The Dampwood Termite Zootermopsis nevadensis. Salmassi and Leadbetter

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1 OVERVIEW Introduction to termites and termite hindgut microbiology Introduction to CO 2 -reductive homoacetogenesis & Treponema primitia strain ZAS-2 Formate dehydrogenase and selenium in T. primitia Comparison of FDH gene diversity in the gut communities in species representing 5 insect families Conclusions - evolutionary signals in community gene diversity

2 The Dampwood Termite Zootermopsis nevadensis Salmassi and Leadbetter

3 Breznak and Pankratz microns

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5 GENERAL SCHEME UNDERLYING SYMBIOSIS BETWEEN ANAEROBES AND WOOD-FEEDING LOWER TERMITES Methanogenic Archaea CH 4 Emitted by insect Polysaccharides fermented by Protozoa plus insect s enzymes CO 2 -Reducing Homoacetogens Wood triturated into small particles by the insect H 2 + CO 2 Acetate Acetate Absorbed by termite for oxidation & biosynthesis Cleveland Hungate Trager Yamin Odelson Breznak Brune ---- Current

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7 Treponema primitia sp. nov. Strains ZAS-1 and ZAS-2 H 2 stimulates growth Classical H 2 :Acetate stoichiometry Fix 14 CO 2 into Acetate Both C Positions in acetate become labeled with 14 CO 2 Exhibit CODH and other key activities of Wood- Ljungdahl Pathway Genomes: 3.4 & 3.9 Mb, respectively 50% G+C compare to 2.8 & 1.1 Mb for T denticola and T. pallidum Leadbetter et al 1999 Graber, Leadbetter & Breznak 2004

8 The Two FDH genes of Strain ZAS-2 Formate Dehydrogenase CO 2 CO 2 H 2 Formyl-THF Synthetase Methenyl-THF Cyclohydrolase Methylene-THF Dehydrogenase Methylene-THF Reductase Methyl Transferase H 2 H 2 Formate ATP ADP Formyl-Tetrahydrofolate Methenyl-THF Methylene-THF Methyl-THF Acetyl-CoA Acetyl - P ADP ATP CO H 2 How are the 2 related to each other? How are they related to other FDHs? Why 2? Why selenocysteine vs cysteine? ACETATE

9 T. primitia ZAS-2 formate dehydrogenases are FdhF-like (H2ase, not NADPH linked) FdhF-like sequences (Hydrogenase linked) Yersinia frederiksenii Yersinia intermedia Erwinia carotovora Rhodopseudomonas palustris Rhodospirillum rubrum Yersinia frederiksenii Yersinia pestis Erwinia carotovora 97 Salmonella enterica Salmonella typhimurium Photobacterium profundum Vibrio angustum Psychromonas sp. Escherichia coli (best studied FdhF) Clostridium difficile Clostridium beijerincki Treponema primitia ZAS-2 (selenoprotein) Treponema primitia ZAS-2 Enterococcus faecalis Mannheimia succiniciproducens Eubacterium acidaminophilum Eubacterium acidaminophilum Vibrio alginolyticus Vibrio vulnificus Photobacterium profundum Moorella thermoacetica NADPH linked (best studied acetogen) Carboxydothermus hydrogenoformans Bacillus cereus Methanococcus maripaludis Methanococcus voltae Methanococcus maripaludis Staphylococcus aureus Ferroplasma acidarmanus Rhodopseudomonas palustris Rhodopseudomonas palustris Pseudomonas fluorescens Eric Matson

10 Summary of fdhf Inventories Species Food Source Alleles Sequenced Location Zootermopsis nevadensis Wood 25 Chilao, CA Reticulitermes hesperus Wood 32 Chilao, CA Incisitermes minor Wood 34 Chilao, CA Nasutitermes sp. 303cost003 Wood 15 Costa Rica Rhynchotermes sp. 303cost004 Litter 23 Costa Rica Microcerotermes sp. 303cost008 Palm 11 Costa Rica Amitermes sp. 303cost010 Sugarcane 13 Costa Rica Cryptocercus punctulatus (Juvenile) Cryptocercus punctulatus (Adult) Wood 20 Wood 15 Mt Collins, NC Mt Collins, NC But this table doesn t reveal much about any patterns seen in host and gene phylogeny Xinning Zhang

11 Protozoan containing Lower Termites and Wood Roach Family Blattidae (Household Roach) Termitidae Rhinotermitidae Kalotermitidae Termopsidae Hodotermitidae * * * Higher termites Lower termites Mastotermitidae Family Cryptocercidae (Wood Roach) * Southern California termite representatives Adapted from Inward et al (2007)

12 Codiversification in phylogenetically distinct lower termites 76 Rhodopseudomonas palustris Rhodospirillum rubrum Yersinia frederiksenii 59 Yersinia pestis Yersinia frederiksenii Yersinia intermedia Erwinia carotovora Erwinia carotovora 99 Serratia grimesii 84 Salmonella enterica 81 Salmonella typhimurium 56 Escherichia coli Citrobacter str TSA-1 Buttiauxella SN1 51 Clostridium difficile Clostridium beijerincki T. primitia ZAS-2 T. primitia ZAS Zootermopsis Incisitermes Reticulitermes Outgrouped with cys FdhF Outgrouped with sel FdhF 0.1 fdhfsel fdhfcys 0.1 Xinning Zhang

