UC Davis EVE161 Lecture 14 by @phylogenomics

50 %
50 %
Information about UC Davis EVE161 Lecture 14 by @phylogenomics
Education

Published on February 27, 2014

Author: phylogenomics

Source: slideshare.net

Description

Slides for Lecture 14 in EVE 161 Course by Jonathan Eisen at UC Davis

Lecture 14: EVE 161:
 Microbial Phylogenomics ! Lecture #14: Era IV: Metagenomics ! UC Davis, Winter 2014 Instructor: Jonathan Eisen Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !1

Where we are going and where we have been • Previous lecture: ! 13: Era III: Sequencing • Current Lecture: ! 14: Era IV: Metagenomics ! Next Lecture: ! 15: Era IV: Metagenomics Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !2

Era IV: Genomes in the environment Era IV: Genomes in the Environment Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

Metagenomics History • Term first use in 1998 paper by Handelsman et al. Chem Biol. 1998 Oct;5(10):R245-9. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products • Collective genomes of microbes in the soil termed the “soil metagenome” • Good review is Metagenomics: Application of Genomics to Uncultured Microorganisms by Handelsman Microbiol Mol Biol Rev. 2004 December; 68(4): 669–685. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

17 GPa. J. Geophys. Res. 102, 12252±12263 (1997). 28. Duffy, T. S. & Vaughan, M. T. Elasticity of enstatite and its relationships to crystal structure. J. Geophys. Res. 93, 383±391 (1988). characteristic of cycle seen with E proteorhodopsin Furthermore, Acknowledgements Bacterial Rhodopsin: M. Ferrero and Boudier spectrum of We thank C. Karger for participation in the measurements, and EvidenceF.for a New Type of the and same isosbes for providing the samplesPhototrophy Papua NewSea respectively. The from Baldissero and in the Guinea, Laboratoire de Tectonophysique's EBSD system was funded by the CNRS/INSU, O ded Béjà, et al. dopsin expressed  Universite of Montpellier Scienceproject , 1902 of an archean craton''. This work II, and NSF 289 ``Anatomy (2000); chemical reactio  was supported by the CNRS/INSU programme ``Action Thematique Innovante''. D O I: 10.1126/science.289.5486.1902 generate a red-s Correspondence should be addressed to A.T. to that of recomb (e-mail: deia@dstu.univ-montp2.fr). is for your personal, non-commercial use only. This copy ¯ash-photolysis http://www.sciencemag.org/content/289/5486/1902.full existence of pro retinal molecules waters. The photocyc If you wish to distribute this article to others, you can order high-quality copies for and, after hydro colleagues, clients, or custom ers by clicking here . adding all-trans indeed derive fro Permission to republish or repurpose articles or portions of articles can be obta Papers for Today ................................................................. Proteorhodopsin phototrophy following ocean in the the guidelines here . The following resources related to this article are available  Á Oded Beja*², Elena N. Spudich²³, John L. Spudich³, Marion Leclerc* online at www.scien (this information is current as of May 18, 2010 ): & Edward F. DeLong* 2 orbance (×10–3) Updated information and services, including high-resolution figures, can be found in 1 * version of this article at: Monterey Bay Aquarium Research Institute, Moss Landing, California 95039, 4 USA http://www.sciencem ag.org/cgi/content/full/289/5486/1902 http://www.nature.com/nature/journal/v411/n6839/abs/411786a0.html 0 ³ Department of Microbiology and Molecular Genetics, The University of A list of selected Houston, Texas 77030, the –1 Texas Medical School,additional articles onUSA S cience W eb sites related to this article c Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 ²found authors contributed equally to this work These at:

Papers for Today Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea O ded Béjà, et al. Science 289, 1902 (2000); D O I: 10.1126/science.289.5486.1902 This copy is for your personal, non-commercial use only. http://www.sciencemag.org/content/289/5486/1902.full If you wish to distribute this article to others, you can order high-quality copies for colleagues, clients, or custom ers by clicking here . Permission to republish or repurpose articles or portions of articles can be obta following the guidelines here . The following resources related to this article are available online at www.scien (this information is current as of May 18, 2010 ): Updated information and services, including high-resolution figures, can be found in version of this article at: http://www.sciencem ag.org/cgi/content/full/289/5486/1902 A list of selected additional articles on the S cience W eb sites related to this article c Slides found at: for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

gene own transducer of light stimuli [for example, leSAR86 (22, 23)]. Although sequence analysis of geHtr idenproteorhodopsin shows moderate statistical roteosupport for a specific relationship with senfrom opsins erent. hereas philes r than rmine l, we a coli presrotein 3A). nes of poprom was (Fig. at 520 banderated odopnce of dth is the kinetics of its photochemical reaction cycle. The transport rhodopsins (bacteriorhodopsins and halorhodopsins) are characterized by cyclic photochemical reaction se- Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !7

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 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !8

http://www.nature.com/nature/journal/v345/n6270/abs/345060a0.html Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

