Linking phylogeny to environmental function

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Linking phylogeny to environmental function

• Dr. William Stafford
• wstafford_at_uwc.ac.za

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Understanding microbial ecology

• Microbial ecologists have the tools to
  quantitatively decipher the processes taking
  place in microbial communities. They can follow
  nitrogen, sulfur, carbon, reducing equivalents,
  energy, etc, etc, as they are transformed from
  one form to another to determine the inputs and
  outputs, and the flux rates between all of the
  forms in a ecological community.
• The limiting factor in microbial ecology is that
  essentially all we know about the organisms that
  make up these communities comes from the study of
  the less than 1 of microbes that are readily
  cultivated.

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Linking phylogeny to function?

• For example, suppose you have an environment in
  which a certain process is taking place, and you
  have a phylogenetic census of the organisms in a
  sample from this environment, how would you
  determine which organisms in your census are the
  ones that carry out the process you're interested
  in?
• Or, suppose you know an organism is abundant in
  an environment, how do you determine what it's
  doing there?

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SAR86 phototrophy in the sea

• Bacterial rhodopsin Evidence for a new type of
  phototrophy in the sea.
• Beja O, et al. 2000 Science 2891902-1906.
• Identifying the environmental contribution of an
  uncultivated species From molecular phylogenetic
  analysis of ocean water members of the "SAR86"
  group within the gamma-proteobacteria are
  abundant worldwide. What is their role in the
  ecology of the seas and oceans?

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SAR86 group

• Made a cosmid bank of DNA isolated from ocean
  water. These cosmid clones contain DNA fragment
  more than 100Kbp in length. They screened these
  cosmids by hybridization to identify those that
  contained the gene for 16S rRNA, so they could
  identify the organism the DNA fragment came from
  (phylogeny). One such clone (EBAC31A08) proved to
  be a member of the SAR86 group, and the authors
  sequenced the entire 130Kbp DNA fragment, hoping
  to find genes that would provide clues about the
  metabolism of the organism.
• One of the genes they found seems to be a gene
  encoding a rhodopsin, presumably acquired by
  horizontal transfer from a halophilic archaeon.
• Could it be that these organisms are using this
  rhodopsin to grow phototrophically?

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Cosmid library from sea metagenome
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• Bioinformatics protein modeling.
• They show that the predicted secondary structure
  of the "proteorhodopsin" is consistent with that
  of a bona fide opsin, and contains the conserved
  amino acids needed to bind it's cofactor, retinal.

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Heterologous expression in E.coli

• In figure 3, they show that if they express this
  protein in E.coli and add retinal (E.coli doesn't
  make retinal,), the cells quickly turn a hue of
  red (Absorbance max of 520nm) consistent with a
  rhodopsin. In other words, the protein as
  expressed by E.coli is correctly folded and
  inserted into the membrane in a form that can
  correctly bind the cofactor.

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But does it pump protons? Figure 4 shows that
E.coli with both rhodopsin and retinal pumps
protons from inside to outside (as measured by
the change in pH of the media) when and only when
provided with light. They use TTP uptake by
rhodopsin/retinal containing vesicles to measure
the electrical potential generated -90mV, which
is consistent with a strong proton pump.
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Proteorhodopsin

• During the pumping process, light energy is
  transformed into chemical gradient potential
  across plasma inner-membrane, the potential
  energy is then used to synthesize ATP.
  Proteorhodopsin is a functional, light-driven
  proton-pump in a group of uncultivated
  gamma-proteobacteria.
• The finding of Proteorhodopsin actually brings to
  light a novel pathway of sunlight utilization in
  contrast to the well-known chlorophyll-dependent
  photosynthesis in the sea (other main
  photosynthetic organisms in the sea are
  phytoplankton and cyanobacteria - Prochlorococcus
  and Synechococcus)

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Significance?

• Since the group of Proteorhodopsin-bearing
  bacteria is one of the numerically richest
  microorganisms on the Earth, accounting for 13
  of the total in ocean surface water they must be
  a key component in energy metabolism and carbon
  cycling in the sea.
• The oceans contribute 40 of the total
  photosynthesis on Earth. This drives the
  biological pump in the surface oceans, which
  exports carbon to the deep sea where it is
  naturally sequestered. If the pump were turned
  off, the concentration of CO2 in the atmosphere
  would more than double (Sarmiento and Orr 1991).

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Questions for thought

• Proteorhodopsin seems to be a major form of
  phototrophhy in the ocean. How could this have
  been missed all these years?
• Do you think you could grow E.coli
  photochemotrophically by expressing in it the
  protorhodopsin and feeding it retinal? How would
  you know it's using the organics only for fixed
  carbon rather than energy? If you did this, would
  you have created a new species of Esherichia?
• Do you know of any other examples where the whole
  ecological niche of an organism is defined by
  genes it acquired horizontally?
• How would you go about trying to cultivate one of
  the members of this group?

