Francisco Rodr

1
Genomes and metagenome of saturated brines

• Francisco Rodríguez-Valera

Universidad Miguel Hernández
Alicante, Spain
2
High throughput, low cost sequencing

• Identify the problems we can approach
• Learn to use the information (bioinformatics,
  databases, systems biology, etc)
• Develop models and/or hypotheses testable with
  the new information
• Funding and patience
• Extreme environments supply pilot scale models
  for more ambitious endeavors (e.g. Tyson et al,
  2004)

3
Outline of the talk

• Part 1.Description of saturated brine as a
  habitat and its microbial major players
• Part 2. Metagenome of the brine and the
  pan-genome of Haloquadratum
• Part 3. Genomes of Haloquadratum and
  Salinibacter and the Habitat Gene Reservoir (HGR)
  model

4
Let me introduce you to one of the most extreme
environments on earth

• The crystallizer of salterns where
• marine salt is harvested

5
Minerals formed when sea water is concentrated by
evaporation
Minerals Precipitated
Original Concentration
1/1
Concentration of sea water
6
Gradient of conditions and habitats with specific
microbiota
7
The precipitation of marine salts by evaporation
is a major geological process that has been going
on a a gigantic scale throughout earth history
8
Evaporites represent 20 of all sedimentary rocks
Microbes have had time and incentive to evolve
into halophiles
9
Physiology of halophilic adaptation
Halomonas
Dunaliella
10
Compatible solutes are great but cannot protect
exposed structures, transport and membrane
bioenergetics need to be compatible with high
salt Hyperhalophiles do not use organic
compatible solutes Haloarchaea, Salinibacter
Haloarchaea Salinibacter
Acidic proteins (low pI)
11
The microbiota of the crystallizers (gt30
salinity)
1 ?m
12
Dominion of the Haloarchaea (Formerly
Halobacteria)
13
People started isolating haloarchaea from
salterns in the 70s
To reach 18 genera by last count! None of them
thrive in the crystallizer
14
biomass reaches 108 cells/ml
However, some things do thrive there
15
Flat square cells making up to 70-80 total
16
Walsbys Square Bacteria Nature 1980
17
Walbys square bacterium is actually a
haloarchaeon With gas vesicles and
bacteriorhodopsin
18
16S r RNA retrieved from the crystallizer
19
The squares hybridize to the FISH probe of SPhT
20
Salinibacter ruber - Hyperhalophilic Bacteria
Phase Contrast
DAPI
FISH Bacteria
21
Salinibacter was easy to grow . . . but colonies
resemble those of haloarchaea
Standard media used for haloarchaea allowed
growth of Salinibacter but their colonies are
morphologically indistinguishable from the
typical red colonies of haloarchaea.
Haloarchaea
That together with the fact that they are always
in lower numbers and grow slowly explains why
members of this phylotype had not been isolated
before from this environment. Once knowing what
we had to look for it was easy.
Salinibacter ruber
22
Salinibacter and Haloarchaea . . . are
similar in more ways than just what catches the
eye
Salinibacter ruber
Halobacterium salinarum


Growth on Saturated NaCl
11.4
Kcontent (cell K/cell Prot.)
12.0
67
GC
67
15-25
Optimum NaCl Requirment
20-25
35-45ºC
Optimum temperature
35-45ºC
Aerobic heterotrophs
Physiology
Aerobic heterotrophs
Yes
Complex Nutrient Requirement
Yes
Yes (acylated C40-carotenoid )
Red Pigment
Yes (C-50 bacterioruberins)
15
Generation Time (hours)
7
23
Finally last year Haloquadratum walsbyi !
24
Salinibacter and Haloquadratum like each other
Salinibacter (inside) and Haloquadratum
(outside) growing on glycerol
25
Both Haloquadratum walsbyi and Salinibacter ruber
are predominant and homogeneous by 16S rRNA seqs.