© 1990 Nature Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, 0099-2240/91/061707-07$02.00/0 Copyright C) 1991, American Society for Microbiology Vol. 57, No. 6 p. 1707-1713 Phylogenetic Analysis of a Natural Marine Bacterioplankton Population by rRNA Gene Cloning and Sequencing THERESA B. BRITSCHGI AND STEPHEN J. GIOVANNONI* Department of Microbiology, Oregon State University, Corvallis, Oregon 97331 VOL. 57, 1991 Received 5 February 1991/Accepted 25 March 1991 MARINE BACTERIOPLANKTON The identification of the prokaryotic species which constitute marine bacterioplankton communities has been a long-standing problem in marine microbiology. To address this question, we used the polymerase chain CZ reaction to construct and analyze a library of 51 small-subunit (16S) rRNA genes cloned from Sargasso Sea bacterioplankton genomic DNA. Oligonucleotides complementary to conserved regions in the 16S rDNAs of 0._ * C) eubacteria were used to direct the synthesis of polymerase chain reaction products, which were then cloned by u blunt-end ligation into the phagemid vector pBluescript. Restriction fragment length polymorphisms and inmm Vibrio Synechococcus phylogeneticC) groups hybridizations to oligonucleotide probes for the SARll and marine anguillarum 10 0 indicated the presence of at least seven classes of genes. The sequences of five unique rDNAs were determined 0 from completely. In addition to 16S rRNA genes - the marine Synechococcus cluster and the previously identified but uncultivated microbial group, the SARll cluster [S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field, Nature (London) 345:60-63], two new gene classes were observed. Phylogenetic comparisons indicated that these belonged to unknown species of a- and -y-proteobacteria. The data confirm the earlier crescentus conclusion that a majority of planktonic bacteria are new species previously unrecognized by bacteriologists. 1711 Escherichia coli Vibrio harveyi P. 4.. titative hybridization data which demonstrated that a novel lineage belonging to the oa-proteobacteria was abundant in and amplification of rDNA. A surface (2-m) 0 Collection SAR139 the Sargasso Sea; we also identified novel lineages which bacterioplankton population was collected from hydrostaI I related to cultivated marine Synechococwere very closely S (32° 4'N, 64° 23'W) in the Sargasso Sea as cus spp. (6). In a 0.05 of a study 0coworkers examined clonedhot-spring ecosystem, Ward and tion 0.15 (8). The polymerase chain reaction (24)previously 0.10 was used described fragments of 16S rRNA cDNAs to produce double-stranded rDNA from the mixed-microbiand found numerous novel lineages but no matches to genes al-population Sargasso Sea to representative, cultivated species (2, 6, 18, from the genomic DNA prepared from the plankton FIG. 4. Phylogenetic tree cultivated hot-spring bacteria (33). the rDNA clones from showing relationships of These results sugsample. The amplification and were 30, 31, 32, 35, 40). Positionsthat natural ecosystems in general may include spe- containingregions in the 5' primers regionscomplementary to gested of uncertain homology in regions of the 16S rRNA conserved insertions and 3' deletions were omitted from the analysis. cies which are unknown to microbiologists. Jukes and Cantor The amplification corrects for the effects of superimposed mutations. of Evolutionary distances were calculated by the methoda genetic marker, genes. (15), which primers were the universal 1406R Although any gene may be used as and eubacterial 68F sequence (ACGGGCGGTGTGTRC)rooted with the (TNANAC of Bacillus subtilis (38). distance matrix method ATGCAAGTCGAKCG) The phylogenetic tree was determineddistinctaadvantages. The extensive use of (20). The tree was (17). The reaction conditions were rRNA genes offer by 16S rRNAs for studies of microbial systematics and evoluR. Woese as the as follows: Sequence data not referenced were provided by C.data bases, such and R. Rossen. 1 ,ug of template DNA, 10 ,ul of 10x reaction tion has resulted in large computer buffer (500 mM KCI, 100 mM Tris HCI [pH 9.0 at 250C], 15 RNA Data Base Project, which encompass the phylogenetic mM MgCl2, 0.1% gelatin, 1% Triton X-100), 1 U of Taq DNA diversity found within culture collections. rRNA genes are polymerase (Promega Biological Research Products, Madison, Wis.), 1 ,uM (each) primer, 200 ,uM (each) dATP, dCTP, C, 1 Slides for UC Davis EVE161 CoursedGTP, and dTTP in aJonathan EisenatWinter 2014 Taught by 100-,ul total volume; 1 min 94°chain * min at 72° We Polymerase conserved phyla were Corresponding author. in the corresponding min at 600C, 4 C to C C; 30 cycles. note with caution that some classes 880 phototroph (G - G). D ownloa de d from a e m .a sm .org a t U N IV r SARIOO D ownloa de d from a e m .a sm .org a t U N IV O F C ALIF D AV IS on M a y 1 8 , 2 0 1 0 Oceanospirillum linum SAR 92 Caulobacter SAR83 .0 -bacte- Erythrobacter and therefore can be 0 to examine OCh 114 used Q highly conserved Within the past decade, it has become evident that - Hyphomicrobium vulgare distant phylogenetic relationships with accuracy. The presrioplankton contribute significantly to biomass and bioC) 0 (4, 36). Until ence of large numbers geochemical activity in planktonic systems Rhodopseudomonas marina of ribosomes within cells and the 0 regulation of their biosyntheses in proportion to cellular recently, progress in identifying the microbial species which Rochalimaearates make these molecules ideally O) for ecologsuited growth quintana constitute these communities was slow, because the majore counts) ical studies with nucleic acid ity of the organisms present (as measured by direct Agrobacterium tumefaciens probes. Here we describe the partial analysis of a 16S rDNA cannot be recovered in cultures (5, 14, 16). The discovery a library prepared from photic zone Sargasso Sea bacteriodecade ago that unicellular cyanobacteria are widely distribSARI more recent observation plankton using a uted in the open ocean (34) and the -Es The general approacha for cloning eubacterial Sargasso 16S rDNAs. SAR95 Sea, central oceanic gyre, of abundant marine prochlorophytes (3) have underscored typifies the oligotrophic conditions of the open ocean, prothe uncertainty of our knowledge about bacterioplankton SAR1I microbial community adapted to viding an example of a community structure. low-nutrient conditions. extremely - Liverwort CP The results extend our Molecular approaches are now providing genetic markers previous observations of high genetic diversity among for the dominant bacterial species in natural microbial pop*Anacystis nidulans closely related lineages within this microbial community and ulations (6, 21, 33). The immediate objectives of these rDNA provide - genetic markers for two novel eubacterial studies are twofold: (i) to identify species, both known and Prochlorothrix lineages. novel, by reference to sequence data bases; and (ii) to - SAR6 construct species-specific probes which can be used in 0 quantitative ecological studies (7, 29). * SAR7 p.0 Previously, we reported rRNA genetic markers and quanMATERIALS AND METHODS -

JUlY 1991, p. 4371-4378 0021-9193/91/144371-08$02.00/0 Copyright C) 1991, American Society for Microbiology JOURNAL OF BACTERIOLOGY, Vol. 173, No. 14 Analysis of a Marine Picoplankton Community by 16S rRNA Gene Cloning and Sequencing THOMAS M. SCHMIDT,t EDWARD F. DELONG,t NORMAN R. PACE* Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, Indiana 47405 AND Received 7 January 1991/Accepted 13 May 1991 The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 104 recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and D ownloa de d from jb.a sm sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the y subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated Slides for UC Davis EVE161 Course Taught by Jonathan Eisen one group within the organisms. Both of these novel phylogenetic clusters are proteobacteria, Winter 2014

