22. Analysis of Haloarchaeal Genomes

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Transcript 22. Analysis of Haloarchaeal Genomes

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-activated
chloride 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
fructose-6P
hexulose-6P
ribulose-5P
+
formaldehyde
The two key enzymes are not in any haloarchaea.
There is one more pathway – nonoxidative PPP
fructose-6P
+
glyceraldehyde-3P
fructose-6P
+
erythrose-4P
+
xylulose-5P
glyceraldehyde-3P
+
sedoheptulose-7P
Function profile – nonoxidative PPP
Halorhabdus has all the enzymes required for nonoxidative PPP.
Why does it have a different pathway?
ribose-5P
+
xylulose-5P
Different pathways of D-xylose catabolism
D-xylose
D-xylose
xylose isomerase, Huta_2443
D-xylulose
D-xylonolactone
xylulokinase, Huta_2446
D-xylulose-5P
non-oxidative
pentose phosphate
pathway
Halorhabdus
D-xylonate
2-keto-3-deoxyxylonate
2-ketoglutarate semialdehyde
2-ketoglutarate
Halorubrum, Haloferax,
Haloterrigena, Haloarcula
L-arabinose metabolism
only in Halorhabdus
L-arabinose
not in any other archaea
L-arabinose isomerase, Huta_1154
L-ribulose
ribulokinase, Huta_1150?
L-ribulose-5P
ribulose-5P 4-epimerase, Huta_1149
D-xylulose-5P
non-oxidative pentose
phosphate pathway
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 metabolism and transport
Glycerol is an important carbon/energy source for halophiles. All
except N. pharaonis have glycerol kinase and glycerol-3P DHase
ATP
glycerol
ADP
quinone quinol
glycerol-3P
DHAP
glycolysis
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
keep haloarchaea close to light and oxygen
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