13 Wood-Roach fdhf Follows Lower Termite Pattern FdhF Sec lower roach FdhF cys Treponema primitia ZAS-2 Treponema primitia ZAS-1 Treponema primitia ZAS-2 Treponema primitia ZAS-1 Termite spirochete group 0.10 Clostridium difficile enteric fdhf Acetonema longum Clostridium beijerinckii enteric fdhf

14 The Higher Termites Family Blattidae (Household Roach) Termitidae Rhinotermitidae Kalotermitidae Termopsidae * Hodotermitidae Higher termites Lower termites Mastotermitidae Family Cryptocercidae (Wood Roach) HIGHER TERMITES: NO protozoa - Dominant: biomass abundance - Dominant: termite species richness Less well studied despite global reach Adapted from Inward et al (2007)

15 Major environmental perturbation Loss of protozoa ==> Extinction of associated bacteria? Loss of FDH genes associated with those bacteria?

16 Cytochrome Oxidase Phylogeny of Termites and Cockroaches Nasutitermes ephratae 88 Nasutitermes sp. cost003 Nasutitermes corniger 67 Nasutitermes nigriceps 86 Nasutitermes pinocchio Bulbitermes makhamensis 94 Hospitalitermes medioflavus Cornitermes bequaerti Cornitermes cumulans 72 Cornitermes walkeri Rhynchotermes sp. cost Microcerotermes annandalei Microcerotermes paracelebensis Microcerotermes crassus 60 Embiratermes chagresi Microcerotermes sp. cost Amitermes dentatus Drepanotermes rubriceps Amitermes sp. cost010 Pericapritermes nitobei Reticulitermes sp AR Reticulitermes hesperus Reticulitermes santonensis Incisitermes sp. ca Bifiditermes improbus Cryptotermes secundus Procryptotermes australiensis Incisitermes immigrans 72 Zootermopsis sp EZ Zootermopsis nevadensis Zootermopsis angusticollis Stolotermes sp. 'Atherton' Cryptocercus punctulatus Cryptocercus relictus Periplaneta americana Polyphaga aegyptiaca Blattella germanica Panesthia cribrata Nasutitermitinae Amitermitinae Rhinotermitidae Kalotermitidae Termopsidae Higher Termites (Family Termitidae) Lower Termites Cockroaches

17 Higher termites possess one single coherent subclade within the fdhf sec line of descent FdhF Sec FdhF cys Amitermes Rhynchotermes Microcerotermes Microcerotermes Nasutitermes Nasutitermes Treponema primitia ZAS-2 Treponema primitia ZAS-1 Treponema primitia ZAS-2 Treponema primitia ZAS-1 Termite spirochete group 0.10 Clostridium difficile Amitermes, Rhynchotermes enteric fdhf lower higher

18 Higher termites: no sequences in fdhf cys clade impact of Se limitation averted by alternative adaptations? FdhF Sec FdhF cys Amitermes Rhynchotermes Microcerotermes Microcerotermes Nasutitermes Nasutitermes Treponema primitia ZAS-2 Treponema primitia ZAS-1 Treponema primitia ZAS-2 Treponema primitia ZAS-1 Termite spirochete group 0.10 Clostridium difficile Amitermes, Rhynchotermes enteric fdhf lower higher

19 Microcerotermes: independent reinvention of Se-independent fdhf cys and subsequent radiation - no evidence for HGT back from lower termites FdhF Sec Amitermes Rhynchotermes Microcerotermes Microcerotermes FdhF cys Nasutitermes Nasutitermes Treponema primitia ZAS-2 Treponema primitia ZAS-1 Treponema primitia ZAS-2 Treponema primitia ZAS-1 Termite spirochete group 0.10 Clostridium difficile Amitermes, Rhynchotermes enteric fdhf lower higher

20 Mystery basal clade outside termite spirochete FdhF cluster recent invasion, or new found success of a minor line in lower termites? FdhF Sec FdhF cys Amitermes Rhynchotermes Microcerotermes Microcerotermes Nasutitermes Nasutitermes Treponema primitia ZAS-2 Treponema primitia ZAS-1 Treponema primitia ZAS-2 Treponema primitia ZAS-1 Termite spirochete group 0.10 Clostridium difficile Amitermes, Rhynchotermes enteric fdhf lower higher

21 Gene signals of major evolutionary events impacting the symbiosis in higher termites Evidence for major, past environmental perturbations to the entire microbial community Loss of protozoa and extinction of protozoa-associated bacteria [subsequent sweeping gene losses, radiation from those left standing] Changes in foraging behavior - relaxation of nutrient limitations [subsequent sweeping gene loss] More recent change in diet or foraging environment by several species [Reemergence of lost-trait though independent convergent evolution] Gene invasion - late entry of genes from a non-termite source, these are not seen in other higher and lower termite lineages There is a lot of talk about co-evolution and co-speciation, but other factors are also at play. There often talk of the possibility of lateral community and gene transfer between termites, but we have yet to see any examples of such.

22 Funding from NSF Ecosystems Studies & NSF Microbial Genomes & DOE Genomes to Life Liz Ottesen Tina Salmassi Jian Yuan Thum Eric Matson Xinning Zhang Kasia Gora Nick Ballor Adam Rosenthal Thank you to my Termite co-workers :