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DNA based Studies of Microbial Diversity
        DNA based Studies of Microbial Diversity
                               Jonathan A. Eisen
                               Jonathan A. Eisen

                         University of California, Davis
                         University of California, Davis



                                                           1
Monday, January 28, 13
Sequencing and Microbes



     • Four major “ERAs” in use of sequencing for
       microbial diversity studies
     • Each area represented by the Eras is being
       revolutionized by new sequencing methods




                                                    2
Monday, January 28, 13
Era I: rRNA Tree of Life




                              Era I:
                         rRNA Tree of Life



                                             3
Monday, January 28, 13
Ernst Haeckel 1866




     Plantae
     Protista
     Animalia


                                         4
                         www.mblwhoilibrary.org

Monday, January 28, 13
Whittaker – Five Kingdoms 1969




    Monera
    Protista
    Plantae
     Fungi
    Animalia
                                 5
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Woese




                         6
Monday, January 28, 13
Woese




Monday, January 28, 13
Woese




Monday, January 28, 13
Woese




Monday, January 28, 13
Woese and Fox


      • Abstract: A phylogenetic analysis based upon ribosomal
        RNA sequence characterization reveals that living
        systems represent one of three aboriginal lines of
        descent: (i) the eubacteria, comprising all typical bacteria;
        (ii) the archaebacteria, containing methanogenic bacteria;
        and (iii) the urkaryotes, now represented in the
        cytoplasmic component of eukaryotic cells.




Monday, January 28, 13
Woese and Fox


      • Propose “three aboriginal lines of descent”
          Eubacteria
          Archaebacteria
          Urkaryotes




Monday, January 28, 13
Woese

Woese 1987 - rRNA




                         Microbiological Reviews 51:221
                                                      12
Monday, January 28, 13
• Appearance of
                                                            microbes not
                                                            informative (enough)
                                                          • rRNA Tree of Life
                                                            identified two major
                                                            groups of organisms
                                                            w/o nuclei
                                                          • rRNA powerful for
                                                            many reasons, though
                                                            not perfect

    Barton, Eisen et al. “Evolution”, CSHL Press. 2007.

    Based on tree from Pace 1997 Science 276:734-740


                                                                                   13
Monday, January 28, 13
Tree of Life


      • Three main kinds of organisms
          Bacteria
          Archaea
          Eukaryotes
      • Viruses not alive, but some call them microbes
      • Many misclassifications occurred before the use of
        molecular methods




                                                             14
Monday, January 28, 13
The Tree of Life
                         2006




                                            adapted from Baldauf, et al., in Assembling the Tree of Life, 2004   15
Monday, January 28, 13
The Tree of Life
                                                                 2006




                         adapted from Baldauf, et al., in Assembling the Tree of Life, 2004

Monday, January 28, 13
Era II: rRNA in the Environment




                        Era II:
               rRNA in the Environment



                                         17
Monday, January 28, 13
Plant/Animal Field Studies




                              18
Monday, January 28, 13
Plant/Animal Field Studies




                              18
Monday, January 28, 13
Plant/Animal Field Studies




                              18
Monday, January 28, 13
Plant/Animal Field Studies




                              18
Monday, January 28, 13
Plant/Animal Field Studies




                              18
Monday, January 28, 13
Plant/Animal Field Studies




                              18
Monday, January 28, 13
Plant/Animal Field Studies




                              18
Monday, January 28, 13
Microbial Field Studies




                          19
Monday, January 28, 13
Microbial Field Studies




                          19
Monday, January 28, 13
Microbial Field Studies




                          19
Monday, January 28, 13
Microbial Field Studies




                          19
Monday, January 28, 13
Microbial Field Studies




                          19
Monday, January 28, 13
Microbial Field Studies




                          19
Monday, January 28, 13
Microbial Field Studies




                          19
Monday, January 28, 13
Culturing Microbes




                         20
Monday, January 28, 13
Great Plate Count Anomaly




                            21
Monday, January 28, 13
Great Plate Count Anomaly




                         Culturing   Microscopy




                                                  22
Monday, January 28, 13
Great Plate Count Anomaly




                         Culturing   Microscopy




                          Count       Count
                                                  23
Monday, January 28, 13
Great Plate Count Anomaly




                         Culturing          Microscopy




                          Count      <<<<    Count
                                                         24
Monday, January 28, 13
Great Plate Count Anomaly


   Problem because
    appearance not
  effective for “who
   is out there?” or
    “what are they
       doing?”


                         Culturing          Microscopy




                          Count      <<<<    Count
                                                         25
Monday, January 28, 13
Great Plate Count Anomaly

                                                     Solution?
   Problem because
    appearance not
  effective for “who
   is out there?” or
    “what are they
       doing?”


                         Culturing          Microscopy




                          Count      <<<<    Count
                                                                 26
Monday, January 28, 13
Great Plate Count Anomaly

                                                     Solution?
   Problem because
    appearance not
  effective for “who
   is out there?” or                                     DNA
    “what are they
       doing?”


                         Culturing          Microscopy




                          Count      <<<<    Count
                                                                 27
Monday, January 28, 13
Analysis of uncultured microbes




                         Collect from
                         environment
                                        28
Monday, January 28, 13
PCR and phylogenetic analysis of rRNA genes

                     DNA
                     extraction                            PCR

                                                       Makes lots                  Sequence
                           PCR                         of copies of               rRNA genes
                                                        the rRNA
                                                        genes in
                                                         sample

                                                                                 rRNA1
                                                                                   5’
                                                                           ...TACAGTATAGGT
     Phylogenetic tree               Sequence alignment = Data matrix      GGAGCTAGCGATC
                                                                               GATCGA... 3’
   rRNA1                 Yeast
                                         rRNA1     A   C   A   C   A   C
                                         Yeast     T   A   C   A G     T
                                         E. coli   A G A       C   A G
  E. coli                   Humans      Humans     T   A   T   A G     T




                                                                                              29
Monday, January 28, 13
PCR and phylogenetic analysis of rRNA genes

                     DNA
                     extraction                            PCR

                                                       Makes lots                 Sequence
                           PCR                         of copies of              rRNA genes
                                                        the rRNA
                                                        genes in
                                                         sample

                                                                                 rRNA1
                                                                                   5’
                                                                           ...ACACACATAGGT
     Phylogenetic tree               Sequence alignment = Data matrix      GGAGCTAGCGATC
                                                                               GATCGA... 3’
   rRNA1                 rRNA2
                                         rRNA1     A   C   A   C   A   C
                                         rRNA2     T   A   C   A G     T         rRNA2
                                                                                   5’
                                         E. coli   A G A       C   A G
                                                                           ...TACAGTATAGGT
  E. coli                   Humans      Humans     T   A   T   A G     T   GGAGCTAGCGATC
                                                                               GATCGA... 3’
                   Yeast                 Yeast     T   A   C   A G     T




                                                                                              30
Monday, January 28, 13
PCR and phylogenetic analysis of rRNA genes

                     DNA
                     extraction                           PCR

                                                      Makes lots                    Sequence
                           PCR                        of copies of                 rRNA genes
                                                       the rRNA
                                                       genes in
                                                        sample

                                                                                    rRNA1
                                                                          5’...ACACACATAGGTGGAGC
                                                                             TAGCGATCGATCGA... 3’
     Phylogenetic tree              Sequence alignment = Data matrix
                                                                                    rRNA2
      rRNA1        rRNA2
                                        rRNA1     A   C   A   C   A   C   5’..TACAGTATAGGTGGAGCT
                           rRNA4                                              AGCGACGATCGA... 3’
rRNA3                                   rRNA2     T   A   C   A G     T
                                                                                    rRNA3
                                        rRNA3     C   A   C   T   G   T   5’...ACGGCAAAATAGGTGGA
  E. coli                  Humans       rRNA4     C   A   C   A G     T     TTCTAGCGATATAGA... 3’

                   Yeast                E. coli   A G A       C   A G               rRNA4
                                                                          5’...ACGGCCCGATAGGTGG
                                       Humans     T   A   T   A G     T
                                                                          ATTCTAGCGCCATAGA... 3’
                                        Yeast     T   A   C   A G     T
                                                                                               31
Monday, January 28, 13
PCR and phylogenetic analysis of rRNA genes




                         PCR




                                              32
Monday, January 28, 13
Major phyla of bacteria & archaea (as of 2002)




                                                  No cultures

                                                  Some cultures
                                                                  33
Monday, January 28, 13
The Hidden Majority                   Richness estimates




                         Hugenholtz 2002         Bohannan and Hughes 2003
                                                                        34
Monday, January 28, 13
Human microbiome example




                           35
Monday, January 28, 13
A: Human biogeography




                         Censored



                         Censored




                                    36
Monday, January 28, 13
A: Human biogeography




                         37
Monday, January 28, 13
A: Human biogeography


                          Naris (R)             Forehead                            Hair                          External nose           Naris (L)




                         Ext. auditory                                                                                                 Ext. auditory
                          canal (R)           Lat. pinna (R)                                                      Lat. pinna (L)        canal (L)




                          Axilla (R)          Dorsal tongue                                                         Oral cavity           Axilla (L)




                            Volar                                                                                                          Volar
                         forearm (R)            Palm (R)                                                             Palm (L)           forearm (L)




                         Palmar index                                                                                                  Palmar index
                          finger (R)                Gut                                                             Umbilicus           finger (L)




                          Popliteal              Plantar                 Glans                  Labia                 Plantar             Popliteal
                          fossa (R)              foot (R)                penis                  minora                foot (L)            fossa (L)




                         Acinetobacter        Actinomycetales    Actinomycineae     Alistipes                     Anaerococcus       Bacteroidales
                         Bacteroides          Bifidobacteriales   Branhamella        Campylobacter                 Capnocytophaga     Carnobacteriaceae1
                         Carnobacteriaceae2   Clostridiales      Coriobacterineae   Corynebacterineae             Faecalibacterium   Finegoldia
                         Fusobacterium        Gemella            Lachnospiraceae    Lachnospiraceae (inc. sed.)   Lactobacillus      Leptotrichia
                         Micrococcineae       Neisseria          Oribacterium       Parabacteroides               Pasteurella        Pasteurellaceae

                                                                                                                                                          38
                         Peptoniphilus        Prevotella         Prevotellaceae     Propionibacterineae           Ruminococcaceae    Staphylococcus
                         Streptococcus        Veillonella        Other


Monday, January 28, 13
Vertebrate Microbiomes

                                          100



                                                                                                                                           Bacteroidetes (red)
                                          80
        16S ribosomal RNA sequences (%)