%&'())%#*+,-###./*/!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 NATURE|Vol more efficiently in large largest populations on the planet. Theory predicts that selection should act437|15 September 2005 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. INSIGHT REVIEW Archaea Crenarchaeota Group I Archaea Euryarchaeota Group II Archaea Group III Archaea Group IV Archaea Chloroflexi SAR202 Planctobacteria Fibrobacter SAR406 Bacteroidetes Marine Actinobacteria Lentisphaerae * Lentisphaera araneosa Bacteria Cyanobacteria * Marine Cluster A (Synechococcus) * Prochlorococcus sp. Certain characteristics of the ocean environment — the prevailing low-nutrient state of the ocean surface, in particular — mean it is sometimes regarded as an extreme ecosystem. Fixed forms of nitrogen, phosphorus and iron are often at very low or undetectable levels in the ocean’s circulatory gyres, which occur in about 70% of the oceans1. Photosynthesis is the main source of metabolic energy and the basis of the food chain; ocean phytoplankton account for nearly 50% of global carbon fixation, and half of the carbon fixed into organic matter is rapidly respired by heterotrophic microorganisms. Most cells are freely suspended in the mainly oxic water column, but some attach to aggregates. In general, these cells survive either by photosynthesizing or by oxidizing dissolved organic matter (DOM) or inorganic compounds, using oxygen as an electron acceptor. α-Proteobacteria cell concentrations are typically about 105 cells mlǁ1 in Microbial * SAR11 - the ocean surface layer (0–300 m) — thymidine uptake into microbial Pelagibacter ubique * Roseobacterindicates average growth rates of about 0.15 divisions per day DNA clade OCS116 (ref. 2). Efficient nutrient recycling, in which there is intense competition for scarce resources, sustains this growth, with predation by ß-Proteobacteria viruses and protozoa keeping populations in check and driving high * OM43 turnover rates3. Despite this competition, steady-state dissolved organic carbon (DOC) concentrations are many times higher than carbon sequestered in living microbial biomass4. However, the average µ-Proteobacteria 5 SAR86 age of the DOC pool in the deep ocean, of about 5,000 years (determined by isotopic dating), suggests that much of the DOM is refrac* OMG Cladeto degradation. Although DOM is a huge resource, rivalling tory * Vibrionaeceae CO2 as a carbon pool6 , chemists have been thwarted by atmospheric the complexity of * Pseudoalteromonas DOM and have characterized it only in broad terms7. The paragraphs above capture prominent features of the ocean * Marinomonas environment, of physical, chemical * Halomonadacae but leave out the complex patternsand diversification of and biological variation that drive the evolution * Colwellia microorganisms. For example, members of the genus Vibrio — which include some * Oceanospirillum of the most common planktonic bacteria that can be isolated on nutrient agar plates — readily grow anaerobically by fermenδ-Proteobacterialife cycles of some Vibrio species have been shown to tation. The include anoxic stages in association with animal hosts, but the broad picture of their ecology in the oceans has barely been characterized8. The story is similar for most of the microbial groups described below: the phylogenetic map is detailed, but the ecological panorama is thinly sketched. New information is rapidly flowing into the field from the cultivation of key organisms, metagenomics and ongoing biogeochemical studies. It seems very likely that the biology of the dominant microbial plankton groups will be unravelled in the years ahead. Here we review current knowledge about marine bacterial and archaeal diversity, as inferred from phylogenies of genes recovered from the ocean water column, and consider the implications of microbial diversity for understanding the ecology of the oceans. Although we leave protists out of the discussion, many of the same issues apply to them. Some of the studies we refer to extend to the abyssal ocean, but we focus principally on the surface layer (0–300 m) — the region of highest biological activity. Phylogenetic diversity in the ocean Small-subunit ribosomal (RNA) genes have become universal phylogenetic markers and are the main criteria by which microbial plankton groups are identified and named9. Most of the marine microbial groups were first identified by sequencing rRNA genes cloned from seawater10–14, and remain uncultured today. Soon after the first reports came in, it became apparent that less than 20 microbial clades accounted for most of the genes recovered15. Figure 1 is a schematic illustration of the phylogeny of these major plankton clades. The taxon names marked with asterisks represent groups for which cultured isolates are available. The recent large-scale shotgun sequencing of seawater DNA is providing much higher resolution 16S rRNA gene phylogenies and biogeographical distributions for marine microbial plankton. Although the main purpose of Venter’s Sorcerer II expedition is to gather wholegenome shotgun sequence (WGS) data from planktonic microorganisms16, thousands of water-column rRNA genes are part of the by-catch. The first set of collections, from the Sargasso Sea, have yielded 1,184 16S rRNA gene fragments. These data are shown in Fig. 2, organized by clade structure. Such data are a rich scientific resource for two reasons. First, they are not tainted by polymerase chain reaction (PCR) artefacts; PCR artefacts rarely interfere with the correct placement of genes in phylogenetic categories, but they are a major problem for reconstructing evolutionary patterns at the population level17. Second, the enormous number of genes provided by the Sorcerer II expedition is revealing the distribution patterns and abundance of microbial groups that compose only a small fraction of the Figure 1 | Schematic illustration of the phylogeny of the major plankton clades. Black letters indicate microbial groups that seem to be ubiquitous in seawater. Gold indicates groups found in the photic zone. Blue indicates groups confined to the mesopelagic and surface waters during polar winters. Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA. Green indicates microbial groups associated with 343 coastal ocean ecosystems. ©2005 Nature Publishing Group 1 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !14

libraries prepared using PCR methods that reduced sequence artefacts17. They concluded that most sequence variation was clustered 35 % of 16S rRNA sequences 30 25 20 15 10 5 0 ) e n II r) tes ia) ria nas ia) era de xi) ria up kto lad cte r r e e la e id cte act mo cte eim r c rofl Ia acte gro lan a C ba o s a a e o i b r h o o up eob sub ytop cter ibr cte eob tino lter eob ein act Chl o b (F Ba rot Ac doa rot Rh eo 2 ( 11 ph ba gr rot s 0 ub γ-P SAR ico teo 06 u e -P -P 4 Ro R2 1s ( P ro (δ arin Pse (α 1 R e 6 6 4 M / SA in α-P SA AR R 8 r as R11 32 S A a R on SA S M red SA m tu ro ul c lte A Un + Ib Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 Phylogenetic clade The ecoty many micro tions in the w best exampl entiated by adapted (hig Phylogeneti gests that th cally distinct Cluster A S of which ca characterist urobilin)37,3 ample suppo teristics that SS120 has a ammonium extreme, Syn nitrate, cyan interesting t seem to pro whereby nu conditions. seasonal spe lular cyanob !15 The obse

DeLong Lab • • Studying Sar86 and other marine plankton Note - published one of first genomic studies of uncultured microbes - in 1996 JOURNAL OF BACTERIOLOGY, Feb. 1996, p. 591–599 0021-9193/96/$04.00 0 Copyright 1996, American Society for Microbiology Vol. 178, No. 3 Characterization of Uncultivated Prokaryotes: Isolation and Analysis of a 40-Kilobase-Pair Genome Fragment from a Planktonic Marine Archaeon JEFFEREY L. STEIN,1* TERENCE L. MARSH,2 KE YING WU,3 HIROAKI SHIZUYA,4 3 AND EDWARD F. DELONG * Recombinant BioCatalysis, Inc., La Jolla, California 920371; Microbiology Department, University of Illinois, Urbana, Illinois 618012; Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, California 931063; and Division of Biology, California Institute of Technology, Pasadena, California 911254 Received 14 July 1995/Accepted 14 November 1995 D ownloa de d from One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated its close relationship to that of previously Slides for UC Davis EVE161 Course coherent group rooted deeply within the Crenarchaeota described planktonic archaea, which form a Taught by Jonathan Eisen Winter 2014

Delong Lab GENOMIC FRAGMENTS FROM PLANKTONIC MARINE ARCHAEA 593 ments isolated from fosmid clones with various restriction endonucle10 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 1he sizes of the separated fragments n standards. The distances of the d SP6 promoter sites on the excised pmol of T7- or SP6-specific oligol) and hybridizing with Southern artial sequences reported in Table the following accession numbers: U40243, U40244, and U40245. The and EF2 have been submitted to and U41261. FIG. 1. Flowchart depicting the construction and screening of an environmental library from a mixed picoplankton sample. MW, molecular weight; PFGE, pulsed-field gel electrophoresis. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 D ownloa de d from jb.a sm .org a t U N IV O 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 digesof the fosmids and their subclones. and DeSoete distance (9) analyses n using GDE 2.2 and Treetool 1.0, (RDP) (23). DeSoete least squares ng pairwise evolutionary distances, to account for empirical base fretained from the RDP, version 4.0 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 swapprotein sequences, the Phylip proaddition and ordinary parsimony !17

Delong Lab J. BACTERIOL. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 D ownloa de FIG. 4. High-density filter replica of 2,304 fosmid clones containing approximately 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 portion of the library. Similar experiments with the remainder of the library yielded similar results. !18

on was des in a cell ward transin proteornd only in (Fig. 4A). edium was ce of a 10 re carbonyl 19). Illumiical potenright-sidence of retilight onset hat proteocapable of physiologe activities containing proteorhomain to be has Proteorhodopsin in Its Genome D ownloa de d from w generated eorhodopSAR86 resence of ndwidth is absorption . The rednm in the ated Schiff ably to the Fig. 1. (A) Phylogenetic tree of bacterial 16S rRNA gene sequences, including that encoded on the 130-kb bacterioplankton BAC clone (EBAC31A08) (16). (B) Phylogenetic analysis of proteorhodopsin with archaeal (BR, HR, and SR prefixes) and Neurospora crassa (NOP1 prefix) rhodopsins (16). Nomenclature: Name_Species.abbreviation_Genbank.gi (HR, halorhodopsin; SR, sensory rhodopsin; BR, bacteriorhodopsin). Halsod, Halorubrum sodomense; Halhal, Halobacterium salinarum (halobium); Halval, Haloarcula vallismortis; Natpha, Natronomonas pharaonis; Halsp, Halobacterium sp; Neucra, Neurospora crassa. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !19