• Haloquadratum by FISH 81.6 of cells that
  hybridize to the archaeal probe ARC 915 (75 DAPI)

• Salinibacter 81.8 of cells that hybridize to
  bacterial probe EUB 338 (12.7 DAPI). 16S rDNA
  indicates possible second species

Second Salininbacter species
Lowest similarity 98.5
26
The two major players have now been retrieved in
pure culture!...
and the genomes have been sequenced!
27
Ca. 3 Mbp
Ca. 50 Kbp
The genome of Haloquadratum walsbyi finished
manual annotation March 05
28
Salinibacter ruber M31 finished by spring 05
29
Part 2. Pan-genome of H. walsbyi
30
Pan-genome E. coli
Pan-genome- term coined by Claire Fraser
31
To what extent do the genomes of strains of
Salinibacter and Haloquadratum represent the pool
of genes these organisms have in the environment
32
Species diversity in the crystallizer as deduced
from 16S rDNA approach 80 Haloquadratum walsbyi
(all within 98.5 16S rDNA similarity) 15
Salinibacter ruber 3 Salinibacter (2nd
species) 2 Other haloarchaea (we know they are
there because they can be isolated and also a few
PCR clones) Reminescent of the acid mine
drainage study Tyson et al, 2004
33
Construction of an environmental genomic library
34
Most DNA belongs toH. walsbyi but not all

• Can we discriminate?
• The GC was a big help (also like in Tyson et al)

35
GC profiles in halophiles and in the crystallizer
36
Fosmid ends 2930 individual reads, ca.3 Mbp
Classification by similarity to the genome of
Haloquadratum walsbyi Martiensried and GC
content
60-80 GClt55 132
No similarity but GClt55 450
80-94 GClt55 254
ANI 97.58
gt94 GClt55 1031
No similarity and GCgt55 1063
37
Fosmids highly related to the Haloquadratum
strain sequenced
Both end reads with gt94 similarity and hits
located at less than 60 Kb in the genome (synteny)
Highlights The vast majority of the genome of
the strain sequenced has been recovered from the
environment in 1500 fosmids
ANI 97.86 GC 47.45
38
Taxonomically relevant fosmids 2B and 7B
39
Conclusions

• The genome of the strain sequenced in
  Martiensried is present in the metagenome, 95 of
  it has been retrieved in a mere 2000 fosmids
  library (reminescent of Tyson et al)
• BUT

40

Some fosmids indicate rearrangements or large
genomic islands in some environmental genomes
Both ends gt94 similarity Average similarity
97.32, GC 47.82
63 fosmids gt 60 Kb (no synteny) Average distance
1.5 Mbp (random distribution)
ANI 97.54 GC48.29
ANI 97.11 GC47.36
41
Borderline with islands of adaptive genes
??No hit
gt94 Haloquadratum
115 fosmids
ANI 97.18 GC 47.97
GC49.57 only 9 seqs gt55
42
2B07
43
Paralog cluster or a second Haloquadratum species?
44
rRNA
rRNA
halomucin
ORI
Discontinity with low GC
Discontinuity with high GC
Discontinuity with 80-94
45
(No Transcript)
46
(No Transcript)
47
Conclusion

• The metagenome contains many genes with the
  Haloquadratum GC and trinucleotide tag that are
  not present in the strain sequenced in
  Martiensried (adaptive pool).
• Very roughly half of the genes with Haloquadratum
  tag in the library are not present in the strain
  sequenced (minimal size of adaptive pool 3 Mb)

48
Conclusion

• About 1/3 of sequences do not have the
  Haloquadratum GC / trinucleotide tag and show
  little similarity to other haloarchaea. But if
  FISH and 16S rDNA data are to be trusted some
  should be in the species gene pool.