                                          60




                                          40




                                          20


                                                                                                                           ANALYSIS        Firmicutes (blue)
                                           0
                      r         s               ts                      n             r                  e t                                     t
                 ate ured rm                 en                  um
                                                                    a              ate t              fac gu                                  gu
            d wcultthwo                    im                 rh                t w men            surrmite                               ate
         xe
       Mi aliner ea
                      r                sed               he                 Sal di               r
                                                                                              ate Te
                                                                                                                                        br
                                   ter                Ot                       se           -w                                  Ve
                                                                                                                                   rte               Worlds within worlds: evolution of
       n-s ts o                hw
                                  a
                                                                         ic o
                                                                             r
                                                                                      Sal
                                                                                          t
    No sec
     In                 rf res                                       n ox                                                                            the vertebrate gut microbiota
                     so                                          e, a
               S oil                                        r fac                                                                                    Ruth E. Ley*‡¶, Catherine A. Lozupone*§¶, Micah Hamady||, Rob Knight § and
                                                        bsu                                                                                          Jeffrey I. Gordon*
                                                     Su                                                                                              Abstract | In this Analysis we use published 16S ribosomal RNA gene sequences to c
   Figure 3 | Relative abundance of phyla in samples. Bar graph showing the proportion of sequences from eachassemblages that are associated withrange of environments. The comp
                                                                                                            the bacterial
                                                                                                                            sample
                                                                                                            and free-living microbial communities that span a
                                                                                                                                                                humans and other mammals, me

   that could be classified at the phylum level. The colour codes for the dominant Firmicutes and Bacteroidetes phyla are microbiota is influenced by diet, host morphology and phyloge
                                                                                                            of the vertebrate gut shown.
                                                                                                   Nature Reviews | Microbiology
   For a complete description of the colour codes see Supplementary information S2 (figure). ‘Other humans’ refersvertebrate gut microbiotacommunity is typical of an omnivorous prima
                                                                                                            in this respect the human gut bacterial
                                                                                                            However, the to body                    is different from free-living communities th
   habitats other than the gut; for example, the mouth, ear, skin, vagina and vulva (see Supplementary information S1 (table)). habitats. We propose that the recently initiated
                                                                                                            not associated with animal body
                                                                                                                                                     international Human Microbiome Project should strive to include a broad represent
                                                                                                                                                     humans, as well as other mammalian and environmental samples, as comparative an
                                                                                                                                                     of microbiotas and their microbiomes are a powerful way to explore the evolutionar
                                                                                                                                                     history of the biosphere.                                                    39
  Genera that cross the divide. Another way to visualize                                     family of the gammaproteobacteria class. This fam-
Monday, January 28, 13
  the vertebrate gut–environment dichotomy is by using a                                     ily contained OTUs from both theDiverse microorganisms and microbial communities are
                                                                                                                              vertebrate gut and
                                                                                                                           Microbiota
                                                                                                                                                                                                  host energy metabolism8–11. Host responses to
40
Monday, January 28, 13
The Built Environment


                                                                                                                                                            Microbial Biogeography of Public Restroom Surfaces
                                                                                                                                                            Gilberto E. Flores1, Scott T. Bates1, Dan Knights2, Christian L. Lauber1, Jesse Stombaugh3, Rob Knight3,4,
                                                                                                                                                            Noah Fierer1,5*
                                                                                                                                                                                                                                                                                            Bacteria of Public Restrooms
                                                                                                                                                            1 Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Computer Science,
                                                                                                                                                            University of Colorado, Boulder, Colorado, United States of America, 3 Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, United
                                                                                                                                                            States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Department of Ecology and Evolutionary
                                                                                                                                                            Biology, University of Colorado, Boulder, Colorado, United States of America



                                                                                                                                                                 Abstract
                                                                                                                                                                  We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the
                                                                                                                                                                  diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited
                                                                                                                                                                  by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of
                                                                 The ISME Journal (2012), 1–11                                                                    the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla:
                                                                 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12              Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those
                                                                 www.nature.com/ismej                                                                             found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with
                                                                                                                                                         Figure 3. Cartoon toilet surfaces, gut-associated taxa were more prevalent, suggesting fecal contamination of Light blue indicates low
                                                                                                                                                                  hands. On illustrations of the relative abundance of discriminating taxa on public restroom surfaces. these surfaces. Floor
           ORIGINAL ARTICLE                                                                                                                              abundance while were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated
                                                                                                                                                                  surfaces dark blue indicates high abundance of taxa. (A) Although skin-associated taxa (Propionibacteriaceae, Corynebacteriaceae,
                                                                                                                                                         Staphylococcaceae especially the Propionibacteriaceae, on all surfaces, they were relatively more abundant on surfaces routinely touched with
                                                                                                                                                                  bacteria, and Streptococcaceae) were abundant dominated surfaces routinely touched with our hands. Certain taxa were more
           Architectural design influences the diversity and                                                                                             hands. (B) Gut-associated taxa (Clostridiales, Clostridiales group XI,vagina-associated Lactobacillaceae were widely Bacteroidaceae)in female
                                                                                                                                                                  common in female than in male restrooms as Ruminococcaceae, Lachnospiraceae, Prevotellaceae and distributed were most
                                                                                                                                                         abundant on toilet surfaces. from urine contamination. Use of the SourceTracker algorithm confirmed Nocardioidaceae) taxonomic
                                                                                                                                                                  restrooms, likely (C) Although soil-associated taxa (Rhodobacteraceae, Rhizobiales, Microbacteriaceae and many of our were in low
                                                                                                                                                         abundance on all restroom surfaces, they were relatively more abundant on the floor of the surfaces. Overall, theseFigure not drawn to scale.
                                                                                                                                                                  observations as human skin was the primary source of bacteria on restroom restrooms we surveyed. results demonstrate that
           structure of the built environment microbiome                                                                                                 doi:10.1371/journal.pone.0028132.g003
                                                                                                                                                                  restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clear
                                                                                                                                                                  linkages between communities on or in different body sites and those communities found on restroom surfaces.Bacteria of P                More
                                1             1              1,2
        Steven W Kembel , Evan Jones , Jeff Kline , Dale Northcutt , Jason Stenson ,           1,2                                   1,2                 the stallgenerally,were likely dispersed manuallypublicwomen used as we Results of human-associated microbes are commonly found
                                                                                                                                                                    in), they this work is relevant to the after health field               show that SourceTracker analysis support the taxonomic
                              1                                                                                                                                   on restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching
                                                                                                                                                         the toilet. Coupling these observations with those of the                       patterns highlighted above, indicating that human skin was the
 time, the M Womack , Brendan JM 100
        Ann                                   Bohannan1, G Z Brown1,2 and Jessica L Green1,3
        1
                                                                                                                          SOURCES                        distribution of gut-associated bacteria demonstrate that we use use high-throughput analyses of bacterial communities to determine
                                                                                                                                                                  of surfaces. Furthermore, we indicate that routine can
                                                                                                                                              Bathroom biogeography. By on indoor surfaces, an approach of                               primary source of bacteria on all public restroom surfaces
          Biology and the Built Environment Center, Institute of Ecology and Evolution, Department of                                                             sources the dispersal of urine- and fecal-associated bacteria
                                                                                                                                                                             of bacteria                                           whichexamined, while the track pathogen transmission and test the
                                                                                                                                                                                                                                           could be used to human gut was an important source on or
un to take
        Biology, University of Oregon, Eugene, OR, USA; 2Energy Studies in Buildings Laboratory,
                                                                                                                              Soil
                                                                                                                                              swabbing toilets results in restroom. While these results are not unexpected,
                                                                                                                                                         different surfaces in
                                                                                                                                                         throughout the of hygiene practices.
                                                                                                                                                                  efficacy                                                               around the toilet, and urine was an important source in women’s
of outside                                      80of Oregon, Eugene, OR, USA and 3Santa Fe Institute,                         Water
                                                         Average contribution (%)




        Department of Architecture, University                                                                                                public restrooms,highlight the importance of hand-hygiene when using
                                                                                                                                                         they do researchers                                                             restrooms (Figure 4, Table S4). Contrary to expectations (see
                                                                                                                              Mouth
om plants Fe, NM, USA
        Santa                                                                                                                                 determined thatCitation: Floressince these Knights D, Lauber CL, Stombaugh J, et al. (2011)above), soil was not identified by the Surfaces. PLoS ONEalgorithm as
                                                                                                                                                         public microbes vary in ST, surfaces could also be potential
                                                                                                                                                                   restrooms GE, Bates                                                     Microbial Biogeography of Public Restroom SourceTracker 6(11): e28132.
                                                                                                                              Urine                             doi:10.1371/journal.pone.0028132
ours after                                      60                                                                                            where they come from depend-
                                                                                                                                                         vehicles for the transmission of human pathogens. Unfortunately,                being a major source of bacteria on any of the surfaces, including
                                                                                                                              Gut                               Editor: Mark R. Liles, Auburn University, United States of America
 ere shut                                                                                                                                     ing on the previous (chart).have documented that college students (who are November 23, 2011 4). Although the floor samples contained family-level
                                                                                                                                                         surface studies                                                                 floors (Figure
                 Buildings are complex ecosystems that house trillions of microorganisms interactingSkin each                   with                     likely Received September 12, 2011; of the studied restrooms) Published
                                                                                                                                                                 the most frequent users Accepted November 1, 2011; are not              taxa that are common in soil, the SourceTracker algorithm
                                                40
ortion of        other, with humans and with their environment. Understanding the ecological and evolutionary                                                   Copyright: diligent of hand-washers open-access article distributed under the terms of the Creative Commons Attribution License, sources, like
                                                                                                                                                         always the most ß 2011 Flores et al. This is an[42,43].                         probably underestimates the relative importance of which permits
                processes that determine the diversity and composition of the built environment microbiome—the                         unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 e human        community of microorganisms that live indoors—is important for understanding the relationship
                                                                                                                         pant in indoor microbial
                                                 20                                                                                    Funding: This work was supported with funding from the Alfred P. Sloan Foundation and their Indoor Environment program, and in part by the National
 ck to pre-     between building design, biodiversity and human health. In this study, we used high-throughputecology research,ofPeccia the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or
                                                                                                                                       Institutes   Health and
                sequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes and
                airborne bacterial communities at 0 health-care facility. We quantified airborne bacterial community
                                                   a                                                                     thinks that the fieldthe manuscript.
                                                                                                                                       preparation of
                                                                                                                                                         has
   wh i c h     structure and environmental conditions in patient rooms exposed to mechanical or window                  yet to gel. And the Sloan The authors have declared that no competing interests exist.
                                                                                                                                       Competing Interests:
                                                                                               Do in

                                                                                                          t
                                                                                                        in

                                                                                       So han t
                                                                                             dis les

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                                                                                               hh t
                                                                                            To ndle

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                                                                                                        or
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                                                                                           lus t sea
                                                                                                        e




                                                                                                                                       * E-mail: noah.fierer@colorado.edu
                                                                                                    ns




                                                                                                     lo

                                                                                                    flo




                ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities was
                                                                                                    or