Bacteriorhodopsin and its relatives 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 Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !20

Halophiles … Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !21

Proteorhodopsin Predicted Secondary Structure RESEARCH ARTICLES Fig. 2. Secondary structure of proteorhodopsin. Singleletter amino acid codes are used (33), and the numbering is as in bacteriorhodopsin. Predicted retinal binding pocket residues are marked in red. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !22

Proteorhodopsin in E. coli 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 membranes (17). A time series of spectra is shown for reconstituted proteorhodopsin membranes (red) and a negative control (black). Time points for spectra after retinal addition, progressing from low to high absorbance values, are 10, 20, 30, and 40 min. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 duce in th occu pigm and ed a tiona sorp in 0. sorp botto nated retin ms d deca shift appe term cay step singl upw amp gene with ms p reco phot !23 prod

both with all-trans retinal. (B) Absorption spectra of retinal-reconstituted proteorhodopsin in E. coli membranes (17). A time series of spectra is shown for reconstituted proteorhodopsin membranes (red) and a negative control (black). Time points for spectra after retinal addition, progressing from low to high absorbance values, are 10, 20, 30, and 40 min. Proteorhodopsin function Fig. 4. (A) Light-driven transport of protons by a proteorhodopsin-expressing E. coli cell suspension. The beginning and cessation of illumination (with yellow light Ͼ485 nm) is indicated by arrows labeled ON and OFF, respectively. The cells were suspended in 10 mM NaCl, 10 mM MgSO4⅐7H2O, and 100 ␮M CaCl2. (B) Transport of 3Hϩ-labeled tetraphenylphosphonium ([3Hϩ]TPP) in E. coli right-side-out vesicles containing expressed proteorhodopsin, reconstituted with (squares) or without (circles) 10 ␮M retinal in the presence of light (open symbols) or in the dark (solid symbols) (20). 1904 geneity in with 87% o ms photocy recovery. A photocycle produces a b this alternat well as a tw component, the total am with a 45amplitude). cycle rate, w teristic of strong evid tions as a tr rhodopsin. Implicat harbor the tributed in bacteria hav ture-indepen oceanic reg Oceans, as (8, 25–29). tribution, pr this ␥-prote 15 SEPTEMBER 2000 VOL 289 SCIENCE www.sciencemag.org Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

Figure 5 Laser flash-induced absorbance changes in suspensions of E. coli membranes containing proteorhodopsin. A 532-nm pulse (6 ns duration, 40 mJ) was delivered at time 0, and absorption changes were monitored at various wavelengths in the visible range in a lab-constructed flash photolysis system as described (34). Sixtyfour transients were collected for each wavelength. (A) Transients at the three wavelengths exhibiting maximal amplitudes. (B) Absorption difference spectra calculated from amplitudes at 0.5 ms (blue) and between 0.5 ms and 5.0 ms (red). http://www.sciencemag.org/content/289/5486/1902/F5.expansion.html Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

Correspondence should be addressed to A.T. Papers for(e-mail: deia@dstu.univ-montp2.fr). Today ................................................................. Proteorhodopsin phototrophy in the ocean to that of recomb ¯ash-photolysis existence of pro retinal molecules waters. The photocyc and, after hydro adding all-trans indeed derive fro  Á Oded Beja*², Elena N. Spudich²³, John L. Spudich³, Marion Leclerc* & Edward F. DeLong* Δ Absorbance (×10–3) 2 1 * Monterey Bay Aquarium Research Institute, Moss Landing, California 95039, USA http://www.nature.com/nature/journal/v411/n6839/abs/411786a0.html 0 ³ Department of Microbiology and Molecular Genetics, The University of –1 Texas Medical School, Houston, Texas 77030, USA ² These authors contributed equally to this work –2 Proteorhodopsin1, a retinal-containing integral membrane protein that functions as a light-driven proton pump, was discovered in the genome of an uncultivated marine bacterium; however, the prevalence, expression and genetic variability of this protein in native marine microbial populations remain unknown. Here we report that photoactive proteorhodopsin is present in oceanic surface waters. We also provide evidence of an extensive family of Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 –3 –4 3 2 0–3) .............................................................................................................................................. 4 0

indeed derive from retinylidene pigmentation. We conclude that hn L. Spudich³, Marion Leclerc* ular Genetics, The University of 30, USA work ................................................................... ining integral membrane pron proton pump, was discovered marine bacterium; however, the c variability of this protein in ons remain unknown. Here we odopsin is present in oceanic idence of an extensive family of psin variants. The protein pign family seem to be spectrally orbing light at different waveavailable in the environment. oteorhodopsin-based phototroic microbial process. taining membrane protein that n pump, was discovered nearly Halobacterium salinarum2. The on contain a high concentration acked in an ordered two-dimention of light, bacteriorhodopsin al shifts (a photocycle), causing a the membrane. The resulting al drives ATP synthesis, through 1 580 nm 400 nm 0 –1 –2 500 nm –3 –4 3 2 ∆ Absorbance (×10–3) ute, Moss Landing, California 95039, ∆ Absorbance (×10–3) 2 0 20 40 Time (ms) 60 80 Proteorhodopsin in E. coli membranes Monterey Bay environmental membranes 1 0 –1 –2 –3 400 450 500 550 600 650 700 Wavelength (nm) Figure 1 Laser ¯ash-induced absorbance changes in suspensions of membranes prepared from the prokaryotic fraction of Monterey Bay surface waters. Top, membrane absorption was monitored at the indicated wavelengths and the ¯ash was at time 0 at 532 nm. Bottom, absorption difference spectrum at 5 ms after the ¯ash for the environmental sample (black) and for E. coli-expressed proteorhodopsin (red). Slides Magazines Ltd NATURE | VOL 411 | 14 JUNE 2001 | www.nature.com © 2001 Macmillan for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

strongly suggests that this protein has a signi®cant role in the M MB M M BA MB M Untreated membranes 10 –3 AU Laser flash B M M MB 10 –1 s PAL E HOT PAL Hydroxylamine-treated membranes PAL E PAL PAL HO PAL PAL HOT PAL B PAL B HOT Retinal-reconstituted membranes 0.01 Figure 2 Laser ¯ash-induced transients at 500 nm of a Monterey Bay bacterioplankton membrane preparation. Top, before addition of hydroxylamine; middle, after 0.2 M hydroxylamine treatment at pH 7.0, 18 8C, with 500-nm illumination for 30 min; bottom, after centrifuging twice with resuspension in 100 mM phosphate buffer, pH 7.0, followed by addition of 5 mM all-trans retinal and incubation for 1 h. Figure 3 Phylogenetic analysis of the inferred proteorhodopsin genes. Distance analysis of 22 by neighbour-joining using the PaupSearch pr 10.0 (Genetics Computer Group; Madison, Wis was used as an outgroup, and is not shown. Sc per site. Bold names indicate the proteorhodop this study. SlidesNATURE | VOL 411 | EVE161 Course Taught by Jonathan Eisen Winter 2014 for UC Davis 14 JUNE 2001 | www.nature.com © 2001 Macmillan Magazines Ltd