49
Comparison with AMD, Tyson et al (2004)

• Similarly low taxonomic diversity but
  crystallizer much higher genomic diversity
• In acid mine all energy derives form
  chemolithotrophic reaction
• Crystallizer is a heterotrophic system (like the
  deep ocean)

ANI 99.92
ANI 97.80
Like a continuous culture with a complex organic
C/E source
50
Part 3. Genomes of Haloquadratum and
Salinibacter and the Habitat Gene Reservoir, HGR)
51
Salinibacter and Haloarchaea Convergence or LGT
52
Significant number apparent HGT events with
haloarchaea but barely 1 of genes show clear
relationship to this group
53
Haloarchaea contain many clear LGT events
54
Lowest average pI value outside haloarchaea
55
Surprise, surprise

• In spite of the similar pIs of proteins and
  lifestyles there is no correlation between pI and
  HGT between Salinibacter and haloarchaea. They do
  not even overlap in which proteins have low pIs

56
However,

• Convergence is not only phenotypic
• HGT from unknown donors has played a role in the
  adaptation of both salinibacter and the
  haloarchea
• HGT or Habitat Gene Reservoir HGR?

57
The remarkable example of the rhodopsins
Halobacterium NRC1
58
Salinibacter has four rhodopsin genes!
Exactly like Halobacterium NRC1
59
(No Transcript)
60
Phototransducer (SR) vs chemoreceptor
61
The transducers
Halobacterium sensory rhodopsin
E. coli chemotaxis transducer
62
Haloarcula marismortui ATCC 43049 YP 135403H
Haloarcula marismortui ATCC 43049 YP 135620H
Halobacterium sp NRC-1 NP 279790H
Haloarcula marismortui ATCC 43049 YP 136942H
Conclusion Haloarchaea and Salinibacter use
their own characteristic family o f Htr proteins
for signal transduction from Sensory rhodopsin.
Halobacterium sp NRC-1 NP 279642H
Halobacterium sp NRC-1 NP 280488H
Halobacterium sp NRC-1 NP 280580H
Haloarcula marismortui ATCC 43049 YP 137199H
Halobacterium sp NRC-1 NP 280509H
Halobacterium sp NRC-1 NP 279786H
Haloarcula marismortui ATCC 43049 YP 137005H
Halobacterium sp NRC-1 NP 279442H
Halobacterium sp NRC-1 NP 279777H
Haloarcula marismortui ATCC 43049 YP 137229H
Haloarcula marismortui Sop2-associated htr (YP
134804)
Halobacterium sp NRC-1 Sop2-associated htr (NP
280332)
Halobacterium sp NRC-1 NP 280506H
Haloarcula marismortui ATCC 43049 YP 135651H
Haloarcula marismortui ATCC 43049 YP 134505H
Halobacterium sp NRC-1 NP 280505H
Haloarcula marismortui Sop1-associated htr (YP
137681)
Halobacterium sp NRC-1 Sop1-associated htr (NP
280433)
Haloarcula marismortui ATCC 43049 YP 135146H
Haloarcula marismortui ATCC 43049 YP 137124H
Halobacterium sp NRC-1 NP 280271H
Haloarcula marismortui ATCC 43049 YP 136650H
Haloarcula marismortui ATCC 43049 YP 134953H
Halobacterium sp NRC-1 NP 280237H
Halobacterium sp NRC-1 NP 280321H
Halobacterium sp NRC-1 NP 279901H
Haloarcula marismortui ATCC 43049 YP 136357H
Pyrococcus abyssi GE5 NP 127232P
Pyrococcus horikoshii OT3 NP 142457P
Thermococcus kodakaraensis KOD1 YP 183043T
ORF00159
ORF01508
ORF00158
ORF00866
ORF00926
ORF00141
ORF02916
Salinibacter flagella-associated Htr ORF02684
Salinibacter SR-1-associated Htr1 ORF02664
Salinibacter SR-associated Htr1 ORF02589
Salinibacter flagella-associated Htr ORF02680
Salinibacter flagella-associated Htr ORF02682
Sinorhizobium meliloti 1021 NP 384553S
Sinorhizobium meliloti 1021 NP 386258S
Sinorhizobium meliloti 1021 NP 386003S
Sinorhizobium meliloti 1021 NP 384983S
Sinorhizobium meliloti 1021 NP 387112S
Sinorhizobium meliloti 1021 NP 384522S
Sinorhizobium meliloti 1021 NP 384815S
Escherichia coli K12 NP 416400E
Escherichia coli K12 NP 418775E
Escherichia coli K12 NP 416399E
Escherichia coli K12 NP 415938E
Bacillus subtilis subsp subtilis str 168 NP
391003B
Bacillus subtilis subsp subtilis str 168 NP
391002B
Bacillus subtilis subsp subtilis str 168 NP
391001B
Bacillus subtilis subsp subtilis str 168 NP
391004B
Bacillus subtilis subsp subtilis str 168 NP
389742B
Methanosarcina mazei Go1 NP 633353M
Methanosarcina acetivorans C2A NP 614993M
ZP 0296669
Thermococcus kodakaraensis KOD1 YP 183051T
Thermococcus kodakaraensis KOD1 YP 184560T
Pyrococcus abyssi GE5 NP 127224P
Pyrococcus horikoshii OT3 NP 142466P
Thermococcus kodakaraensis KOD1 YP 182569T
Pyrococcus abyssi GE5 NP 126114P
Pyrococcus horikoshii OT3 NP 143680P
Pyrococcus horikoshii OT3 NP 142424P
Pyrococcus abyssi GE5 NP 127264P
Methanococcus maripaludis S2 NP 988049M
Methanococcus maripaludis S2 NP 987533M
Methanococcus maripaludis S2 NP 987607M
Methanococcus maripaludis S2 NP 987908M
Methanosarcina mazei Go1 NP 632357M
Methanosarcina acetivorans C2A NP 617964M
Methanosarcina mazei Go1 NP 633682M
ZP 0147913
Archaeoglobus fulgidus DSM 4304 NP 069867A
Archaeoglobus fulgidus DSM 4304 NP 069878A
Helicobacter pylori J99 NP 222797H
Helicobacter pylori J99 NP 222812H
Synechocystis sp PCC 6803 NP 442715S
Synechocystis sp PCC 6803 NP 442716S
0.1
63
Isoprenoid biosynthesis
Haloarchaea mevalonate pathway
Salinibacter Deoxyxylulose pathway like in
Chlorobium
64
(No Transcript)
65
(No Transcript)
66
(No Transcript)
67
(No Transcript)
68
D-rich domain
D-rich domain
Leader peptide
VGGL(x)35 repeat
D/S repeat
69
(No Transcript)
70
(No Transcript)
71
Sialic acid decoration
Haloquadratum one ORF for N-acetyl neuramicin
acid synthase in a high GC island
Salinibacter two ORFS for the N-acetylneuraminic
acid synthase in a low GC island
72
Respiratory chain and microaerophilic metabolism
73
(No Transcript)
74
Nitrate reduction
75
Habitat Gene Reservoir of saturated brines (at
least)