                                                                                                    all



                                                                                                     d




 26 Janu-       lower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbialFoundation’s Olsiewski
                                                                                                  or




                                                                                                  tf
                                                                                                   a
                                                                                                 Do




                                                                                                 pe
                                                                                                 St
                                                                                       Fa Sta




                                                                                                nk
                                                                                               ile
                                                                                             T




  Journal,      communities than did window-ventilated rooms. Bacterial communities in indoor environments               shares some of his con-
                                                                                         uc

                                                                                         ap



                                                                                         tf




                contained many taxa that are absent or rare outdoors, including taxa closely related to potential                    Introduction                                                                         communities and revealed a greater diversity of bacteria on
hanically                                                                                                                cern. “Everybody’s gen-
                                                                                    To




                human pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relative                                                                                                 indoor surfaces than captured using cultivation-based techniques
had lower       humidity and temperature, were correlated with the diversity and composition of indoor bacterial         erating vastMore than ever, individuals across the globe spend a large [10–13]. Most of the organisms identified in these studies are
                                                                                                                                         amounts of
                communities. The relative abundance of bacteria closely related to human pathogens was higher
 y than ones with openthan outdoors, and higher in rooms withquantify those con- lower relative humidity. looking acrossofdata lives of indooryet relatively littleOf known aboutthat related to human commensals suggesting that the organisms are
                indoors win-        they move around. But to lower airflow rates and data,” she says, but                            portion their
                                                                                                                                     microbial diversity
                                                                                                                                                         setsindoors, environments. is the studies the
                                                                                                                                                          Figure 2. Relationship between bacterial communities associated with tenon the surfaces but rather Communities were
                                                                                                                                                                                                                          not actively growing public restroom surfaces. were deposited
bility of fresh air translated tributions, Peccia’s team has had to develop diversity suggests that
                The observed relationship between building design and airborne bacterial can be difficult because groups choose dif- of the unweighted UniFrac distance matrix. Each point represents a single sample. Note that the floor (triangles) and toilet (as
                                                                                                                                                                                               rg on February 9, 2012

                                                                                                                                                          PCoA
                                                                                                                                     have examined microorganisms associated with indoor environ-                         directly (i.e. touching) or indirectly (e.g. shedding of skin cells) by
                we can manage indoor environments, altering through building design and operation the community
rtions of microbes associ- new methods to collect airborne bacteria and our timeanalytical tools. With Sloan support,
                of microbial species that potentially colonize the human microbiome during ferent indoors.                           ments, most have relied upon cultivation-based techniques hands. humans. Despite these efforts, we still have an incomplete
                                                                                                                                                          form clusters distinct from surfaces touched with to
                                                                                                                                                          doi:10.1371/journal.pone.0028132.g002
                The ISME Journal advance online publication, 26the microbes are much                                                 detect organisms residing on a variety of household surfaces [1–5].
                                                                January 2012; doi:10.1038/ismej.2011.211 a data archive and integrated analyt-                                                                            understanding of bacterial communities associated with indoor
 an body, and consequently, microbial their DNA, as
                Subject Category:
                                    extract population and community ecology                    though,                              Not surprisingly, these studies have identified surfaces in kitchens                 environments because limitations of traditional 16 S rRNA gene
pathogens. Although this less abundant in air than on surfaces.
                Keywords: aeromicrobiology; bacteria; built environment microbiome; community ecology; are in the works. and restrooms as being hot of floorof bacterial contamination. the frequency of sequencing differences in themade replicate samplings
                                                                                                ical tools dispersal;                                     high diversity spots communities is likely due to
                                                                                                                                                                                                                          cloning and
                                                                                                                                                                                                                                             related
                                                                                                                                                                                                                                                       techniques have
                                                                                                                                                                                                                                                                         relative abundances of
                  environmental filtering In one recent study, they used air filters
hat having natural airflow                                                                   To foster collaborations between micro- with the bottom aofvariety ofto survive on inanddiversity characterizations of the communities Most surfaces
                                                                                                                     Because several contact
                                                                                                                                       pathogenic bacteria are known which would track a in-depth some surfaces (Figure 1B, Table S2). prohibitive.
                                                                                                                                      of microorganisms from
                                                                                                                                                                   shoes,
                                                                                                                                                                          sources including soil, which is were clearly more abundant on certain
                                                                                                                                                                                                                                                     notably
                                                                                                                     surfaces for extended periods of time [6–8], these studies are of       With the advent of high-throughput restrooms (Figure 1B). Some
                                                                                                                                                                                                                                 sequencing techniques, we
 Green says answering that to sample airborne particles and microbes biologists, architects, and building scientists,in preventing the spread of human habitat [27,39]. Indeed, restrooms than male
                                                                                                                                      known to be a highly-diverse microbial disease.
                                                                                                                     obvious importance
                                                                                                                                                                                                                                                  41
                                                                                                                                                                                             can now investigate are
                                                                                                                                                                                                           family indoor microbial communitiesmost abu
                                                                                                                                                                                                                                             often at an
          Introduction
 clinical data; she’s hoping in a classroom during 4 days during which human pathogensalso sponsored a symposium widely recognized that the majority of Rhodobacteraceae, depth and the most common, andthe relationship
                                                                 microbiome—includes the foundation and com-         However, it is bacteria commonly associated with soil (e.g.
                                                                                                                                       now                                                   unprecedented found in the vagina of healthy reproductive age w
                                                                                                                                                                                                                       begin to understand
                                                                 mensals interacting with each other and with their  microorganisms Rhizobiales, Microbacteriaceae and[9] and thus, the
                                                                                                                                      cannot be readily cultivated Nocardioidaceae) were, on average,
 ital to participate in a study 90% of theirwere present and 4 days during et on the microbiome of the built environment abundant on floor surfaces (Figure 3C, Table S2). and are relatively less abundant in male urine
          Humans spend up to students lives indoors              environment (Eames     al., 2009). There have been                                                                          between humans, microbes and the built environment.
                                                                                                                     overall diversitymore microorganisms associated with indoor
                                                                                                                                         of
    Monday,etJanuary which 13 the was vacant. They measured at the 2011 Indoor Air conference in Austin, largelysome of the Recentflush handles harbored In order to begin to of female urine samples collected as part
                                        28, the
dence of hospital-acquired Consequently,roomway we few attempts to comprehensively survey the built
          (Klepeis     al., 2001).
           design and operate the indoor environment has a
                                                                                                                                      Interestingly, unknown. toilet use of cultiva-
                                                                                                                     environments remains                                                        bacterial analysis comprehensively describe the microbial
                                                                                                                                                                                communities similar to those found on the floor diversity of indoor environments, 1A), characterized the bacterial
                                                                                            environment microbiome (Rintala et al., 2008;                                                                                        (Figure 2,      study [26] (Figure we found that Lactobacillaceae we
Era III: Genome Sequencing




                          Era III:
                     Genome Sequencing



                                         42
Monday, January 28, 13
1st Genome Sequence




                         Fleischmann
                         et al. 1995 43
Monday, January 28, 13
Genomes Revolutionized Microbiology


      • Predictions of metabolic processes
      • Better vaccine and drug design
      • New insights into mechanisms of evolution
      • Genomes serve as template for functional studies
      • New enzymes and materials for engineering and
        synthetic biology




                                                           44
Monday, January 28, 13
45
Monday, January 28, 13
Metabolic Predictions




                         46
Monday, January 28, 13
Lateral Gene Transfer




   Perna et al. 2003
                         47
Monday, January 28, 13
Network of Life


                         Bacteria




                                                                Archaea




                          Eukaryotes

                             Figure from Barton, Eisen et al.
                                “Evolution”, CSHL Press.
                           Based on tree from Pace NR, 2003.
                                                                          48
Monday, January 28, 13
Using the Core




                         49
Monday, January 28, 13
Whole Genome Phylogeny




  Whole genome tree
  built using
  AMPHORA
  by Martin Wu and
  Dongying Wu
                         50
Monday, January 28, 13
Microbial genomes




                         From http://genomesonline.org   51
Monday, January 28, 13
GEBA as example




                         52
Monday, January 28, 13
Phylogenetic Diversity


    • Phylogenetic diversity poorly
      sampled
    • GEBA project at DOE-JGI
      correcting this




                                      53
Monday, January 28, 13
54
Monday, January 28, 13
http://www.jgi.doe.gov/programs/GEBA/pilot.html   55
Monday, January 28, 13
GEBA Lesson 1: rRNA utility in IDing novel genomes




   From Wu et al. 2009 Nature 462, 1056-1060         56
Monday, January 28, 13
GEBA Lesson 2: rRNA Tree is not perfect



                     16s                           WGT, 23S




  Badger et al. 2005 Int J System Evol Microbiol 55: 1021-1026.   57
Monday, January 28, 13
GEBA Lesson 3: Phylogenetic sampling improves annotation



        • Took 56 GEBA genomes and compared results vs. 56
          randomly sampled new genomes
        • Better definition of protein family sequence “patterns”
        • Greatly improves “comparative” and “evolutionary”
          based predictions
        • Conversion of hypothetical into conserved hypotheticals
        • Linking distantly related members of protein families
        • Improved non-homology prediction




                                                                    58
Monday, January 28, 13
GEBA Lesson 4 : Metadata Important




                                     59
Monday, January 28, 13
GEBA Lesson 5:Improves discovering new genetic diversity




                                                           60
Monday, January 28, 13
Protein Family Rarefaction Curves


      • Take data set of multiple complete genomes
      • Identify all protein families using MCL
      • Plot # of genomes vs. # of protein families




                                                      61
Monday, January 28, 13
Wu et al. 2009 Nature 462, 1056-1060
                                       62
Monday, January 28, 13
Wu et al. 2009 Nature 462, 1056-1060
                                       62
Monday, January 28, 13
Wu et al. 2009 Nature 462, 1056-1060
                                       62
Monday, January 28, 13
Wu et al. 2009 Nature 462, 1056-1060
                                       62
Monday, January 28, 13
Wu et al. 2009 Nature 462, 1056-1060
                                       62
Monday, January 28, 13
Synapomorphies exist




Wu et al. 2009 Nature 462, 1056-1060
                                                63
Monday, January 28, 13
III: Epidemiology & Forensics




                                64
Monday, January 28, 13
Era IV: Genomes in the environment




                Era IV:
       Genomes in the Environment



                                     65
Monday, January 28, 13
Marine Microbe Background


      • rRNA PCR studies of marine microbes have been
        extensive
      • Comparative analysis had revealed many lineages, some
        very novel, some less so, that were dominant in many, if
        not all, open ocean samples
      • Lineages given names based on specific clones: e.g.,
        SAR11, SAR86, etc




                                                                   66
Monday, January 28, 13
%&'())%#*+,-###./*/!0##*1""#23##4(56#,!
                                                                                          Molecular diversity and ecology
                                                                                          of microbial plankton
                                                                                          Stephen J. Giovannoni1 & Ulrich Stingl1

                                                                                          The history of microbial evolution in the oceans is probably as old as the history of life itself. In contrast to
                                                                                          terrestrial ecosystems, microorganisms are the main form of biomass in the oceans, and form some of the
  INSIGHT REVIEW                                                                          largest populations on the planet. Theory predicts that selection should act437|15 September 2005
                                                                                                                                                    NATURE|Vol more efficiently in large
                                                                                          populations. But whether microbial plankton populations harbour organisms that are models of adaptive
                                                                                          sophistication remains to be seen. Genome sequence data are piling up, but most of the key microbial
                                                                                          plankton clades have no cultivated representatives, and information about their ecological activities is sparse.