nt to produce substantial amounts of efore, the high density of proteorhorane indicated by our calculations otein has a signi®cant role in the 10 –1 s PAL E6 HOT 75m3 PAL B1 e-treated membranes PAL E7 PAL B2 PAL B8 HOT 75m1 PAL B7 PAL E1 HOT 75m4 PAL B5 PAL B6 HOT 75m8 stituted membranes 0.01 at 500 nm of a Monterey Bay bacterioplankton tion of hydroxylamine; middle, after 0.2 M C, with 500-nm illumination for 30 min; bottom, n in 100 mM phosphate buffer, pH 7.0, followed incubation for 1 h. nature.com Antarctica and deep HOT 10 –3 AU ed membranes Monterey Bay and shallow HOT MB 0m2 MB 40m5 BAC 40E8 HOT 0m1 MB 20m2 MB 40m12 MB 100m10 MB 20m12 BAC 31A8 MB 40m1 MB 100m5 MB 20m5 BAC 64A5 MB 100m7 MB 0m1 MB 100m9 Figure 3 Phylogenetic analysis of the inferred amino-acid sequence of cloned proteorhodopsin genes. Distance analysis of 220 positions was used to calculate the tree by neighbour-joining using the PaupSearch program of the Wisconsin Package version 10.0 (Genetics Computer Group; Madison, Wisconsin). H. salinarum bacteriorhodopsin was used as an outgroup, and is not shown. Scale bar represents number of substitutions per site. Bold names indicate the proteorhodopsins that were spectrally characterized in this study. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 787 © 2001 Macmillan Magazines Ltd !29

nes from the HOT station were m samples. Most proteorhodopsurface waters belonged to the (on the amino-acid level) to the 40E8. In contrast, most of the ) fell within the Antarctic clade es from the HOT station, HOT d in E. coli and their absorption d from their primary sequences, ng with the Antarctic clade gave um, whereas the proteorhodopclade gave a green (527-nm) central North Paci®c gyre, most ge, with maximal intensity near eak is maintained over depth, At the surface, the energy peak th between 400 and 650 nm. In narrows and the half bandwidth 00 nm (Fig. 5). Considering the members of the two different 0.06 0.05 Absorbance rom Antarctica (E. F. DeLong, dopsin primers, and sequenced minary sequence analyses based indicate that, despite differences orption spectra, the Antarctic a bacterium highly related to AR86 bacteria of Monterey Bay10 dopsin genetic variants in mixed populations with a selective advantage at different points along the depth-dependent light 0.04 HOT 75 m Antarctica 0.03 HOT 0 m 0.02 Monterey Bay 0.01 1.0 Relative irradiance mechanisms of wavelength regurhodopsins. Furthermore, palE6 larly to its Monterey Bay diated transport of protons in . Spudich et al., manuscript in 5m 0.8 0.6 75 m 0.4 0.2 0 350 400 450 500 550 600 650 Wavelength (nm) Figure 5 Absorption spectra of retinal-reconstituted proteorhodopsins in E. coli membranes. All-trans retinal (2.5 mM) was added to membrane suspensions in 100 mM phosphate buffer, pH 7.0, and absorption spectra were recorded. Top, four spectra for palE6 (Antarctica), HOT 75m4, HOT 0m1, and BAC 31A8 (Monterey Bay) at 1 h after retinal addition. Bottom, downwelling irradiance from HOT station measured at six wavelengths (412, 443, 490, 510, 555 and 665 nm) and at two depths, for the same depths and date that the HOT samples were collected (0 and 75 m). Irradiance is plotted relative to irradiance at 490 nm. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !30

whereas the total energy decreases. At the surface, the energy peak is very broad with a half bandwidth between 400 and 650 nm. In deeper water below 50 m, the peak narrows and the half bandwidth is restricted to between 450 and 500 nm (Fig. 5). Considering the different wavelengths absorbed by members of the two different palE6 (Antarctica), HOT 75m4, HOT 0m1, and BAC 31A8 (Monterey Bay) at 1 h after retinal addition. Bottom, downwelling irradiance from HOT station measured at six wavelengths (412, 443, 490, 510, 555 and 665 nm) and at two depths, for the same depths and date that the HOT samples were collected (0 and 75 m). Irradiance is plotted relative to irradiance at 490 nm. Figure 4 Multiple alignment of proteorhodopsin amino-acid sequences. The secondary structure shown (boxes for transmembrane helices) is derived from hydropathy plots. Residues predicted to form the retinal-binding pocket are marked in red. 788 © 2001 Macmillan Magazines Ltd NATURE | VOL 411 | 14 JUNE 2001 | www.nature.com Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !31

(e-mail: denning@atmos.colostate.edu). More on Large Insert Metagenomics 6 ................................................................. 98 Unsuspected diversity among marine aerobic anoxygenic phototrophs  Á Oded Beja*², Marcelino T. Suzuki*, John F. Heidelberg³, William C. Nelson³, Christina M. Preston*, Tohru Hamada§², Jonathan A. Eisen³, Claire M. Fraser³ & Edward F. DeLong* * Monterey Bay Aquarium Research Institute, Moss Landing, http://www.nature.com/nature/journal/v415/n6872/full/415630a.html California 95039-0628, USA ³ The Institute for Genomic Research, Rockville, Maryland 20850, USA § Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi City, Iwate 026-0001, Japan .............................................................................................................................................. Aerobic, anoxygenic, phototrophic bacteria containing bacteriochlorophyll a (Bchla) require oxygen for both growth and Bchla synthesis1±6. Recent reports suggest that these bacteria are widely distributed in marine plankton, and that they may account for up to 5% of Slides for UCocean photosyntheticby Jonathan Eisen Winter 2014and surface Davis EVE161 Course Taught electron transport7 8 b

