare stable-isotopes (e.g. >99%, 13C or 15N) are added to complex environments, and the labeled isotopes tracked by finding the mass increase due to the single additional neutron. Idealized structure of the two most common stable isotopes of carbon, 12C (left) and 13C (right). + = protons, which are positively charged, O = neutrons (neutral), and the path taken by the six negatively charged electrons as they orbit the nucleus, balancing the charge of the protons. SIP relies on the fact that DNA synthesized during microbial growth on a substrate enriched with a ‘heavy’ stable-isotope becomes sufficiently heavy to be separated from unlabeled DNA by equilibrium centrifugation in a CsCl density-gradient. Escherichia coli grown on 15NH +. 4 The buoyant density of DNA varies with its guanine-cytosine content, but a high proportion of a naturally rare stableisotope (2H, 15N or 13C) into DNA increases the density difference between labeled and unlabeled DNA fractions. Eg. bacterial cultures grown on 13CH3OH and 13CO2 as a carbon source, and soil microorganisms that actively used methanol and methane. To see if SIP is a suitable technique we must see whether each DNA molecule in the target microorganisms will contain enough stable-isotope (13C) to collect a 13C-labeled ‘heavy’ DNA fraction ([13C]DNA). There might be: 13C dilution because of naturally occurring carbon substrates (i.e. 12C-labeled), 13C turnover due to substrate co-oxidation or eating of the target microorganisms, or 13C assimilation without DNA replication. All might change the proportion of a microbial genome that will become labeled. 13C- SIP has been used to target metabolically restricted groups [methylotrophs and ammoniaoxidizing bacteria (AOB)] that grow in the presence of high concentrations of labeled substrate. Figure 7.1 CsCl/ethidium bromide density gradients of DNA fractions extracted from Methylobacterium extorquens grown on either [12C]- or [13C]methanol as the carbon source. ultracentrifugation at (A) 265 000 g for 16 h at 20°C and (B) 140 000 g for 60 h at 20°C. Bar=1 cm. Figure 2 | DNA-based stable isotope probing (SIP) and 13C-phospholipid fatty acids (PLFA) analyses. a A 13C-labelled substrate is added to an environmental sample, and incubated so that the labelled carbon from the substrate can be incorporated into the biomass of the active microorganisms in the sample. b Total DNA that has been purified from the incubated sample should represent those microorganisms that grew using the 13Clabelled substrate. This genomic DNA — enriched with the 13C isotope — can be separated from the community DNA (12C-DNA) by CsCl gradient centrifugation. Figure 2 | DNA-based stable isotope probing (SIP) and 13C-phospholipid fatty acids (PLFA) analyses. Phylogenetic analyses of sequence data produced by PCR amplification of the isolated 13C-labelled DNA using selected primers sets (chosen by the researcher based on their knowledge of probable community members) such as 16S rRNA, pmoA (particulate methane monooxygenase), mmoX (soluble methane monooxygenase), cmuA (chloromethane utilization) and mxaF (methanol dehydrogenase) can help to identify organisms that are active in the soil sample Microarrays can also be used to find which of the amplified genes are the most numerous. c PLFA can also be purified, and PLFA profiles can reveal which microorganisms incorporated the 13C isotope. 7.3 Application of stable-isotope probing PCR primers that are universal for the smallsubunit rRNA genes of Bacteria, Archaea and Eukarya are needed to use SIP for identification of microorganisms involved in a specific function. More selective PCR primers, such as those targeting functional genes, can also be applied to study populations that are known to be involved in specific processes. 7.3.1 Methylotroph populations Methylotrophs are microorganisms that can use reduced one-carbon compounds as a sole source of carbon and energy. Although the known methylotrophs include a variety of Bacteria, Archaea and Eukarya, most aerobic strains are Bacteria belonging to the class Proteobacteria. A specialized subgroup of methylotrophs is the methane oxidizing bacteria (methanotrophs). 7.3.1 Methylotroph populations Proteobacterial methylotrophs have common features of their biochemistry So we can design PCR primers that target key functional genes of methylotrophs and methanotrophs; those encoding the active-site subunits of methanol dehydrogenase (MDH) and methane monooxygenase (MMO). 