     Archaea                                                                           Certain characteristics of the ocean environment — the prevailing           cultivation of key organisms, metagenomics and ongoing biogeo-
                                                                                       low-nutrient state of the ocean surface, in particular — mean it is         chemical studies. It seems very likely that the biology of the dominant
           Crenarchaeota                                  Euryarchaeota                sometimes regarded as an extreme ecosystem. Fixed forms of nitrogen,        microbial plankton groups will be unravelled in the years ahead.
           Group I Archaea                               Group II Archaea              phosphorus and iron are often at very low or undetectable levels in the         Here we review current knowledge about marine bacterial and
                                                                                       ocean’s circulatory gyres, which occur in about 70% of the oceans1.         archaeal diversity, as inferred from phylogenies of genes recovered
                                                         Group III Archaea             Photosynthesis is the main source of metabolic energy and the basis of      from the ocean water column, and consider the implications of micro-
                                                         Group IV Archaea              the food chain; ocean phytoplankton account for nearly 50% of global        bial diversity for understanding the ecology of the oceans. Although
                                                                                       carbon fixation, and half of the carbon fixed into organic matter is        we leave protists out of the discussion, many of the same issues apply
                                                                                       rapidly respired by heterotrophic microorganisms. Most cells are freely     to them. Some of the studies we refer to extend to the abyssal ocean,
                                                                                       suspended in the mainly oxic water column, but some attach to aggre-        but we focus principally on the surface layer (0–300 m) — the region
                                                                                       gates. In general, these cells survive either by photosynthesizing or by    of highest biological activity.
                                                                                       oxidizing dissolved organic matter (DOM) or inorganic compounds,
                                                                             α-Proteobacteria as an electron acceptor.
                                                                                       using oxygen                                                                Phylogenetic diversity in the ocean
                                                                                                                                                 5
                                                                             * SAR11 - theMicrobial cell concentrations are thymidine uptake into microbial
                                                                                        Pelagibacterlayer (0–300 m) — typically about 10 cells ml in               Small-subunit ribosomal (RNA) genes have become universal phylo-
                                                                                                                                                           ǁ1

                                                                                           ocean surface ubique                                                    genetic markers and are the main criteria by which microbial plank-
                                                                             * Roseobacterindicates average growth rates of about 0.15 divisions per day
                                                                                       DNA clade                                                                   ton groups are identified and named9. Most of the marine microbial
                    Chloroflexi                                              OCS116 (ref. 2). Efficient nutrient recycling, in which there is intense competi-     groups were first identified by sequencing rRNA genes cloned from
                                                                                       tion for scarce resources, sustains this growth, with predation by          seawater10–14, and remain uncultured today. Soon after the first reports
                     SAR202                                                  ß-Proteobacteria
                                                                                       viruses and protozoa keeping populations in check and driving high          came in, it became apparent that less than 20 microbial clades
                                                                             * OM43 turnover rates3. Despite this competition, steady-state dissolved              accounted for most of the genes recovered15. Figure 1 is a schematic
                                                                                       organic carbon (DOC) concentrations are many times higher than              illustration of the phylogeny of these major plankton clades. The taxon
                                                                             µ-Proteobacteria
                                                                                       carbon sequestered in living microbial biomass4. However, the average       names marked with asterisks represent groups for which cultured iso-
     Planctobacteria                                                         SAR86 age of the DOC pool in the deep ocean, of about 5,000 years5 (deter-            lates are available.
                                                                             * OMG Clade by isotopic dating), suggests thatismuch of the DOM is refrac-
                                                                                       mined
                                                                                       tory to degradation. Although DOM a huge resource, rivalling
                                                                                                                                                                       The recent large-scale shotgun sequencing of seawater DNA is pro-
                                                                                                                                                                   viding much higher resolution 16S rRNA gene phylogenies and bio-
                                                                             * Vibrionaeceae CO2 as a carbon pool6 , chemists have been thwarted by
                                                                                       atmospheric                                                                 geographical distributions for marine microbial plankton. Although
                                                                             * Pseudoalteromonas DOM and have characterized it only in broad terms7.
                                                                                       the complexity of                                                           the main purpose of Venter’s Sorcerer II expedition is to gather whole-
      Fibrobacter                                                            * Marinomonas The paragraphs above capture prominent features of the ocean            genome shotgun sequence (WGS) data from planktonic microorgan-
        SAR406                                                               * Halomonadacae but leave out the complex patternsand diversification of
                                                                                       environment,                                        of physical, chemical   isms16, thousands of water-column rRNA genes are part of the
                                                                                       and biological variation that drive the evolution                           by-catch. The first set of collections, from the Sargasso Sea, have
                                  Bacteroidetes                              * Colwellia
                                                                                       microorganisms. For example, members of the genus Vibrio — which Figure 1 | Schematic illustration of the phylogeny of
                                                                                                                                                                   yielded 1,184 16S rRNA gene fragments. These data are shown in
                                                                             * Oceanospirillum of the most common planktonic bacteria that can be iso-
                                                                                       include some                                                                Fig. 2, organized by clade structure. Such data are a rich scientific
       Marine Actinobacteria                                                                                                                            the major plankton clades. Black letters indicate
                                                                                       lated on nutrient agar plates — readily grow anaerobically by fermen-       resource for two reasons. First, they are not tainted by polymerase
                                                                             δ-Proteobacterialife cycles of some Vibrio species have been shown to
                                                                                       tation. The
                                                                                                                                                        microbial groups that seem to be ubiquitous in
                                                                                       include anoxic stages in association with animal hosts, but the broad
                                                                                                                                                                   chain reaction (PCR) artefacts; PCR artefacts rarely interfere with the
                                                                                                                                                                   correct placement of genes in phylogenetic categories, but they are a
                                                         Cyanobacteria                                                                                  seawater. Gold indicates groups found in the photic
                                                                                       picture of their ecology in the oceans has barely been characterized8.      major problem for reconstructing evolutionary patterns at the popu-
                               Lentisphaerae          * Marine Cluster A               The story is similar for most of the microbial groups described below:      lation level17. Second, the enormous number of genes provided by the
                           * Lentisphaera araneosa      (Synechococcus)                                                                                 zone. Blue indicates groups confined to the
                                                                                       the phylogenetic map is detailed, but the ecological panorama is thinly     Sorcerer II expedition is revealing the distribution patterns and abun-
                                                                                       sketched. New information is rapidly flowing into the field from the        dance of microbial groups that compose only a small fraction of the
                                                     * Prochlorococcus sp.                                                                              mesopelagic and surface waters during polar winters.
                                                                                          Department of Microbiology, Oregon State University, Corvallis, Oregon 97331,indicates microbial groups associated with
                                                                                                                                                        Green USA.
     Bacteria                                                                             1




                                                                                                                                                        coastal ocean ecosystems.
                                                                                                                                                                  ©2005 Nature Publishing Group
                                                                                                                                                                                                                  343

                                                                                                                                                                                                                                   67
  community. As discussed below, some opportunistic strains that                                  cyanobacteria. As obligate phototrophs, these cyanobacteria are con-
                                                                                                                     ! !               © !""# Nature Publishing Group!
 Monday, January 28, 13
facts17. They concluded that most sequence variation was clustered                                      many microbia
                                                                                                                   tions in the wat
                                                                                                                   best example. T
                                          35
                                                                                                                   entiated by the
                                          30                                                                       adapted (high-b
                % of 16S rRNA sequences
                                                                                                                   Phylogenetic e
                                          25                                                                       gests that the h
                                                                                                                   cally distinct lin
                                          20                                                                       Cluster A Syne
                                                                                                                   of which can b
                                          15                                                                       characteristics
                                                                                                                   urobilin)37,38. S
                                          10                                                                       ample support
                                                                                                                   teristics that aff
                                           5
                                                                                                                   SS120 has a mu
                                          0
                                                                                                                   ammonium an
                                               )    II
                                                   Ib     n       e      r) tes ia) ria nas ia) era de xi)         extreme, Synec
                                            ria up kto lad cte
                                               +                               e      r   e     r         la   e
                               Ia acte gro lan a C ba                        id cte act mo cte eim r c rofl        nitrate, cyanate
                             s                             i     o        ro      a     b   o a   h     e    o
                         oup eob sub ytop cter ibr cte eob tino lter eob ein act Chl                               interesting to n
                       r         t
                     bg Pro AR1 op ob
                                          1      h     a     (F Ba rot Ac doa rot Rh eob 2 (
                    u      -                  c    e     0 6              P
                                                                       δ- rine seu (α-
                                                                                            P     s
                                                                                                Ro R2
                                                                                                       0           seem to prospe
               1 1 s (γ            S        Pi rot R4                 ( a
             AR R8
                      6                   e      P SA                4 M            /P 6            SA             whereby nutrie
            S A                     a rin α-                      32             as R11
                S                 M red                        AR              on SA                               conditions. Th
                                        tu                    S             om
                                   c ul                                 ter                                        seasonal specia
                                Un                                    Al
                                                                                                                   lular cyanobact
                                                           Phylogenetic clade                                          The observa
                                                                                                                                68
Monday, January 28, 13                                                                                             diverged into e
Delong GENOMIC FRAGMENTS FROM PLANKTONIC MARINE ARCHAEA
          Lab                                                                                                 593


ments isolated from fosmid clones
 with various restriction endonucle-
10 kb, the F-factor-based vector
  the fosmid subfragments. Partial
  of restriction enzyme to 1 ⇥g of
 mixture. The reaction mixture was
  removed at 10, 40, and 60 min.
dding 1 ⇥l of 0.5 M EDTA to the
e. The partially digested DNA was
 s described above except using a 1-
he sizes of the separated fragments
n standards. The distances of the
d SP6 promoter sites on the excised
 pmol of T7- or SP6-specific oligo-
 l) and hybridizing with Southern

 fosmid and pBAC clones digested
  probed with labeled T7 and SP6
eled subclones and PCR fragments
 otgun sequencing described above.
e estimates from the partial diges-
of the fosmids and their subclones.
  and DeSoete distance (9) analyses
 n using GDE 2.2 and Treetool 1.0,
 (RDP) (23). DeSoete least squares




                                                                                                                     D ownloaded from jb.asm .org at U N IV O F C
ng pairwise evolutionary distances,
 to account for empirical base fre-
 tained from the RDP, version 4.0
u rRNA sequences were performed
 the RDP. For distance analyses of
 lutionary distances were estimated
d tree topology was inferred by the
n addition and global branch swap-
protein sequences, the Phylip pro-
  addition and ordinary parsimony
                                         FIG. 1. Flowchart depicting the construction and screening of an environ-
 artial sequences reported in Table    mental library from a mixed picoplankton sample. MW, molecular weight;
 the following accession numbers:      PFGE, pulsed-field gel electrophoresis.
U40243, U40244, and U40245. The
and EF2 have been submitted to
and U41261.                                                                                                                                                         69
                                         Recombinant fosmids, each containing ca. 40 kb of pico-
  Monday, January 28, 13
Delong Lab                                                                    J. BACTERIOL.