esearch, Rockville, Maryland 20850, USA ute, Kamaishi Laboratories, Kamaishi City, ........................................................................................ 100 100 67 99 envHOT2 envHOT3 cDNA 20m10 BAC 29C02 BAC 39B11 BAC 52B02 100 54 BAC 24D02 BAC 65D09 rRNA PUF Chloroflexus aurantiacus ototrophic bacteria containing bacterioa BAC 56B12 quire oxygen for both growth and Bchla BAC 60D04 b Roseobacter denitrificans* 100 0.1 BAC 30G07 rts suggest that these bacteria are widely 0.1 100 cDNA 0m13 Roseobacter litoralis* 52 ankton, and that they may account cDNAup for 20m11 91 a, b, Evolutionary distances for the R2A84* and photosynthetic electron transport7env20m1 pufM genes (a) were determined from 100 76 R2A62* env20m5 α-3 obial community8. Known planktonic20m22 an alignment of 600 nucleotide cDNA MBIC3951* 100 env0m2 99 positions, and for rRNA genes (b) from belong to only a few restricted groups Rhodobacter capsulatus cDNA 20m8 95 R2A84* an alignment of 860 nucleotide Rhodobacter sphaeroides 72 ia a-subclass. Here we report genomic R2A62* R2A163* Rhodovulum sulfidophilum* sequence positions. Evolutionary 100 thetic gene content and operon organizaRhodobacter capsulatus 95 relationships were determined by Erythromicrobium ramosum α-3 ng marine bacteria. TheseRhodobacter sphaeroides photosynthetic Rhodovulum sulfidophilum* Erythrobacter longus* 82 neighbour-joining analysis (see 69 ome that most closely resembled those of Roseobacter denitrificans* 100 α-4 Erythrobacter litoralis* Methods). The green non-sulphur Roseobacter litoralis* 73 b-subclass, which have never before been MBIC3019* MBIC3951* 100 bacterium Chloroflexus aurantiacus 61 82 cDNA ronments. Furthermore, thesecDNA0m1 photosynSphingomonas natatoria 61 was used as an outgroup. pufM genes 20m21 65 100 env0m1 Rhodomicrobium vannielii α-2 ly distributed in marine plankton, and 99 that were amplified by PCR in this envHOT1 Rhodopseudomonas palustris 98 itic bacterioplankton assemblages, indistudy are indicated by the env prefix, Erythrobacter longus* 100 Rhodospirillum rubrum α-1 69 Erythromicrobium ramosum nti®ed phototrophs were photosynthetiwith 'm' indicating Monterey, and HOT 100 96 Erythrobacter litoralis* Rubrivivax gelatinosus 100 α-4 ta demonstrate that planktonic bacterial indicating Hawaii ocean time series. 98 MBIC3019* Roseateles depolymerans 100 Sphingomonas natatoria Cultivated aerobes are marked in light β ply composed of one uniform, widespread Rhodoferax fermentans 100 cDNA 0m20 blue, bacteria cultured from sea water otrophs, as previously proposed8; rather, Rhodocyclus tenuis Thiocystisge latinosa 96 100 γ are marked with an asterisk, and Allochromatium vinosum ain multiple, distantly related, photoThiocystis gelatinosa 100 γ Rhodopseudomonas palustris environmental cDNAs are marked in Allochromatium vinosum erial groups, including some unrelated α–1 Rhodospirillum rubrum α-2 Chloroflexus aurantiacus red. Photosynthetic -, - and Rhodomicrobium vannielii 56 types. Rhodocyclus tenuis proteobacterial groups are indicated 52 Rubrivivax gelatinosus uired for the formation of bacteriochloro- β by the vertical bars to the right of the Roseateles depolymerans AAP ystems in aerobic, anoxygenic, photo- Figure 1 Phylogenetic relationships of pufM gene (a) and rRNA (b) sequences of Bootstrap values greater than Rhodoferax fermentans tree. re clustered in a contiguous, 45-kilobase bacteria. a, b, Evolutionary distances for the pufM genes (a) were determined from an indicated above the envHOT2 100 50% are envHOT3 100 (superoperon)6. These include bch and crt alignment of 600 nucleotide positions, and for rRNA genes (b) from an alignment of 860 The scale bar represents cDNA 20m10 branches. 67 BAC 29C02 by neighbournzymes of the bacteriochlorophyll and nucleotide sequence positions. Evolutionary relationships were determined number of substitutions per site. BAC 39B11 99 BAC 52B02 pathways, and the puf genes coding for joining analysis (see Methods). The green non-sulphur bacterium Chloro¯exus 100 54 BAC 24D02 harvesting complex (pufB and pufA) and aurantiacus was used as an outgroup. pufM genes that were ampli®ed by PCR in this BAC 65D09 Chloroflexus aurantiacus ex (pufL and pufM). To better describe the study are indicated by the env pre®x, with `m' indicating Monterey, and HOT indicating planktonic, anoxygenic, photosynthetic Hawaii ocean time series. Cultivated aerobes are marked in light blue, bacteria cultured b 100 surface-water bacterial Roseobacter denitrificans* arti®cial chromo- from sea water are marked with an asterisk, and environmental cDNAs are marked in red. 0.1 Roseobacter litoralis* 52 Slides for UC Davis EVE161and g-proteobacterial by Jonathan Eisen Winter bars to Course Taught groups are indicated by the vertical 2014 91 Photosynthetic a-, b-

complementary DNA. However, a different primer targeting a from which the BACs originated are functional. Rubrivivax gelatinosus M C A D C M L B H F N B I B C E F C X Z L M C B A M L puf bch idi crt Y H orf276 orf154 orf227 puhA I hemN BAC 65D09 B N G P G F P orf358 B A L orf440 Z ppsR Y lhaA E F C X bch bch cycA Rhodobacter sphaeroides X M L A BQ Z Y X C F E D C B I A I D G J P O E F N B H L M BAC 60D04 E F C X crt Y bch Z QB A L puf Figure 2 Schematic comparison of photosynthetic operons from R. gelatinosus (bproteobacteria), R. sphaeroides (a-proteobacteria) and uncultured environmental BACs. ORF abbreviations use the nomenclature de®ned in refs 13, 14 and 24. Predicted ORFs are coloured according to biological category: green, bacteriochloropyll biosynthesis NATURE | VOL 415 | 7 FEBRUARY 2002 | www.nature.com G P M bch M L H B bch N F tspO C D ppa ppsR B lhaA I orf292 orf277 orf128 orf213 puhA bch I A idi D orf641 O genes; orange, carotenoid biosynthesis genes; red, light-harvesting and reaction centre genes; and blue, cytochrome c2. White boxes indicate non-photosynthetic and hypothetical proteins with no known function. Homologous regions and genes are connected by shaded vertical areas and lines, respectively. © 2002 Macmillan Magazines Ltd Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 631

letters to nature a b BchB Rhodobacter capsulatus BAC 60D04 Acidiphilium rubrum Rhodopseudomonas palustris Rubrivivax gelatinosus Rhodobacter sphaeroides 100/94 Acidiphilium rubrum BchH Rubrivivax gelatinosus 100/66 100/100 Rhodopseudomonas palustris BAC 60D04 BAC 29C02 100/100 BAC 65D09 Rhodobacter capsulatus 100/96 Rhodobacter sphaeroides 100/100 100/100 100/100 0.1 100/100 BAC 29C02 100/100 100/99 BAC 65D09 100/100 0.1 100/100 Heliobacillus mobilis Heliobacillus mobilis Chloroflexus aurantiacus Chlorobium tepidum Figure 3 Phylogenetic analyses of BchB and BchH proteins. a, Phylogenetic tree for the BchB protein. b, Phylogenetic tree for the BchH protein. The BchH sequences from Chlorobium vibrioforme25and BchH2 and BchH3 from C. tepidum18 were omitted from the tree because these genes potentially encode an enzyme for bacteriochlorophyll c Chloroflexus aurantiacus Chlorobium tepidum biosynthesis and are probably of distinct origin (J. Xiong, personal communication). Bootstrap values (neighbour-joining/parsimony method) greater than 50% are indicated next to the branches. The scale bar represents number of substitutions per site. The position of Acidiphilium rubrum (bold branch) was not well resolved by both methods. We compared several photosynthetic genes found on the oceanic operon) to cultivated Erythrobacter species. bacteriochlorophyll superoperons with characterized homologues Recently, it has been suggested that cultivated Erythrobacter from cultured photosynthetic bacteria. The relationships among species (a-4 subclass of proteobacteria) may represent the preBchla biosynthetic proteins BchB (a subunit of light-independent dominant AAPs in the upper ocean8,20. Surprisingly, we were not protochlorophyllide reductase) and BchH (magnesium chelatase) able to retrieve photosynthetic operon genes belonging to this group Slides for UC relationships, BchB and in any Jonathan Eisen Winter 2014 were determined18 (Fig. 3). Similar to pufMDavis EVE161 Course Taught byof the samples analysed. Furthermore, very few sequences

