Advanced Environmental Biotechnology II

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Advanced Environmental Biotechnology II

• Week 09 - Stable-isotope probing

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• Based on Chapter 7Stable-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

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• 7.1 Introduction
• Determining the metabolic function of individual
  taxa within microbial communities is a major
  challenge in microbial ecology. One to do this
  has first involved the isolation, identification
  and characterization of microorganisms to which a
  particular function can be attributed. A
  functional group can sometimes be defined by
  small subunit rRNA gene similarities, enabling
  the subsequent use of molecular biological
  techniques to investigate these closely related
  populations in situ.

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• An analogous approach has defined functional
  groups on the basis of similarities between genes
  that encode key enzymes in metabolic pathways,
  functional genes. It is likely that many
  microorganisms will share metabolic functions,
  and therefore some uncultivated taxa will be
  detected using these molecular approaches.
  However, not all uncultivated taxa will
  necessarily share the genetic similarities used
  to define an individual functional group, and so
  the metabolic function and identity of these
  organisms will remain unclear.

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• A different way to link metabolic function with
  taxonomic identity is first to establish the
  function of uncultivated microbial populations
  and then determine their identity using molecular
  biological techniques. Several techniques
  involving the use of substrates labeled with
  radioisotopes or stable-isotopes can achieve this
  goal and simultaneously link identity, activity
  and function under conditions which approach
  those occurring in situ. The technique of
  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.

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• Figure 1 Cultivation-independent identification
  of microorganisms using radioisotopes.
• a FISH (fluorescence in situ hybridization)micr
  oautoradiography. An environmental sample is
  incubated with labelled substrates such as
  3H-acetate, 14C-pyruvate, 14C-butyrate or
  14C-bicarbonate, and then fixed onto a glass
  slide. Samples are analysed by FISH using
  fluorescently labelled oligonucleotide probes
  specific for various 16S rRNA sequences chosen by
  the researcher. The slides are treated with an
  autoradiographic emulsion, and silver grains
  associated with radioactive cells are visualized
  by inverse confocal laser scanning microscopy.
  FISH together with detection of radioactivity can
  identify those microorganisms that are present
  and metabolizing the specific radiolabelled
  substrate under the conditions tested.
• b Isotope array. The environmental sample is
  incubated with a 14C-labelled substrate, after
  which the 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. Hybridization
  to the array (fluorescence) can show
  microorganisms are present in the sample, and
  radioactivity indicates which of the
  microorganisms have metabolized the labelled
  substrate and incorporated the isotope into their
  RNA.

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• Substrates labeled with 14C or 13C have also been
  added to environmental samples and recovered as
  labeled lipid fractions that can be compared with
  the lipid fractions of cultivated strains. More
  recently, the technique of stable-isotope probing
  (SIP) was described, which used substrates highly
  enriched with 13C to selectively recover the DNA
  of functional groups of microorganisms, enabling
  later identification 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.

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• 7.2 Stable-isotope labeling of DNA
• Stable-isotope labeling of biomarkers uses the
  physical properties of atoms. 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). Therefore, substrates
  that are highly enriched in the rare
  stable-isotopes (e.g. gt99, 13C or 15N) can be
  added to complex environments, and the labeled
  isotopes can be tracked using techniques that
  detect the mass increase due to the single
  additional neutron.

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• 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 seperated from unlabeled
  DNA by equilibrium centrifugation in a CsCl
  density-gradient.
• Meselson and Stahl showed this with Escherichia
  coli grown on 15NH4. Although the buoyant
  density of DNA varies with its guanine-cytosine
  content, the incorporation of a high proportion
  of a naturally rare stable-isotope (2H, 15N or
  13C) into DNA increases the density difference
  between labeled and unlabeled DNA fractions. This
  principle has been used with bacterial cultures
  grown on 13CH3OH and 13CO2 as a carbon source,
  and has helped identify microorganisms in soil
  that actively assimilated methanol and methane.

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• 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 (13CDNA).
• Factors including 13C dilution due to the
  simultaneous assimilation of naturally occurring
  carbon substrates (i.e. 12C-labeled), 13C
  turnover due to substrate co-oxidation or
  predation of the target microorganisms, or 13C
  assimilation without DNA replication, might
  influence the proportion of a microbial genome
  that will become 13C-labeled.
• To date, SIP has been used to target
  metabolically restricted groups methylotrophs
  and ammonia-oxidizing bacteria (AOB) that grow
  in the presence of high concentrations of labeled
  substrate.

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• Figure 7.1
• CsCl/ethidium bromide density gradients of DNA
  fractions extracted from Methylobacterium
  extorquens grown on either 12C- or
  13Cmethanol as the carbon source. Visualization
  of 13C- and 12C-labeled DNA bands (arrows) with
  long-wavelength UV light following equilibrium
  ultracentrifugation at (A) 265 000 g for 16 h at
  20C and (B) 140 000 g for 60 h at 20C. Bar1 cm.

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• 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, such as soil, water or
  plant material, either in situ in the field, or
  in a serum vial (as depicted). The sample is
  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 13C-labelled
  substrate. This genomic DNA enriched with the
  13C isotope can be separated from the community
  DNA (12C-DNA) by CsCl gradient centrifugation.
• 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 identify 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.