                FIG. 4. High-density filter replica of 2,304 fosmid clones containing approx-
             imately 92 million bp of DNA cloned from the mixed picoplankton community.
             The filter was probed with the labeled insert from clone 4B7 (dark spot). The
             lack of other hybridizing clones suggests that contigs of 4B7 are absent from this




                                                                                                  D ownloaded f
             portion of the library. Similar experiments with the remainder of the library
             yielded similar results.

                                                                                                                  70
Monday, January 28, 13
l gene  own transducer of light stimuli [for example,   the kinetics of its photochemical reaction cy-
 leDelong Lab
    ge- Htr (22, 23)]. Although sequence analysis of    cle. The transport rhodopsins (bacteriorho-
  iden- proteorhodopsin shows moderate statistical      dopsins and halorhodopsins) are character-
 roteo- support for a specific relationship with sen-   ized by cyclic photochemical reaction se-
  from
opsins
ferent.
hereas
 philes
 r than

 rmine
 l, we
a coli
  pres-
 rotein
   3A).
 nes of
popro-
m was
  (Fig.
at 520
 band-
 erated
 odop-
nce of
 dth is                                                                                           71
 rption January 28, 13
   Monday,
generated




                                                                                                                    D ownloaded from w
  Delong Lab
 eorhodop-
resence of
ndwidth is
absorption
. The red-
  nm in the
ated Schiff
ably to the

 on was de-
s in a cell
ward trans-
 in proteor-
nd only in
 (Fig. 4A).
edium was
ce of a 10
re carbonyl
19). Illumi-
ical poten-
  right-side-
nce of reti-
light onset
hat proteo-
  capable of    Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the
 physiolog-     130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodop-
                sin with archaeal (BR, HR, and SR prefixes) and Neurospora crassa (NOP1 prefix) rhodopsins (16).
e activities    Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin;
 containing     BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halo-
 proteorho-     bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp;
main to be      Neucra, Neurospora crassa.
                                                                                                                                         72
   www.sciencemag.org
  Monday, January 28, 13    SCIENCE VOL 289 15 SEPTEMBER 2000                                                   1903
73
Monday, January 28, 13
Figure 3. Phylogenetic tree based on the amino acid sequences of 25 archaeal rhodopsins. (a) NJ-tree. The numbers at each node are clustering
probabilities generated by bootstrap resampling 1000 times. D1 and D2 represent gene duplication points. The four shaded rectangles indicate the
speciation dates when halobacteria speciation occurred at the genus level. (b) ML-tree. Log likelihood value for ML-tree was −6579.02 (best
score) and that for topology of the NJ-tree was −6583.43. The stippled bars indicate the 95% confidence limits. Both trees were tentatively rooted
at the mid-point of the longest distance, although true root positions are unknown.

From Ihara et al. 1999                                                                                                                       74
Monday, January 28, 13
RESEARCH ARTICLES
    Fig. 2. Secondary
    structure of proteo-
    rhodopsin. Single-
    letter amino acid
    codes are used (33),
    and the numbering
    is as in bacteriorho-
    dopsin.     Predicted
    retinal binding pock-
    et    residues    are
    marked in red.




                                                75
Monday, January 28, 13
duce
                                                                                                                  in th
                                                                                                                  occu
                                                                                                                  pigm
                                                                                                                  and t
                                                                                                                  ed at
                                                                                                                  tiona
                                                                                                                  sorpt
                                                                                                                  in 0.
                                                                                                                  sorpt
                                                                                                                  botto
                                                                                                                  nated
                                                                                                                  retin
                                                                                                                  ms d
                                                                                                                  deca
                                                                                                                  shift
                                                                                                                  appe
                                                                                                                  term
                                                                                                                  cay o
                                                                                                                  step
                                                                                                                  singl
                                                                                                                  upwa
        Fig. 3. (A) Proteorhodopsin-expressing E. coli cell suspension (ϩ) compared to control cells (Ϫ),
        both with all-trans retinal. (B) Absorption spectra of retinal-reconstituted proteorhodopsin in E. coli
                                                                                                                  ampl
        membranes (17). A time series of spectra is shown for reconstituted proteorhodopsin membranes             gene
        (red) and a negative control (black). Time points for spectra after retinal addition, progressing from    with
        low to high absorbance values, are 10, 20, 30, and 40 min.                                                ms p
                                                                                                                  recov
                                                                                                                  phot
                                                                                                                  prod
                                                                                                                   76
Monday, January 28, 13                                                                                            this
Eisen Lecture for Ian Korf genomics course
Eisen Lecture for Ian Korf genomics course
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Eisen Lecture for Ian Korf genomics course