om the HOT station were mples. Most proteorhodopce waters belonged to the he amino-acid level) to the . In contrast, most of the within the Antarctic clade om the HOT station, HOT E. coli and their absorption m their primary sequences, th the Antarctic clade gave whereas the proteorhodope gave a green (527-nm) al North Paci®c gyre, most with maximal intensity near s maintained over depth, he surface, the energy peak tween 400 and 650 nm. In ows and the half bandwidth m (Fig. 5). Considering the mbers of the two different 0.06 0.05 Absorbance Antarctica (E. F. DeLong, in primers, and sequenced ary sequence analyses based ate that, despite differences on spectra, the Antarctic cterium highly related to bacteria of Monterey Bay10 advantage at different points along the depth-dependent light 0.04 HOT 75 m Antarctica 0.03 HOT 0 m 0.02 Monterey Bay 0.01 1.0 Relative irradiance opsins. Furthermore, palE6 to its Monterey Bay d transport of protons in udich et al., manuscript in 5m 0.8 0.6 75 m 0.4 0.2 0 350 400 450 500 550 600 650 Wavelength (nm) Figure 5 Absorption spectra of retinal-reconstituted proteorhodopsins in E. coli membranes. All-trans retinal (2.5 mM) was added to membrane suspensions in 100 mM phosphate buffer, pH 7.0, and absorption spectra were recorded. Top, four spectra for palE6 (Antarctica), HOT 75m4, HOT 0m1, and BAC 31A8 (Monterey Bay) at 1 h after retinal addition. Bottom, downwelling irradiance from HOT station measured at six wavelengths (412, 443, 490, 510, 555 and 665 nm) and at two depths, for the same depths and date that the HOT samples were collected (0 and 75 m). Irradiance is plotted relative to irradiance at 490 nm. Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014

articles Community structure and metabolism through reconstruction of microbial genomes from the environment Gene W. Tyson1, Jarrod Chapman3,4, Philip Hugenholtz1, Eric E. Allen1, Rachna J. Ram1, Paul M. Richardson4, Victor V. Solovyev4, Edward M. Rubin4, Daniel S. Rokhsar3,4 & Jillian F. Banfield1,2 1 Department of Environmental Science, Policy and Management, 2Department of Earth and Planetary Sciences, and 3Department of Physics, University of California, Berkeley, California 94720, USA 4 Joint Genome Institute, Walnut Creek, California 94598, USA ........................................................................................................................................................................................................................... RESEARCH ARTICLE Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment. 1 1 3 Environmental Genome Shotgun Sequencing of the Sargasso Sea J. Craig Venter, * Karin Remington, John F. Heidelberg, Aaron L. Halpern,2 Doug Rusch,2 Jonathan A. Eisen,3 The study of microbial evolution and ecology has been revolutio- fluorescence in situ hybridization (FISH) revealed that all biofilms Dongying Wu,3 Ian Paulsen,3 Karen E. Nelson,3 William Nelson,3 nized by DNA sequencing and analysis1–3. However, isolates have contained mixtures of bacteria (Leptospirillum, Sulfobacillus and, in Derrick few cases, 3 Samuel Levy,2 archaea (Ferroplasma 6 been the main source of sequence data, and only a small fraction of aE. Fouts, Acidimicrobium) andAnthony H. Knap,and other 4–6 has members of the Thermoplasmatales). The genome of one microorganisms have been cultivated . Consequently, focusMichael W. Lomas,6 Ken Nealson,5 Owen White,3 of these shifted towards the analysis of uncultivated microorganisms via archaea, Ferroplasma acidarmanus 1 Rachel Parsons,6 Jeremy Peterson,3 Jeff Hoffman, fer1, isolated from the Richmond cloning of conserved genes5 and genome fragments directly from mine, has been sequenced previously (http://www.jgi.doe.gov/JGI_ 4 Holly Baden-Tillson,1 Cynthia Pfannkoch,1 Yu-Hui the environment7–9. To date, only a small fraction of genes have been microbial/html/ferroplasma/ferro_homepage.html).Rogers, Slides for UC Davis EVE161 Course biofilm (Fig.Jonathan 1 of AMD communities was Hamilton 1a) typical recovered from individual environments, limiting the analysis of A pink Taught by O. Smith Eisen Winter 2014 chlorococcus, tha photosynthetic bio Surface water were collected ab from three sites o February 2003. A lected aboard the S station S” in May are indicated on F S1; sampling prot one expedition to was extracted from genomic libraries w 2 to 6 kb were m !37 prepared plasmid

Shotgun metagenomics shotgun sequence Slides for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !38

clones). The first step in assignment of scaffolds to organism types was to Acid Mine Drainage 2004 Figure 1 The pink biofilm. a, Photograph of the biofilm in the Richmond mine (hand included for scale). b, FISH image of a. Probes targeting bacteria (EUBmix; fluorescein isothiocyanate (green)) and archaea (ARC915; Cy5 (blue)) were used in combination with a probe targeting the Leptospirillum genus (LF655; Cy3 (red)). Overlap of red and green (yellow) indicates Leptospirillum cells and shows the dominance of Leptospirillum. c, Relative microbial abundances determined using quantitative FISH counts. 2 represent a nearly complete genome of uncultured Ferroplasma species distinct this as Ferroplasma type II. The dominan was unexpected before the genomic analy We assigned the roughly 3£ coverage Leptospirillum group III on the basis of rRN up to 31 kb, totalling 2.66 Mb). Comparis those assigned to Leptospirillum group sequence divergence and only locally co firming that the scaffolds belong to a rel Leptospirillum group II. A partial 16S rR Sulfobacillus thermosulfidooxidans was assembled reads, suggesting very low cov any Sulfobacillus scaffolds .2 kb were a grouped with the Leptospirillum group II We compared the 3£ coverage, low Gþ 4.12 Mb) to the fer1 genome in order to types (Supplementary Fig. S6). Scaffold identity to fer1 were assigned to an enviro I genome (170 scaffolds up to 47 kb in 1.48 Mb of sequence). The remaining scaffolds are tentatively assigned to G-pla in this bin (62 kb) contains the G-plasma scaffolds assigned to G-plasma comprise partial 16S rRNA gene sequence from A-pl unassembled reads, suggesting low covera scaffolds from A-plasma .2 kb would be bin. Although eukaryotes are present in th in low abundance in the biofilm studied. eukaryotes have been detected. As independent evidence that the Lep Ferroplasma type II genomes are nearly co complement of transfer RNA synthetases An almost complete set of these genes Leptospirillum group III. The G-plasma bin set of tRNA synthetases, consistent with in scaffolds. In addition, we established group II, Leptospirillum group III, Ferrop type II and G-plasma bins contained onl 2004 Nature Publishing Slides for UC Davis EVE161 Course Taught by Jonathan©Eisen Winter 2014 Group NATURE | doi:10.1038/n !39

genes needed to fix carbon by means of the Calvin–Benson– Bassham cycle (using type II ribulose 1,5-bisphosphate carboxylase–oxygenase). All genomes recovered from the AMD system fixation via the reductive acetyl coenzyme A (acetyl-CoA) pathway by some, or all, organisms. Given the large number of ABC-type sugar and amino acid transporters encoded in the Ferroplasma type Figure 4 Cell metabolic cartoons constructed from the annotation of 2,180 ORFs identified in the Leptospirillum group II genome (63% with putative assigned function) and 1,931 ORFs in the Ferroplasma type II genome (58% with assigned function). The cell drainage stream (viewed in cross-section). Tight coupling between ferrous iron oxidation, Slides for UC Davis EVE161 Course pyrite dissolution and acid generation is indicated. Rubisco, ribulose 1,5-bisphosphate Taught by Jonathan Eisen Winter 2014 carboxylase–oxygenase. THF, tetrahydrofolate. !40