7.3.1.1 Methanol assimilation Stable-isotope probing shows the active methanol using microorganisms in an acidic forest soil. Soil in a microcosm (small container) was exposed to 13CH3OH (0.5% v/w) for 44 days. Then a [13C]DNA fraction was found from total community DNA using a CsCl density-gradient. 7.3.1.1 Methanol assimilation Domain level PCR primers only found bacterial sequences in the [13C]DNA fraction. 16S rDNA sequences showed that three closely related genera within the α-Proteobacteria had taken up the [13C] methanol. This was also seen in genes coding MDH. Other 16S rDNA sequences from the [13C]DNA fraction seemed to be from the Acidobacterium division. Acidobacterium don’t often grow in culture. Finding these bacteria take up methanol or the by-products of methylotrophic carbon metabolism tells us about the metabolic function of a diverse, poorly studied and maybe important group of bacteria. 7.3.1.2 Methane assimilation The active methanotrophs in a peat soil with 13CH4 (8% v/v) were studied using a ‘heavy’ [13C]DNA fraction. The [13C]DNA fraction was a ‘smear’, possibly intermediate density, from: Pieces of DNA from the growth of methanotrophs and bacteria using 13C-labeled intermediates or by-products of methanotroph metabolism. 7.3.1.2 Methane assimilation PCR of 16S rRNA genes and functional genes for MDH and MMO had many methanotroph sequences, showing the activity of these bacteria in situ. The [13C]DNA fraction also had a large proportion of 16S rDNA sequences of bacteria not known as methanotrophs or methylotrophs. Maybe these other groups of bacteria are also involved in cycling the carbon derived from CH4 (possibly in the form of 13C-labeled metabolites or biomass). So SIP can identify the microbial population involved in the cycling of a specific compound, even though their function is unclear. 7.3.2 Ammonia-oxidizing populations Autotrophic ammonia-oxidizing bacteria (AOB) are slow-growing and difficult to study in culture, but are important in the global cycling of nitrogen. Phylogenetic analysis of rRNA gene sequences places nearly all AOB in a group within the β-Proteobacteria. Now there is wide use of selective 16S rDNA PCR primers to study their ecology. 7.3.2 Ammonia-oxidizing populations In enrichment cultures inoculated with a freshwater sediment, SIP identified AOB species which used 13CO2. Although several types of AOB were detected in total DNA extracted from the enrichment cultures, only some subgroups of AOB were present in the [13C]DNA fraction. This suggests that certain subgroups of AOB are out competed in laboratory culture. It also shows that ‘heavy’ isotope labeled DNA can be used to identify which members of a metabolically defined population are active under set conditions. 7.4 Future SIP is able to enrich and isolate the combined ‘genome’ of a microbial population that is involved in a specific function. So SIP helps us to investigate the ecology of these microorganisms in situ using a variety of molecular biological techniques. 7.4 Future We could use cloning and hybridization techniques to find entire gene clusters from uncultivated bacteria whose function has been defined. Maybe then biases in the use of selective PCR primers may be avoided and improved PCR primers could be designed. 7.4 Future Also in the future, we could look for molecules that do not need replication of the chromosome for 13C-labeling (e.g. rRNA). Ribosomes are naturally amplified in active cells, so this would improve the sensitivity of SIP. We could use less label but still be able to link identity with function. DNA-SIP has a long incubation time for DNA replication and incorporation of the 13C-label into newly synthesized DNA. Because there is much more RNA synthesis than DNA synthesis, it is possible to get 13C-RNA more quickly than 13C-DNA. RNA-SIP has helped to identify bacteria that degrade phenol in an aerobic industrial bioreactor. A pulse of 13C-phenol was added to a bioreactor sludge sample and RNA was collected for analysis 8 hours later. The 13C-labelled RNA was separated from 12C-RNA by density gradient centrifugation. Reverse transcription (RT-) PCR amplification of 13C-labelled 16S rRNA showed that a Thauera species was important in phenol degradation in this bioreactor. 13C-labelled RNA with a specific buoyant density is found in several fractions in density gradients. We must analyse each gradient fraction by RT-PCR and denaturing gradient gel electrophoresis (DGGE). Shifts in band intensities that occurred during the pulse of 13C-phenol in the bioreactor showed which bacteria were being labelled.