  • 1. DNA based Studies of Microbial Diversity DNA based Studies of Microbial Diversity Jonathan A. Eisen Jonathan A. Eisen University of California, Davis University of California, Davis 1 Monday, January 28, 13
  • 2. Sequencing and Microbes • Four major “ERAs” in use of sequencing for microbial diversity studies • Each area represented by the Eras is being revolutionized by new sequencing methods 2 Monday, January 28, 13
  • 3. Era I: rRNA Tree of Life Era I: rRNA Tree of Life 3 Monday, January 28, 13
  • 4. Ernst Haeckel 1866 Plantae Protista Animalia 4 www.mblwhoilibrary.org Monday, January 28, 13
  • 5. Whittaker – Five Kingdoms 1969 Monera Protista Plantae Fungi Animalia 5 Monday, January 28, 13
  • 6. Woese 6 Monday, January 28, 13
  • 10. Woese and Fox • Abstract: A phylogenetic analysis based upon ribosomal RNA sequence characterization reveals that living systems represent one of three aboriginal lines of descent: (i) the eubacteria, comprising all typical bacteria; (ii) the archaebacteria, containing methanogenic bacteria; and (iii) the urkaryotes, now represented in the cytoplasmic component of eukaryotic cells. Monday, January 28, 13
  • 11. Woese and Fox • Propose “three aboriginal lines of descent”  Eubacteria  Archaebacteria  Urkaryotes Monday, January 28, 13
  • 12. Woese Woese 1987 - rRNA Microbiological Reviews 51:221 12 Monday, January 28, 13
  • 13. • Appearance of microbes not informative (enough) • rRNA Tree of Life identified two major groups of organisms w/o nuclei • rRNA powerful for many reasons, though not perfect Barton, Eisen et al. “Evolution”, CSHL Press. 2007. Based on tree from Pace 1997 Science 276:734-740 13 Monday, January 28, 13
  • 14. Tree of Life • Three main kinds of organisms  Bacteria  Archaea  Eukaryotes • Viruses not alive, but some call them microbes • Many misclassifications occurred before the use of molecular methods 14 Monday, January 28, 13
  • 15. The Tree of Life 2006 adapted from Baldauf, et al., in Assembling the Tree of Life, 2004 15 Monday, January 28, 13
  • 16. The Tree of Life 2006 adapted from Baldauf, et al., in Assembling the Tree of Life, 2004 Monday, January 28, 13
  • 17. Era II: rRNA in the Environment Era II: rRNA in the Environment 17 Monday, January 28, 13
  • 18. Plant/Animal Field Studies 18 Monday, January 28, 13
  • 19. Plant/Animal Field Studies 18 Monday, January 28, 13
  • 20. Plant/Animal Field Studies 18 Monday, January 28, 13
  • 21. Plant/Animal Field Studies 18 Monday, January 28, 13
  • 22. Plant/Animal Field Studies 18 Monday, January 28, 13
  • 23. Plant/Animal Field Studies 18 Monday, January 28, 13
  • 24. Plant/Animal Field Studies 18 Monday, January 28, 13
  • 25. Microbial Field Studies 19 Monday, January 28, 13
  • 26. Microbial Field Studies 19 Monday, January 28, 13
  • 27. Microbial Field Studies 19 Monday, January 28, 13
  • 28. Microbial Field Studies 19 Monday, January 28, 13
  • 29. Microbial Field Studies 19 Monday, January 28, 13
  • 30. Microbial Field Studies 19 Monday, January 28, 13
  • 31. Microbial Field Studies 19 Monday, January 28, 13
  • 32. Culturing Microbes 20 Monday, January 28, 13
  • 33. Great Plate Count Anomaly 21 Monday, January 28, 13
  • 34. Great Plate Count Anomaly Culturing Microscopy 22 Monday, January 28, 13
  • 35. Great Plate Count Anomaly Culturing Microscopy Count Count 23 Monday, January 28, 13
  • 36. Great Plate Count Anomaly Culturing Microscopy Count <<<< Count 24 Monday, January 28, 13
  • 37. Great Plate Count Anomaly Problem because appearance not effective for “who is out there?” or “what are they doing?” Culturing Microscopy Count <<<< Count 25 Monday, January 28, 13
  • 38. Great Plate Count Anomaly Solution? Problem because appearance not effective for “who is out there?” or “what are they doing?” Culturing Microscopy Count <<<< Count 26 Monday, January 28, 13
  • 39. Great Plate Count Anomaly Solution? Problem because appearance not effective for “who is out there?” or DNA “what are they doing?” Culturing Microscopy Count <<<< Count 27 Monday, January 28, 13
  • 40. Analysis of uncultured microbes Collect from environment 28 Monday, January 28, 13
  • 41. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots Sequence PCR of copies of rRNA genes the rRNA genes in sample rRNA1 5’ ...TACAGTATAGGT Phylogenetic tree Sequence alignment = Data matrix GGAGCTAGCGATC GATCGA... 3’ rRNA1 Yeast rRNA1 A C A C A C Yeast T A C A G T E. coli A G A C A G E. coli Humans Humans T A T A G T 29 Monday, January 28, 13
  • 42. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots Sequence PCR of copies of rRNA genes the rRNA genes in sample rRNA1 5’ ...ACACACATAGGT Phylogenetic tree Sequence alignment = Data matrix GGAGCTAGCGATC GATCGA... 3’ rRNA1 rRNA2 rRNA1 A C A C A C rRNA2 T A C A G T rRNA2 5’ E. coli A G A C A G ...TACAGTATAGGT E. coli Humans Humans T A T A G T GGAGCTAGCGATC GATCGA... 3’ Yeast Yeast T A C A G T 30 Monday, January 28, 13
  • 43. PCR and phylogenetic analysis of rRNA genes DNA extraction PCR Makes lots Sequence PCR of copies of rRNA genes the rRNA genes in sample rRNA1 5’...ACACACATAGGTGGAGC TAGCGATCGATCGA... 3’ Phylogenetic tree Sequence alignment = Data matrix rRNA2 rRNA1 rRNA2 rRNA1 A C A C A C 5’..TACAGTATAGGTGGAGCT rRNA4 AGCGACGATCGA... 3’ rRNA3 rRNA2 T A C A G T rRNA3 rRNA3 C A C T G T 5’...ACGGCAAAATAGGTGGA E. coli Humans rRNA4 C A C A G T TTCTAGCGATATAGA... 3’ Yeast E. coli A G A C A G rRNA4 5’...ACGGCCCGATAGGTGG Humans T A T A G T ATTCTAGCGCCATAGA... 3’ Yeast T A C A G T 31 Monday, January 28, 13
  • 44. PCR and phylogenetic analysis of rRNA genes PCR 32 Monday, January 28, 13
  • 45. Major phyla of bacteria & archaea (as of 2002) No cultures Some cultures 33 Monday, January 28, 13
  • 46. The Hidden Majority Richness estimates Hugenholtz 2002 Bohannan and Hughes 2003 34 Monday, January 28, 13
  • 47. Human microbiome example 35 Monday, January 28, 13
  • 48. A: Human biogeography Censored Censored 36 Monday, January 28, 13
  • 49. A: Human biogeography 37 Monday, January 28, 13
  • 50. A: Human biogeography Naris (R) Forehead Hair External nose Naris (L) Ext. auditory Ext. auditory canal (R) Lat. pinna (R) Lat. pinna (L) canal (L) Axilla (R) Dorsal tongue Oral cavity Axilla (L) Volar Volar forearm (R) Palm (R) Palm (L) forearm (L) Palmar index Palmar index finger (R) Gut Umbilicus finger (L) Popliteal Plantar Glans Labia Plantar Popliteal fossa (R) foot (R) penis minora foot (L) fossa (L) Acinetobacter Actinomycetales Actinomycineae Alistipes Anaerococcus Bacteroidales Bacteroides Bifidobacteriales Branhamella Campylobacter Capnocytophaga Carnobacteriaceae1 Carnobacteriaceae2 Clostridiales Coriobacterineae Corynebacterineae Faecalibacterium Finegoldia Fusobacterium Gemella Lachnospiraceae Lachnospiraceae (inc. sed.) Lactobacillus Leptotrichia Micrococcineae Neisseria Oribacterium Parabacteroides Pasteurella Pasteurellaceae 38 Peptoniphilus Prevotella Prevotellaceae Propionibacterineae Ruminococcaceae Staphylococcus Streptococcus Veillonella Other Monday, January 28, 13
  • 51. Vertebrate Microbiomes 100 Bacteroidetes (red) 80 16S ribosomal RNA sequences (%) 60 40 20 ANALYSIS Firmicutes (blue) 0 r s ts n r e t t ate ured rm en um a ate t fac gu gu d wcultthwo im rh t w men surrmite ate xe Mi aliner ea r sed he Sal di r ate Te br ter Ot se -w Ve rte Worlds within worlds: evolution of n-s ts o hw a ic o r Sal t No sec In rf res n ox the vertebrate gut microbiota so e, a S oil r fac Ruth E. Ley*‡¶, Catherine A. Lozupone*§¶, Micah Hamady||, Rob Knight § and bsu Jeffrey I. Gordon* Su Abstract | In this Analysis we use published 16S ribosomal RNA gene sequences to c Figure 3 | Relative abundance of phyla in samples. Bar graph showing the proportion of sequences from eachassemblages that are associated withrange of environments. The comp the bacterial sample and free-living microbial communities that span a humans and other mammals, me that could be classified at the phylum level. The colour codes for the dominant Firmicutes and Bacteroidetes phyla are microbiota is influenced by diet, host morphology and phyloge of the vertebrate gut shown. Nature Reviews | Microbiology For a complete description of the colour codes see Supplementary information S2 (figure). ‘Other humans’ refersvertebrate gut microbiotacommunity is typical of an omnivorous prima in this respect the human gut bacterial However, the to body is different from free-living communities th habitats other than the gut; for example, the mouth, ear, skin, vagina and vulva (see Supplementary information S1 (table)). habitats. We propose that the recently initiated not associated with animal body international Human Microbiome Project should strive to include a broad represent humans, as well as other mammalian and environmental samples, as comparative an of microbiotas and their microbiomes are a powerful way to explore the evolutionar history of the biosphere. 39 Genera that cross the divide. Another way to visualize family of the gammaproteobacteria class. This fam- Monday, January 28, 13 the vertebrate gut–environment dichotomy is by using a ily contained OTUs from both theDiverse microorganisms and microbial communities are vertebrate gut and Microbiota host energy metabolism8–11. Host responses to
  • 53. The Built Environment Microbial Biogeography of Public Restroom Surfaces Gilberto E. Flores1, Scott T. Bates1, Dan Knights2, Christian L. Lauber1, Jesse Stombaugh3, Rob Knight3,4, Noah Fierer1,5* Bacteria of Public Restrooms 1 Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder, Colorado, United States of America, 2 Department of Computer Science, University of Colorado, Boulder, Colorado, United States of America, 3 Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, United States of America, 4 Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, United States of America, 5 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado, United States of America Abstract We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of The ISME Journal (2012), 1–11 the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla: & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those www.nature.com/ismej found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with Figure 3. Cartoon toilet surfaces, gut-associated taxa were more prevalent, suggesting fecal contamination of Light blue indicates low hands. On illustrations of the relative abundance of discriminating taxa on public restroom surfaces. these surfaces. Floor ORIGINAL ARTICLE abundance while were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated surfaces dark blue indicates high abundance of taxa. (A) Although skin-associated taxa (Propionibacteriaceae, Corynebacteriaceae, Staphylococcaceae especially the Propionibacteriaceae, on all surfaces, they were relatively more abundant on surfaces routinely touched with bacteria, and Streptococcaceae) were abundant dominated surfaces routinely touched with our hands. Certain taxa were more Architectural design influences the diversity and hands. (B) Gut-associated taxa (Clostridiales, Clostridiales group XI,vagina-associated Lactobacillaceae were widely Bacteroidaceae)in female common in female than in male restrooms as Ruminococcaceae, Lachnospiraceae, Prevotellaceae and distributed were most abundant on toilet surfaces. from urine contamination. Use of the SourceTracker algorithm confirmed Nocardioidaceae) taxonomic restrooms, likely (C) Although soil-associated taxa (Rhodobacteraceae, Rhizobiales, Microbacteriaceae and many of our were in low abundance on all restroom surfaces, they were relatively more abundant on the floor of the surfaces. Overall, theseFigure not drawn to scale. observations as human skin was the primary source of bacteria on restroom restrooms we surveyed. results demonstrate that structure of the built environment microbiome doi:10.1371/journal.pone.0028132.g003 restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clear linkages between communities on or in different body sites and those communities found on restroom surfaces.Bacteria of P More 1 1 1,2 Steven W Kembel , Evan Jones , Jeff Kline , Dale Northcutt , Jason Stenson , 1,2 1,2 the stallgenerally,were likely dispersed manuallypublicwomen used as we Results of human-associated microbes are commonly found in), they this work is relevant to the after health field show that SourceTracker analysis support the taxonomic 1 on restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching the toilet. Coupling these observations with those of the patterns highlighted above, indicating that human skin was the time, the M Womack , Brendan JM 100 Ann Bohannan1, G Z Brown1,2 and Jessica L Green1,3 1 SOURCES distribution of gut-associated bacteria demonstrate that we use use high-throughput analyses of bacterial communities to determine of surfaces. Furthermore, we indicate that routine can Bathroom biogeography. By on indoor surfaces, an approach of primary source of bacteria on all public restroom surfaces Biology and the Built Environment Center, Institute of Ecology and Evolution, Department of sources the dispersal of urine- and fecal-associated bacteria of bacteria whichexamined, while