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• 7.3 Application of stable-isotope probing
• The availability of PCR primers that are
  universal for the small-subunit rRNA genes of
  Bacteria, Archaea and Eukarya is needed to use
  SIP for identification, a priori, 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.

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• 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 these methylotrophs is
  the methane oxidizing bacteria (methanotrophs).
  Characterization of proteobacterial methylotrophs
  has identified certain common features of their
  biochemistry, which has permitted the design of
  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), respectively.

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• 7.3.1.1 Methanol assimilation
• Stable-isotope probing was first applied to
  identify the active methanol assimilating
  microorganisms in an acidic forest soil. Soil in
  a microcosm (small-scale experimental chamber
  that attempts to mimic environmental conditions)
  was exposed to 13CH3OH (0.5 v/w) for 44 days,
  after which a distinct 13CDNA fraction was
  resolved from total community DNA using a CsCl
  density-gradient. Domain level PCR primers only
  detected bacterial sequences in the 13CDNA
  fraction. Analysis of 16S rDNA sequences
  identified that three closely related genera
  within the a-Proteobacteria had assimilated the
  13C methanol, which was reflected in a parallel
  analysis of genes encoding MDH.

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• Other 16S rDNA sequences retrieved from the
  13CDNA grouped with the Acidobacterium
  division, which is poorly represented by
  cultivated strains. Association of these bacteria
  with assimilation of methanol or the by-products
  of methylotrophic carbon metabolism provides
  information about the metabolic function of a
  diverse, poorly studied and potentially important
  group of bacteria.

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• 7.3.1.2 Methane assimilation
• The active population of methanotrophs in a peat
  soil microcosm with a gas headspace containing
  13CH4 (8 v/v) was characterized following
  recovery of a heavy 13CDNA fraction. In
  contrast to the discrete 13CDNA band observed
  in the previous methanol experiment, the 13CDNA
  fraction was observed as a smear, indicating
  intermediate density DNA species that probably
  resulted from the growth of methanotrophs and
  additionally of bacteria using 13C-labeled
  intermediates/by-products of methanotroph
  metabolism. PCR amplification products of 16S
  rRNA genes and functional genes for MDH and MMO
  identified many sequences related to those of
  methanotrophs, demonstrating the activity of
  these bacteria in situ. The 13CDNA fraction
  also contained a large proportion of 16S rDNA
  sequences of bacteria not recognized as
  methanotrophs or methylotrophs, suggesting that
  other groups of bacteria are also involved in
  cycling the carbon derived from CH4 (possibly in
  the form of 13C-labeled metabolites or biomass)
  under these experimental conditions. SIP can thus
  identify the microbial population involved in the
  cycling of a specific compound, even though their
  function is unclear.

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• 7.3.2 Ammonia-oxidizing populations
• Autotrophic ammonia-oxidizing bacteria (AOB) are
  a specialized group of bacteria that are
  slow-growing and relatively 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 monophyletic
  group within the ß-Proteobacteria, which has
  resulted in the wide use of selective 16S rDNA
  PCR primers to study their ecology. SIP was
  recently used to identify the active
  13CO2-assimilating species of AOB in enrichment
  cultures inoculated with a fresh-water sediment.
  Although several types of AOB were detected in
  total DNA extracted from the enrichment cultures,
  only some subgroups of AOB were present in the
  13CDNA fraction. These results not only support
  previous observations that certain subgroups of
  AOB are out competed in laboratory culture, but
  also illustrate that heavy isotope labeled DNA
  can be used to identify which members of a
  metabolically defined population are active under
  a specific set of conditions.

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• 7.4 Future prospects
• SIP is able to enrich and isolate the combined
  genome of a microbial population that is
  involved in a specific function, thereby
  providing a reason to investigate the ecology of
  these microorganisms in situ using a variety of
  molecular biological techniques. One attractive
  option is the use of cloning and hybridization
  techniques to retrieve entire gene clusters from
  uncultivated bacteria whose function has been
  defined. In this manner, biases implicit in the
  use of selective PCR primers may be circumvented
  and improved PCR primers could be designed. A
  further development which could be envisaged is
  the targeting of molecules that do not rely on
  replication of the chromosome for 13C-labeling
  (e.g. rRNA). As ribosomes are naturally amplified
  in active cells, this would improve the
  sensitivity of SIP by reducing the amount of
  label required for linking identity with
  function.

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• One of the drawbacks of DNA-SIP is the relatively
  long incubation times that are required for DNA
  replication and incorporation of the 13C-label
  into newly synthesized DNA. Because RNA synthesis
  occurs at a faster rate than DNA synthesis, it is
  possible to obtain 13C-RNA more quickly than
  13C-DNA. RNA-SIP was used 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 caesium
  trifluoroacetate density gradient centrifugation.
  Reverse transcription (RT-) PCR amplification of
  13C-labelled 16S rRNA revealed that a Thauera
  species was a key player in phenol degradation in
  this bioreactor.

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• Unlike DNA-SIP, 13C-labelled RNA with a specific
  buoyant density is distributed in several
  fractions in density gradients, so it is
  necessary in RNA-SIP to analyse each gradient
  fraction by RT-PCR and denaturing gradient gel
  electrophoresis (DGGE). Analysis of the shifts in
  band intensities that occurred during the pulse
  of 13C-phenol in the bioreactor made it possible
  to determine which bacteria were being labelled.
  The first use of RNA-SIP in soil has recently
  been shown using 13C-pentachlorophenol