RESEARCH ARTICLE Environmental Genome Shotgun Sequencing of the Sargasso Sea J. Craig Venter,1* Karin Remington,1 John F. Heidelberg,3 Aaron L. Halpern,2 Doug Rusch,2 Jonathan A. Eisen,3 Dongying Wu,3 Ian Paulsen,3 Karen E. Nelson,3 William Nelson,3 Derrick E. Fouts,3 Samuel Levy,2 Anthony H. Knap,6 Michael W. Lomas,6 Ken Nealson,5 Owen White,3 Jeremy Peterson,3 Jeff Hoffman,1 Rachel Parsons,6 Holly Baden-Tillson,1 Cynthia Pfannkoch,1 Yu-Hui Rogers,4 Hamilton O. Smith1 We have applied “whole-genome shotgun sequencing” to microbial populations collected en masse on tangential flow and impact filters from seawater samples collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs http://www.sciencemag.org/content/304/5667/66 of nonredundant sequence was generated, annotated, and analyzed to elucidate the gene content, diversity, and relative abundance of the organisms within these environmental samples. These data are estimated to derive from at least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes. We have identified over 1.2 million previously unknown genes represented in these samples,by Jonathanmore than 782 new Slides for UC Davis EVE161 Course Taught including Eisen Winter 2014 chlorococcus, th photosynthetic bi Surface wate were collected a from three sites February 2003. A lected aboard the station S” in Ma are indicated on S1; sampling pro one expedition to was extracted fro genomic libraries 2 to 6 kb were prepared plasmid both ends to prov Craig Venter Sc nology Center on ers (Applied Bi Whole-genome ra the Weatherbird II 4) produced 1.66 in length, for a tota microbial DNA se sequences were g !41

two groups of scaffolds representing two disSargasso Sea related to the published tinct strains closely at depths ranging from 4ϫ to 36ϫ (indicated with shading in table S3 with nine depicted in Fig. 1. MODIS-Aqua satellite image of ocean chlorophyll in the Sargasso Sea grid about the BATS site from 22 February 2003. The station locations are overlain with their respective identifications. Note the elevated levels of chlorophyll (green color shades) around station 3, which are not present around stations 11 and 13. http://www.sciencemag.org/content/304/5667/66 Fig. 2. Gene conserSlides vation among closely for UC Davis EVE161 Course Taught by Jonathan Eisen Winter 2014 !42

sequence when they would otherwise be labeled as repetitive. We evaluated our final assembly Table 1. Gene count results in a tiered fashion, looking at well-sampled breakdown by TIGR role genomic those found on ascategory. Gene set includes regions separately from those barely sampled at our current level of sequencing. semblies from samples 1 to 41.66 million sequencesreads the The and fragment from from samples 5 to 7. AWeatherbird II samples (table S1; samples 1 to more detailed table, sep4; stations 3, 11, and 13), were pooled and arating Weatherbird II samplesto providethe Sorcerer II assembled from a single master assembly samples is presented in for comparative purposes. The assembly generthe SOM (table S4). Note ated 64,398 scaffolds ranging size from 826 that there are 28,023 genes which were 256inMbp of unique bp to 2.1 Mbp, containing classified sequence and in more than one role category.spanning 400 Mbp. After assembly, there remained 217,015 paired-end reads, or “mini-scaffolds,” spanning 820.7 Mbp as well as an additional 215,038 unassembled sinTotal gleton TIGR role category reads covering 169.9 Mbp (table S2, genes column 1). The Sorcerer II samples provided almost no assembly, so we consider for these samples only the 153,458 mini-scaffolds, spanAmino acid biosynthesis 518.4 Mbp, and the remaining 18,692 37,118 ning singleton reads (table S2, column 2). In total, Biosynthesis of cofactors, 25,905 1.045 Gbp of nonredundant sequence was genprosthetic groups, and carriers of overlapping reads within the erated. The lack unassembled set indicates that lack of additionCell envelope 27,883 al assembly was not due to algorithmic limitaCellular processes 17,260 tions but to the relatively limited depth of sequencing coverage Central intermediary metabolism given the level of diversity 13,639 within the sample. DNA metabolism 25,346 The whole-genome shotgun (WGS) assembly has been deposited at DDBJ/EMBL/GenBank Energy metabolism 69,718 under Fatty acid and phospholipidthe project accession AACY00000000, 18,558 and all traces have been deposited in a corresponding TraceDB trace archive. The version metabolism described paper is the first version, Mobile and extrachromosomalin this Unlike a conventional WGS 1,061 AACY01000000. element functions entry, we have deposited not just contigs and scaffolds but the unassembled paired singletons Protein fate 28,768 and individual singletons in order to accurateProtein synthesis 48,012 ly reflect the diversity in the sample and allow searches Purines, pyrimidines, nucleosides,across the entire sample with19,912 in a single database. and nucleotides Genomes and large assemblies. Our analysis first focused on the well-sampled geRegulatory functions nomes

Add a comment

Related presentations

Related pages

UC Davis EVE161 Lecture 10 by @phylogenomics - Education

Share UC Davis EVE161 Lecture 10 by @phylogenomics. ... the finding of a conserved core of euryarchaeal genomes21,22 and than those from bacteria13,14.
Read more

UC Class #2 lecture - Education - documents

UC Davis EVE161 Lecture 9 by @phylogenomics ... Sport PR at UC Lecture #2, 2 4-14. UC Sport Lecture, 2-18. Technical analysis lecture thursday mba class (2)
Read more

EVE161 Class at UCDavis Winter 2014 Lecture 3 - Jonathan ...

EVE161 Class at UCDavis Winter 2014 Lecture 3 ... Lecture 9 SectionB BIS2C UC Davis Spring ... talk by Jonathan Eisen @phylogenomics at # ...
Read more

EVE161 Class at UCDavis Winter 2014 Lecture 4 - YouTube

EVE161 Class at UCDavis Winter 2014 Lecture 4 ... Talk by Jonathan Eisen @phylogenomics: ... Lecture 9 SectionB BIS2C UC Davis Spring 2014 ...
Read more

The Tree of Life: On my evolving thoughts on the #UCDavis ...

On my evolving thoughts on the #UCDavis saga involving Chancellor Linda Katehi ... UC Davis Storer Lecture ... (@phylogenomics) April 14, ...
Read more

UC Davis – microBEnet: the microbiology of the Built ...

Lecture 4 was fun and a bit scary for me. You see, I finally decided to read in ... And since UC Davis is so big (some 2500 or so faculty I think) ...
Read more

Uc Davis | LinkedIn

UC Davis Health System is a major driver of economic prosperity in the... Mario Eguizabal, PHR. Student Services Coordinator at UC Davis School of Nursing.
Read more

Uc Davis | LinkedIn

View 83456 Uc Davis posts, presentations, experts, and more. Get the professional knowledge you need on LinkedIn. LinkedIn Home What is LinkedIn? Join Today
Read more