the track pathogen transmission and test the could be used to human gut was an important source on or un to take Biology, University of Oregon, Eugene, OR, USA; 2Energy Studies in Buildings Laboratory, Soil swabbing toilets results in restroom. While these results are not unexpected, different surfaces in throughout the of hygiene practices. efficacy around the toilet, and urine was an important source in women’s of outside 80of Oregon, Eugene, OR, USA and 3Santa Fe Institute, Water Average contribution (%) Department of Architecture, University public restrooms,highlight the importance of hand-hygiene when using they do researchers restrooms (Figure 4, Table S4). Contrary to expectations (see Mouth om plants Fe, NM, USA Santa determined thatCitation: Floressince these Knights D, Lauber CL, Stombaugh J, et al. (2011)above), soil was not identified by the Surfaces. PLoS ONEalgorithm as public microbes vary in ST, surfaces could also be potential restrooms GE, Bates Microbial Biogeography of Public Restroom SourceTracker 6(11): e28132. Urine doi:10.1371/journal.pone.0028132 ours after 60 where they come from depend- vehicles for the transmission of human pathogens. Unfortunately, being a major source of bacteria on any of the surfaces, including Gut Editor: Mark R. Liles, Auburn University, United States of America ere shut ing on the previous (chart).have documented that college students (who are November 23, 2011 4). Although the floor samples contained family-level surface studies floors (Figure Buildings are complex ecosystems that house trillions of microorganisms interactingSkin each with likely Received September 12, 2011; of the studied restrooms) Published the most frequent users Accepted November 1, 2011; are not taxa that are common in soil, the SourceTracker algorithm 40 ortion of other, with humans and with their environment. Understanding the ecological and evolutionary Copyright: diligent of hand-washers open-access article distributed under the terms of the Creative Commons Attribution License, sources, like always the most ß 2011 Flores et al. This is an[42,43]. probably underestimates the relative importance of which permits processes that determine the diversity and composition of the built environment microbiome—the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. e human community of microorganisms that live indoors—is important for understanding the relationship pant in indoor microbial 20 Funding: This work was supported with funding from the Alfred P. Sloan Foundation and their Indoor Environment program, and in part by the National ck to pre- between building design, biodiversity and human health. In this study, we used high-throughputecology research,ofPeccia the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or Institutes Health and sequencing of the bacterial 16S rRNA gene to quantify relationships between building attributes and airborne bacterial communities at 0 health-care facility. We quantified airborne bacterial community a thinks that the fieldthe manuscript. preparation of has wh i c h structure and environmental conditions in patient rooms exposed to mechanical or window yet to gel. And the Sloan The authors have declared that no competing interests exist. Competing Interests: Do in t in So han t dis les ile oile r hh t To ndle Si or or ou et ll ou lus t sea e * E-mail: noah.fierer@colorado.edu ns lo flo ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial communities was or all d 26 Janu- lower indoors than outdoors, and mechanically ventilated rooms contained less diverse microbialFoundation’s Olsiewski or tf a Do pe St Fa Sta nk ile T Journal, communities than did window-ventilated rooms. Bacterial communities in indoor environments shares some of his con- uc ap tf contained many taxa that are absent or rare outdoors, including taxa closely related to potential Introduction communities and revealed a greater diversity of bacteria on hanically cern. “Everybody’s gen- To human pathogens. Building attributes, specifically the source of ventilation air, airflow rates, relative indoor surfaces than captured using cultivation-based techniques had lower humidity and temperature, were correlated with the diversity and composition of indoor bacterial erating vastMore than ever, individuals across the globe spend a large [10–13]. Most of the organisms identified in these studies are amounts of communities. The relative abundance of bacteria closely related to human pathogens was higher y than ones with openthan outdoors, and higher in rooms withquantify those con- lower relative humidity. looking acrossofdata lives of indooryet relatively littleOf known aboutthat related to human commensals suggesting that the organisms are indoors win- they move around. But to lower airflow rates and data,” she says, but portion their microbial diversity setsindoors, environments. is the studies the Figure 2. Relationship between bacterial communities associated with tenon the surfaces but rather Communities were not actively growing public restroom surfaces. were deposited bility of fresh air translated tributions, Peccia’s team has had to develop diversity suggests that The observed relationship between building design and airborne bacterial can be difficult because groups choose dif- of the unweighted UniFrac distance matrix. Each point represents a single sample. Note that the floor (triangles) and toilet (as rg on February 9, 2012 PCoA have examined microorganisms associated with indoor environ- directly (i.e. touching) or indirectly (e.g. shedding of skin cells) by we can manage indoor environments, altering through building design and operation the community rtions of microbes associ- new methods to collect airborne bacteria and our timeanalytical tools. With Sloan support, of microbial species that potentially colonize the human microbiome during ferent indoors. ments, most have relied upon cultivation-based techniques hands. humans. Despite these efforts, we still have an incomplete form clusters distinct from surfaces touched with to doi:10.1371/journal.pone.0028132.g002 The ISME Journal advance online publication, 26the microbes are much detect organisms residing on a variety of household surfaces [1–5]. January 2012; doi:10.1038/ismej.2011.211 a data archive and integrated analyt- understanding of bacterial communities associated with indoor an body, and consequently, microbial their DNA, as Subject Category: extract population and community ecology though, Not surprisingly, these studies have identified surfaces in kitchens environments because limitations of traditional 16 S rRNA gene pathogens. Although this less abundant in air than on surfaces. Keywords: aeromicrobiology; bacteria; built environment microbiome; community ecology; are in the works. and restrooms as being hot of floorof bacterial contamination. the frequency of sequencing differences in themade replicate samplings ical tools dispersal; high diversity spots communities is likely due to cloning and related techniques have relative abundances of environmental filtering In one recent study, they used air filters hat having natural airflow To foster collaborations between micro- with the bottom aofvariety ofto survive on inanddiversity characterizations of the communities Most surfaces Because several contact pathogenic bacteria are known which would track a in-depth some surfaces (Figure 1B, Table S2). prohibitive. of microorganisms from shoes, sources including soil, which is were clearly more abundant on certain notably surfaces for extended periods of time [6–8], these studies are of With the advent of high-throughput restrooms (Figure 1B). Some sequencing techniques, we Green says answering that to sample airborne particles and microbes biologists, architects, and building scientists,in preventing the spread of human habitat [27,39]. Indeed, restrooms than male known to be a highly-diverse microbial disease. obvious importance 41 can now investigate are family indoor microbial communitiesmost abu often at an Introduction clinical data; she’s hoping in a classroom during 4 days during which human pathogensalso sponsored a symposium widely recognized that the majority of Rhodobacteraceae, depth and the most common, andthe relationship microbiome—includes the foundation and com- However, it is bacteria commonly associated with soil (e.g. now unprecedented found in the vagina of healthy reproductive age w begin to understand mensals interacting with each other and with their microorganisms Rhizobiales, Microbacteriaceae and[9] and thus, the cannot be readily cultivated Nocardioidaceae) were, on average, ital to participate in a study 90% of theirwere present and 4 days during et on the microbiome of the built environment abundant on floor surfaces (Figure 3C, Table S2). and are relatively less abundant in male urine Humans spend up to students lives indoors environment (Eames al., 2009). There have been between humans, microbes and the built environment. overall diversitymore microorganisms associated with indoor of Monday,etJanuary which 13 the was vacant. They measured at the 2011 Indoor Air conference in Austin, largelysome of the Recentflush handles harbored In order to begin to of female urine samples collected as part 28, the dence of hospital-acquired Consequently,roomway we few attempts to comprehensively survey the built (Klepeis al., 2001). design and operate the indoor environment has a Interestingly, unknown. toilet use of cultiva- environments remains bacterial analysis comprehensively describe the microbial communities similar to those found on the floor diversity of indoor environments, 1A), characterized the bacterial environment microbiome (Rintala et al., 2008; (Figure 2, study [26] (Figure we found that Lactobacillaceae we
  • 54. Era III: Genome Sequencing Era III: Genome Sequencing 42 Monday, January 28, 13
  • 55. 1st Genome Sequence Fleischmann et al. 1995 43 Monday, January 28, 13
  • 56. Genomes Revolutionized Microbiology • Predictions of metabolic processes • Better vaccine and drug design • New insights into mechanisms of evolution • Genomes serve as template for functional studies • New enzymes and materials for engineering and synthetic biology 44 Monday, January 28, 13
  • 58. Metabolic Predictions 46 Monday, January 28, 13
  • 59. Lateral Gene Transfer Perna et al. 2003 47 Monday, January 28, 13
  • 60. Network of Life Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. 48 Monday, January 28, 13
  • 61. Using the Core 49 Monday, January 28, 13
  • 62. Whole Genome Phylogeny Whole genome tree built using AMPHORA by Martin Wu and Dongying Wu 50 Monday, January 28, 13
  • 63. Microbial genomes From http://genomesonline.org 51 Monday, January 28, 13
  • 64. GEBA as example 52 Monday, January 28, 13
  • 65. Phylogenetic Diversity • Phylogenetic diversity poorly sampled • GEBA project at DOE-JGI correcting this 53 Monday, January 28, 13
  • 68. GEBA Lesson 1: rRNA utility in IDing novel genomes From Wu et al. 2009 Nature 462, 1056-1060 56 Monday, January 28, 13
  • 69. GEBA Lesson 2: rRNA Tree is not perfect 16s WGT, 23S Badger et al. 2005 Int J System Evol Microbiol 55: 1021-1026. 57 Monday, January 28, 13
  • 70. GEBA Lesson 3: Phylogenetic sampling improves annotation • Took 56 GEBA genomes and compared results vs. 56 randomly sampled new genomes • Better definition of protein family sequence “patterns” • Greatly improves “comparative” and “evolutionary” based predictions • Conversion of hypothetical into conserved hypotheticals • Linking distantly related members of protein families • Improved non-homology prediction 58 Monday, January 28, 13
  • 71. GEBA Lesson 4 : Metadata Important 59 Monday, January 28, 13
  • 72. GEBA Lesson 5:Improves discovering new genetic diversity 60 Monday, January 28, 13
  • 73. Protein Family Rarefaction Curves • Take data set of multiple complete genomes • Identify all protein families using MCL • Plot # of genomes vs. # of protein families 61 Monday, January 28, 13
  • 74. Wu et al. 2009 Nature 462, 1056-1060 62 Monday, January 28, 13
  • 75. Wu et al. 2009 Nature 462, 1056-1060 62 Monday, January 28, 13
  • 76. Wu et al. 2009 Nature 462, 1056-1060 62 Monday, January 28, 13
  • 77. Wu et al. 2009 Nature 462, 1056-1060 62 Monday, January 28, 13
  • 78. Wu et al. 2009 Nature 462, 1056-1060 62 Monday, January 28, 13
  • 79. Synapomorphies exist Wu et al. 2009 Nature 462, 1056-1060 63 Monday, January 28, 13
  • 80. III: Epidemiology & Forensics 64 Monday, January 28, 13
  • 81. Era IV: Genomes in the environment Era IV: Genomes in the Environment 65 Monday, January 28, 13
  • 82. Marine Microbe Background • rRNA PCR studies of marine microbes have been extensive • Comparative analysis had revealed many lineages, some very novel, some less so, that were dominant in many, if not all, open ocean samples • Lineages given names based on specific clones: e.g., SAR11, SAR86, etc 66 Monday, January 28, 13