• Rhodopsins as alternative energy sources and
  phototactic behaviour
• Ion transport
• Cell envelope
• Respiratory chain with anaerobic/microaerophilic
  components

76
Habitat Gene Reservoir

• Genes relevant for survival in a specific habitat
  
• They do not associate to any phylogenetic pattern
  
• Inserted into different cell backgrounds
  (phylogenies) by molecular tinkering

77

• The evolutionary species concept Ernst Mayr
• The shape of biodiversity

...an entity composed of organisms which
maintains its identity from other such entities
through time and over space, and which has its
own independent evolutionary fate and historical
tendencies. Wiley and Mayden, 1997.
Species are seen as time-space worms that
exchange different allelles for the same genes
78
HGR habitat B
Global Genome
The shape of prokaryotic biodiversity
HGR habitat A
Pan-genome species A
Species A core
Pan-genome species B
Species B core
Species C core
HGR habitat C
79
Co-workers

• My lab
• Boris Legault
• Arantxa López-López
• Jose Carlos Alba
• Doolittles lab
• Thane Papke
• Ford Doolittle
• David Walsh
• Adrian Sharma

• TIGR (Salinibacter team)
• Emmanuel Mongodin
• Karen Nelson
• MPI Martiensried (H walsbyi team)
• Henk Bolhuis
• Dieter Oesterhelt

Thanks!