1. Archaeal extreme halophiles (halobacteria, haloarchaea) require high concentration of NaCl for growth found in Great Salt Lake, Dead Sea, evaporation ponds bacteriorhodopsin – light-activated proton pump – covalently attached retinal – red color 1 family, 34 genera heterotrophs mostly aerobic

2. Adaptation to high salt high internal concentration of KCl halorhodopsin – light-activated chloride pump, pumps chloride in protein adaptations to high salt excess acidic amino acids fewer hydrophobic amino acids

3. Halophiles with sequenced genomes Halobacterium salinarum NRC-1 and R1 Haloarcula marismortui Haloquadratum walsbyi Natronomonas pharaonis Haloferax volcanii New genomes in our study Halorubrum lacusprofundi - CSP Halogeometricum borinquense - GEBA Halomicrobium mukohataei - GEBA Halorhabdus utahensis - GEBA Haloterrigena turkmenica – GEBA

4. IsolationGrows on Halobacteriumsalted cow hide complex medium or amino acids HaloarculaDead Sea water Glc, Frc, Scr, Gly, Ac, Suc, Mal (Rib, Xyl) Haloquadratum saltern water, Spain, Australia Pyruvate, DHA Natronomonas desert lake water, Egypt Pyr, Fum, Form, But HaloferaxDead Sea sediment Yeast extract, tryptone, Glc, Xyl, Gly, Ac, Lac HalorubrumDeep Lake, Antarctica Glc, Gal, Man, Rib, Lct, Gly, Suc, Lac, Ac, Prop, Eth, Form Halogeometricum saltern water, Puerto Rico Glc, Frc, Man, Xyl, Mlt, Tre, Cel, Raf, Gly (Ara, Rib, Lac, Scr) Halomicrobiumsalt flat soil, Argentina Glc, Gal, Scr, Mlt, Gly (Man, Rib) Halorhabdus Great Salt Lake sediment Glc, Xyl, Frc, Xylan Haloterrigenasaline soil, TurkmeniaGlc, Frc, Man, Scr, Mlt

5. Questions ● What genes are conserved in all ten genomes? ● What are the haloarchaeal signature genes (in all haloarchaea but not in other archaea)? ● What are the organism-specific genes in each genome? ● Have some genomes recently lost genes? ● Are there genes found in water halophiles but not in soil/sediment halophiles? ● Are there genes found in soil/sediment halophiles but not in water halophiles? All of these questions can be answered using the phylogenetic profiler.

6. Genes conserved in all halophiles (core genes)

7. 40% of total genes in most genomes 50% of total genes in Halobacterium NRC-1 This % of conserved genes is about the same as other families with multiple genomes from different genera

8. Genes conserved in all halophiles (core genes) Part of gene list from phylogenetic profiler results.

9. Core genes - results Transcription, Translation, DNA replication RNA processing – sRNPs, RNAse P, methyltransferases Protein processing – chaperones, proteases Protein secretion – signal-dependent secretion, twin arginine Signal transduction – kinases, phosphatases, response regulators Amino acid synthesis – most, but not Leu, Ile, Val, Lys Purine/pyrimidine synthesis – except guanylate kinase, thymidylate synthase Cofactor synthesis – heme, pyridoxal, flavins, F 420 Lipid synthesis – mevalonate pathway, phospholipid metabolism Central metabolism – TCA cycle, most of glycolysis

10. Haloarchaeal signature – genes present in all haloarchaea but not in other archaea First, select all Archaea Second, select halophiles (I also removed draft genomes)

11. Haloarchaeal signature genes

12. Most have unknown functions (even the ones with COGs) but a few are involved in gamma-glutamylcysteine metabolism

13. Central metabolism in the IMG network

14. Pentose phosphate pathway (PPP) ● haloarchaea are thought to use the oxidative PPP ● produces pentoses for RNA, DNA synthesis ● produces NADPH for biosynthesis of amino acids, etc.

15. Add the IMG terms to the function cart, do a profile with the ten haloarchaea. key enzyme is missing in Halorhabdus utahensis Check COGs also

16. Some archaea use the reverse ribulose monophosphate pathway The two key enzymes are not in any haloarchaea. There is one more pathway – nonoxidative PPP fructose-6P hexulose-6P ribulose-5P + formaldehyde

17. Function profile – nonoxidative PPP Halorhabdus has all the enzymes required for nonoxidative PPP. Why does it have a different pathway? fructose-6P + glyceraldehyde-3P erythrose-4P + xylulose-5P fructose-6P + glyceraldehyde-3P + sedoheptulose-7P ribose-5P + xylulose-5P

18. Different pathways of D-xylose catabolism D-xylose D-xylulose D-xylulose-5P xylose isomerase, Huta_2443 xylulokinase, Huta_2446 non-oxidative pentose phosphate pathway D-xylose D-xylonolactone D-xylonate 2-keto-3-deoxyxylonate 2-ketoglutarate semialdehyde 2-ketoglutarate Halorhabdus Halorubrum, Haloferax, Haloterrigena, Haloarcula

19. L-arabinose metabolism L-arabinose L-ribulose L-arabinose isomerase, Huta_1154 L-ribulose-5P ribulokinase, Huta_1150? D-xylulose-5P ribulose-5P 4-epimerase, Huta_1149 non-oxidative pentose phosphate pathway not in any other archaea only in Halorhabdus Conclusion: Non-oxidative PPP is reversible, may make it easier to grow on pentoses.

20. How does Halorhabdus generate NADPH? In other halophiles, oxidative PPP produces NADPH NADP + + NADH NAD + + NADPH Halorhabdus is the only haloarchaeon to have NAD/NADP transhydrogenase.

21. glycerol glycerol-3P DHAP ATP ADP quinone quinol glycolysis Glycerol metabolism and transport Glycerol is an important carbon/energy source for halophiles. All except N. pharaonis have glycerol kinase and glycerol-3P DHase But only Halomicrobium mukohataei has an identified glycerol transporter

22. glycerol-3P DHase glycerol kinase predicted membrane protein glycerol transporter, MIP family Hbor has 2 glycerol kinases, both adj to conserved membrane protein

23. In two bacteria, the same membrane protein is adjacent to glycerol kinase glycerol kinase In two bacteria, the membrane protein is adjacent to 1,3-propanediol dehydrogenase 1,3-propanediol dehydrogenase

24. In five bacteria, the membrane protein is close to glycerol dehydratase small subunit large subunit We have used a not well characterized group of organisms to predict gene function in model organisms (E. coli, Rhodobacter)

25. Motility in haloarchaea Flagellar proteins, chemotaxis proteins

26. Gas vesicle proteins H. walsbyi lacks flagellar motility but has gas vesicle proteins Sediment halophiles lack gas vesicle proteins Haloterrigena turkmenica was isolated from soil. Why does it have gas vesicle proteins? Isolated from upper crust of solonchak, poorly drained soil keep haloarchaea close to light and oxygen