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

Adaptation to high salt

high internal concentration of KCl

halorhodopsin – light-activatedchloride pump, pumps chloride in

protein adaptations to high salt

excess acidic amino acids

fewer hydrophobic amino acids

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

Isolation Grows on

Halobacterium salted cow hide complex medium or amino acids

Haloarcula Dead 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

Haloferax Dead Sea sediment Yeast extract, tryptone, Glc, Xyl, Gly, Ac, Lac

Halorubrum Deep 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)

Halomicrobium salt flat soil, Argentina Glc, Gal, Scr, Mlt, Gly (Man, Rib)

Halorhabdus Great Salt Lake sediment Glc, Xyl, Frc, Xylan

Haloterrigena saline soil, Turkmenia Glc, Frc, Man, Scr, Mlt

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.

Genes conserved in all halophiles (core genes)

Genes conserved in all halophiles (core genes)

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

Genes conserved in all halophiles (core genes)

Part of gene list from phylogenetic profiler results.

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, F420

Lipid synthesis – mevalonate pathway, phospholipid metabolism

Central metabolism – TCA cycle, most of glycolysis

Haloarchaeal signature – genes present in all haloarchaea but not in other archaea

First, select all Archaea

Second, select halophiles (I also removed draft genomes)

Haloarchaeal signature genes

Haloarchaeal signature genes

Most have unknown functions (even the ones with COGs)

but a few are involved in gamma-glutamylcysteine metabolism

Central metabolism in the IMG network

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.

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

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

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

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-ketoglutarateHalorhabdus

Halorubrum, Haloferax, Haloterrigena, Haloarcula

L-arabinose metabolism

L-arabinose

L-ribuloseL-arabinose isomerase, Huta_1154

L-ribulose-5Pribulokinase, Huta_1150?

D-xylulose-5Pribulose-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.

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.

glycerol glycerol-3P DHAPATP 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

glycerol-3P DHase

glycerol kinase

predicted membrane

protein

glycerol transporter, MIP family

Hbor has 2 glycerol kinases, both adj to conserved membrane protein

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

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)

Motility in haloarchaea

Flagellar proteins, chemotaxis proteins

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