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Industrial wastewater treatment facility

• RNA stable isotope probing, a novel means of
  linking
• microbial community function to phylogeny
• Manefield M, Whitely AS, Griffiths RI and Bailey
  MJ 2002. Appl. Environ. Microbiol. 685367-5373
• Identifying the organisms that are responsible
  for a particular process What are the
  microorganisms in this environment that actually
  degrade the phenol?

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• This industrial wastewater treatment facility
  uses an aerobic digester to reduce the
  concentration of phenolics in the waste flow to
  levels that can be "released" into a public
  waterway. The reactors contain a continuous-flow
  microbial sludge (1010 cells/ml) with flow rate
  of 1800 liters of wastewater per minute and a
  volume of 1.7 million liters.
• The retention time is about 100 minutes, during
  which time more than 95 of the phenol is
  removed- 200ug/ml phenol per liter at the
  iinflow must be decreased to 10ug/ml so that it
  can be dumped into waterways. About 200 thousand
  kilograms of phenol per year is removed!
• The microbial population seems to turn over
  quickly - growth is continuous but controlled by
  grazing protists, and therefore the carbon that
  goes in as phenol presumably ends up as CO2 from
  respiration by the protists.

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Stable isotope Probing (SIP)

• Add 13C (heavy) labelled phenol to a culture for
  1-3 days, then extract RNA. The RNA is then
  fractionated by density-centrifugation in cesium
  TFA (tetrafluoroacetate). The more 13C
  incorporated, the denser the RNA, and therefore
  the lower in the gradient the RNA bands. The
  presumption here is that the organisms that
  actually eat phenol will incorporate the 13C from
  the labelled phenol into their RNA.
• Ribosomal RNA from gradient fractions is
  converted to DNA using reverse transcriptase, and
  PCR using rRNA-specific primers is used to
  amplify rDNAs from each fraction of the density
  gradients. The rDNAs are separated by denaturing
  gradient gel electrophoresis (DGGE). rDNA bands
  in the DGGE gels that are enriched in 13C can be
  re-amplified by PCR and sequenced to determine
  their identity.

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The two gels are DGGE's of rDNAs in fractions
from CsTFA gradients from PCRs of the
phenol-degraading community labeled for A 1
hour (too soon to get significant labelling) and
B 8 hours. Fraction 4 is from the bottom of
the gradient (most dense), fraction 13 is from
the top (least dense). The authors identify 5
bands as the "major" bands in the samples, and
label them A - E. As you can see, bands A, B, and
D are shifted to the bottom (left, dense) of the
gradient after 8 hours of growth with 13C phenol.
Each of these bands presumably represents a
species that can quickly utilize phenol for
growth.
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• The most intense such band, band "D", and show
  that it specifically gets more abundant in the
  heavy fraction - the amount of it in the light
  fraction doesn't change much. Newly-made RNA,
  then, all goes to the heavy fraction. They also
  use mass spectroscopy to confirm that RNA in the
  denser fractions really is enriched in 13C

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Determining phylogeny of the phenol degraders

• All 5 of the most abundant rDNAs (bands A - E)
  were cut out of the DGGE gel, reamplified, and
  sequenced to determine the identity of the
  organisms they represent. The apparent
  phenol-degraders were an alpha-proteobacterium
  (band A), and two beta-proteobacteria (B and D).
  The single most adundant phenol degrader (band D)
  turns out to be a beta-proteobacterium in the
  genus Thauera. This was a surprise, because if
  you do enrichments and pure cultures, the phenol
  degraders you isolate from this environment are
  gamma-proteobacteria, members of the genus
  Pseudomonas. Thauera is not very well studied, it
  is know to be involved in the degradation of
  aromatic compounds.
• This would seem, then, to be a novel species of
  Thauera, and the authors say they're trying to
  isolate it for further study.

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Questions for thought

• Although rRNA seems to label much better than DNA
  with 13C in these feding experiments, can you
  think of any reasons why it might be more useful
  to be able to isolate the DNA (rather than rRNA)
  of organisms that can eat the labeled substrate?
• Do you see any other bands in the DGGE gels that
  might represent less abundant phenol eaters?
• How could you make adjustments in the RT-PCR to
  make them more quantitative?
• Can you think of systems in which adding a
  labeled substrate and assuming the rRNAs that get
  labeled represent the organisms that use the
  substrate directly might be mistaken?
• How would you go about trying to cultivate one of
  the members of this group?

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