  • 83. %&'())%#*+,-###./*/!0##*1""#23##4(56#,! Molecular diversity and ecology of microbial plankton Stephen J. Giovannoni1 & Ulrich Stingl1 The history of microbial evolution in the oceans is probably as old as the history of life itself. In contrast to terrestrial ecosystems, microorganisms are the main form of biomass in the oceans, and form some of the INSIGHT REVIEW largest populations on the planet. Theory predicts that selection should act437|15 September 2005 NATURE|Vol more efficiently in large populations. But whether microbial plankton populations harbour organisms that are models of adaptive sophistication remains to be seen. Genome sequence data are piling up, but most of the key microbial plankton clades have no cultivated representatives, and information about their ecological activities is sparse. Archaea Certain characteristics of the ocean environment — the prevailing cultivation of key organisms, metagenomics and ongoing biogeo- low-nutrient state of the ocean surface, in particular — mean it is chemical studies. It seems very likely that the biology of the dominant Crenarchaeota Euryarchaeota sometimes regarded as an extreme ecosystem. Fixed forms of nitrogen, microbial plankton groups will be unravelled in the years ahead. Group I Archaea Group II Archaea phosphorus and iron are often at very low or undetectable levels in the Here we review current knowledge about marine bacterial and ocean’s circulatory gyres, which occur in about 70% of the oceans1. archaeal diversity, as inferred from phylogenies of genes recovered Group III Archaea Photosynthesis is the main source of metabolic energy and the basis of from the ocean water column, and consider the implications of micro- Group IV Archaea the food chain; ocean phytoplankton account for nearly 50% of global bial diversity for understanding the ecology of the oceans. Although carbon fixation, and half of the carbon fixed into organic matter is we leave protists out of the discussion, many of the same issues apply rapidly respired by heterotrophic microorganisms. Most cells are freely to them. Some of the studies we refer to extend to the abyssal ocean, suspended in the mainly oxic water column, but some attach to aggre- but we focus principally on the surface layer (0–300 m) — the region gates. In general, these cells survive either by photosynthesizing or by of highest biological activity. oxidizing dissolved organic matter (DOM) or inorganic compounds, α-Proteobacteria as an electron acceptor. using oxygen Phylogenetic diversity in the ocean 5 * SAR11 - theMicrobial cell concentrations are thymidine uptake into microbial Pelagibacterlayer (0–300 m) — typically about 10 cells ml in Small-subunit ribosomal (RNA) genes have become universal phylo- ǁ1 ocean surface ubique genetic markers and are the main criteria by which microbial plank- * Roseobacterindicates average growth rates of about 0.15 divisions per day DNA clade ton groups are identified and named9. Most of the marine microbial Chloroflexi OCS116 (ref. 2). Efficient nutrient recycling, in which there is intense competi- groups were first identified by sequencing rRNA genes cloned from tion for scarce resources, sustains this growth, with predation by seawater10–14, and remain uncultured today. Soon after the first reports SAR202 ß-Proteobacteria viruses and protozoa keeping populations in check and driving high came in, it became apparent that less than 20 microbial clades * OM43 turnover rates3. Despite this competition, steady-state dissolved accounted for most of the genes recovered15. Figure 1 is a schematic organic carbon (DOC) concentrations are many times higher than illustration of the phylogeny of these major plankton clades. The taxon µ-Proteobacteria carbon sequestered in living microbial biomass4. However, the average names marked with asterisks represent groups for which cultured iso- Planctobacteria SAR86 age of the DOC pool in the deep ocean, of about 5,000 years5 (deter- lates are available. * OMG Clade by isotopic dating), suggests thatismuch of the DOM is refrac- mined tory to degradation. Although DOM a huge resource, rivalling The recent large-scale shotgun sequencing of seawater DNA is pro- viding much higher resolution 16S rRNA gene phylogenies and bio- * Vibrionaeceae CO2 as a carbon pool6 , chemists have been thwarted by atmospheric geographical distributions for marine microbial plankton. Although * Pseudoalteromonas DOM and have characterized it only in broad terms7. the complexity of the main purpose of Venter’s Sorcerer II expedition is to gather whole- Fibrobacter * Marinomonas The paragraphs above capture prominent features of the ocean genome shotgun sequence (WGS) data from planktonic microorgan- SAR406 * Halomonadacae but leave out the complex patternsand diversification of environment, of physical, chemical isms16, thousands of water-column rRNA genes are part of the and biological variation that drive the evolution by-catch. The first set of collections, from the Sargasso Sea, have Bacteroidetes * Colwellia microorganisms. For example, members of the genus Vibrio — which Figure 1 | Schematic illustration of the phylogeny of yielded 1,184 16S rRNA gene fragments. These data are shown in * Oceanospirillum of the most common planktonic bacteria that can be iso- include some Fig. 2, organized by clade structure. Such data are a rich scientific Marine Actinobacteria the major plankton clades. Black letters indicate lated on nutrient agar plates — readily grow anaerobically by fermen- resource for two reasons. First, they are not tainted by polymerase δ-Proteobacterialife cycles of some Vibrio species have been shown to tation. The microbial groups that seem to be ubiquitous in include anoxic stages in association with animal hosts, but the broad chain reaction (PCR) artefacts; PCR artefacts rarely interfere with the correct placement of genes in phylogenetic categories, but they are a Cyanobacteria seawater. Gold indicates groups found in the photic picture of their ecology in the oceans has barely been characterized8. major problem for reconstructing evolutionary patterns at the popu- Lentisphaerae * Marine Cluster A The story is similar for most of the microbial groups described below: lation level17. Second, the enormous number of genes provided by the * Lentisphaera araneosa (Synechococcus) zone. Blue indicates groups confined to the the phylogenetic map is detailed, but the ecological panorama is thinly Sorcerer II expedition is revealing the distribution patterns and abun- sketched. New information is rapidly flowing into the field from the dance of microbial groups that compose only a small fraction of the * Prochlorococcus sp. mesopelagic and surface waters during polar winters. Department of Microbiology, Oregon State University, Corvallis, Oregon 97331,indicates microbial groups associated with Green USA. Bacteria 1 coastal ocean ecosystems. ©2005 Nature Publishing Group 343 67 community. As discussed below, some opportunistic strains that cyanobacteria. As obligate phototrophs, these cyanobacteria are con- ! ! © !""# Nature Publishing Group! Monday, January 28, 13
  • 84. facts17. They concluded that most sequence variation was clustered many microbia tions in the wat best example. T 35 entiated by the 30 adapted (high-b % of 16S rRNA sequences Phylogenetic e 25 gests that the h cally distinct lin 20 Cluster A Syne of which can b 15 characteristics urobilin)37,38. S 10 ample support teristics that aff 5 SS120 has a mu 0 ammonium an ) II Ib n e r) tes ia) ria nas ia) era de xi) extreme, Synec ria up kto lad cte + e r e r la e Ia acte gro lan a C ba id cte act mo cte eim r c rofl nitrate, cyanate s i o ro a b o a h e o oup eob sub ytop cter ibr cte eob tino lter eob ein act Chl interesting to n r t bg Pro AR1 op ob 1 h a (F Ba rot Ac doa rot Rh eob 2 ( u - c e 0 6 P δ- rine seu (α- P s Ro R2 0 seem to prospe 1 1 s (γ S Pi rot R4 ( a AR R8 6 e P SA 4 M /P 6 SA whereby nutrie S A a rin α- 32 as R11 S M red AR on SA conditions. Th tu S om c ul ter seasonal specia Un Al lular cyanobact Phylogenetic clade The observa 68 Monday, January 28, 13 diverged into e
  • 85. Delong GENOMIC FRAGMENTS FROM PLANKTONIC MARINE ARCHAEA Lab 593 ments isolated from fosmid clones with various restriction endonucle- 10 kb, the F-factor-based vector the fosmid subfragments. Partial of restriction enzyme to 1 ⇥g of mixture. The reaction mixture was removed at 10, 40, and 60 min. dding 1 ⇥l of 0.5 M EDTA to the e. The partially digested DNA was s described above except using a 1- he sizes of the separated fragments n standards. The distances of the d SP6 promoter sites on the excised pmol of T7- or SP6-specific oligo- l) and hybridizing with Southern fosmid and pBAC clones digested probed with labeled T7 and SP6 eled subclones and PCR fragments otgun sequencing described above. e estimates from the partial diges- of the fosmids and their subclones. and DeSoete distance (9) analyses n using GDE 2.2 and Treetool 1.0, (RDP) (23). DeSoete least squares D ownloaded from jb.asm .org at U N IV O F C ng pairwise evolutionary distances, to account for empirical base fre- tained from the RDP, version 4.0 u rRNA sequences were performed the RDP. For distance analyses of lutionary distances were estimated d tree topology was inferred by the n addition and global branch swap- protein sequences, the Phylip pro- addition and ordinary parsimony FIG. 1. Flowchart depicting the construction and screening of an environ- artial sequences reported in Table mental library from a mixed picoplankton sample. MW, molecular weight; the following accession numbers: PFGE, pulsed-field gel electrophoresis. U40243, U40244, and U40245. The and EF2 have been submitted to and U41261. 69 Recombinant fosmids, each containing ca. 40 kb of pico- Monday, January 28, 13
  • 86. Delong Lab J. BACTERIOL. FIG. 4. High-density filter replica of 2,304 fosmid clones containing approx- imately 92 million bp of DNA cloned from the mixed picoplankton community. The filter was probed with the labeled insert from clone 4B7 (dark spot). The lack of other hybridizing clones suggests that contigs of 4B7 are absent from this D ownloaded f portion of the library. Similar experiments with the remainder of the library yielded similar results. 70 Monday, January 28, 13
  • 87. l gene own transducer of light stimuli [for example, the kinetics of its photochemical reaction cy- leDelong Lab ge- Htr (22, 23)]. Although sequence analysis of cle. The transport rhodopsins (bacteriorho- iden- proteorhodopsin shows moderate statistical dopsins and halorhodopsins) are character- roteo- support for a specific relationship with sen- ized by cyclic photochemical reaction se- from opsins ferent. hereas philes r than rmine l, we a coli pres- rotein 3A). nes of popro- m was (Fig. at 520 band- erated odop- nce of dth is 71 rption January 28, 13 Monday,
  • 88. generated D ownloaded from w Delong Lab eorhodop- resence of ndwidth is absorption . The red- nm in the ated Schiff ably to the on was de- s in a cell ward trans- in proteor- nd only in (Fig. 4A). edium was ce of a 10 re carbonyl 19). Illumi- ical poten- right-side- nce of reti- light onset hat proteo- capable of Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the physiolog- 130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodop- sin with archaeal (BR, HR, and SR prefixes) and Neurospora crassa (NOP1 prefix) rhodopsins (16). e activities Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin; containing BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halo- proteorho- bium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp; main to be Neucra, Neurospora crassa. 72 www.sciencemag.org Monday, January 28, 13 SCIENCE VOL 289 15 SEPTEMBER 2000 1903
  • 90. Figure 3. Phylogenetic tree based on the amino acid sequences of 25 archaeal rhodopsins. (a) NJ-tree. The numbers at each node are clustering probabilities generated by bootstrap resampling 1000 times. D1 and D2 represent gene duplication points. The four shaded rectangles indicate the speciation dates when halobacteria speciation occurred at the genus level. (b) ML-tree. Log likelihood value for ML-tree was −6579.02 (best score) and that for topology of the NJ-tree was −6583.43. The stippled bars indicate the 95% confidence limits. Both trees were tentatively rooted at the mid-point of the longest distance, although true root positions are unknown. From Ihara et al. 1999 74 Monday, January 28, 13
  • 91. RESEARCH ARTICLES Fig. 2. Secondary structure of proteo- rhodopsin. Single- letter amino acid codes are used (33), and the numbering is as in bacteriorho- dopsin. Predicted retinal binding pock- et residues are marked in red. 75 Monday, January 28, 13
  • 92. duce in th occu pigm and t ed at tiona sorpt in 0. sorpt botto nated retin ms d deca shift appe term cay o step singl upwa Fig. 3. (A) Proteorhodopsin-expressing E. coli cell suspension (ϩ) compared to control cells (Ϫ), both with all-trans retinal. (B) Absorption spectra of retinal-reconstituted proteorhodopsin in E. coli ampl membranes (17). A time series of spectra is shown for reconstituted proteorhodopsin membranes gene (red) and a negative control (black). Time points for spectra after retinal addition, progressing from with low to high absorbance values, are 10, 20, 30, and 40 min. ms p recov phot prod 76 Monday, January 28, 13 this