1. GENOMIC VARIABILITY WITHIN A
POPULATION OF Alteromonas
macleodii FROM A SINGLE DEEP
MEDITERRANEAN SAMPLE
Francisco Rodriguez-Valera

2. Introducción
Welch et al, 2002
Ussery, D

3. Human The great divide: constreined
sex (a.k.a. meiosis)
70%
Drosophila
70% genes in
common

4. All this diversity only with
different alleles of the same
genes

6. Metagenome B +viriome
Global Genome
The shape of prokaryotic biodiversity
Metagenome A+ viriome
Pan-genome species A
Species A core
Pan-genome species B
Species B core
Species C core
Metagenome C +viriome

7. Periodic-Selection (Atwood et al, 1951)
The unit of
selection:
The clone?
Giovannoni
Nature 430: 515 (2004)

8. Periodic selection clonal sweep

9. • How to reconcile PS with large pan-
genomes?
• Study the genes found in the
accessory pool

10. Solar Saltern
Evapora. Carbonate Gypsum Halite
Ratio Domain Domain Domain No
Intermediate
(Vi/Vo) 36-70 g/l 70-140 g/l 220-290 g/l >290 g/l life
140-220 g/l
Diatoms
Haloarchaea
Cyanobacteria
Haloquadratum
Green Algae Sulphur Phototrophic Bacteria
Artenia Salina
walsbyi
Dunaliella
up to 70-95% of the
population
Along many years:
“unique” low GC
content

11. H. walsbyi is a unique specie
Num. Mbp over 95% id (in 70% length) = 398.8
A
100
95
90
%id
85
80
75
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Haloquadratum walsbyi HSBQ001 chr. (3132494 bp)
B C House-keeping genes
Metagenome
AtpB
SecY
Tef2
RadA
Num. Seqs.
Num. Seqs.
%id 398.8Mp %id

12. Environmental pan-genome of H. walsbyi
GI 1 GI 2 GI 3 GI 4
(promer)
(BLASTN)
H. Walsbyi (3132494 bp)

13. e-fosmids of H. walsbyi Variability of GI 1
Cell surface glycoprotein 100 90 80 70 60 50 40 30 %sim.
Cell surface adhesin
Putative S-layer protein
Dentin-sialophosphoprotrein
2'3'-cyclic-nucleotide 2'-phosphodiesterase
IS element 240631 279189
Ig-like domain-containing protein H.w. C23
Protease
ABC-type cobalamin/iron(III)
eHw-1
Hmu2 H.w.
239281 312087 HBSQ001
eHw-4
eHw-7
eHw-559
eHw-5
eHw-12
eHw-6
eHw-9

14. METAGENOMIC ISLANDS
GOS marine metagenomes
GENOMIC ISLANDS
O-chain LPS Giant protein Transporters EXTRACELLULAR COMPONENTS IN GENOMIC ISLANDS
Pili Exo-polysaccharide Other
Prochlorococcus marinus MED4 Prochlorococcus marinus MIT9301
Pelagibacter ubique HTCC1062
100%
% sim.
Pelagibacter ubique HTCC1062 (1308759 bp)
50% Burkholderia sp. 383 chr.1 Shewanella sp. MR-4
Pili O-chain LPS Giant Exo- Pili
Transporters protein polysaccharide
related
Pelagibacter ubique HTCC1002 contigs
100%
Aeromonas hydrophila subs. hydrophila
ATCC 7966 Synechococcus sp. WH812
% sim.
50%
Transmembrane/ O-chain LPS Pili Respiratory system Giant
Outer membrane Transporters Fe transporter protein
proteins Pili
Solar salter metagenomes
Pelagibacter ubique HTCC7211
100%
Salinibacter ruber DSM 13855 Haloquadratum walsbyi DSM 16790
100 100
% sim.
95 95
Y Axis Title
Y Axis Title
90 90
85 85
50%
80 80
O-chain LPS Pili Zn transporter, Phosphonate Pili
O-chain 75 Exo-polysaccharide 75 Cell surface Cell surface
and Phosphate metabolism LPS 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 glycoproteins
0 500000 1000000 1500000 2000000
glycoproteins
2500000 3000000
X Axis Title
X Axis Title

15. Low recruiting genes (MGIs and
islets)
• Coding for exposed features: O-chain of LPS,
glycoproteins etc, pili, flagella
• Transporters
• Sensors and regulators (behaviour)
• Sub-niche specialization e.g. microaerophilic
growth

18. Alteromonas macleodii
•  Proteobacterium isolated by Baumann
from surface waters near Hawaii in the
‘60s
• Common isolate
• Heterotrophic aerobe, wide range of
substrates as C,E source, 2-3 m long,
typical copiotroph r strategist
• Mostly found in the particulate fraction

19. 160 isolates on MA characterized by 16S
rRNA sequencing , 28% A. macleodii
2%
Alteromonas macleodii DE strain genome
4% 2% 2%
Alteromonas sp. Sequenced
Sample from 6%
Bacillus sp.
1000 m deep 5% 28%
Sulfitobacter sp.
South Adriatic 10%
Rhodobacteraceae
28% 5% bacterium SCSWE04
8% Oceanobacillus
iheyensis
3 other strains
17/05/2003; 22:14 R/V Urania
Bottom ca. 1100 m sequenced now
200 L retrieved by rossete
(single cast) from 1000 m AD1000 Metagenomic
fosmid library ca. 38,000
clones (1.75 Gbp or 600
prokaryotic genomes)
Sampling site

20. Variation of A. macleodii in a
single sample of 200 L
If we consider that the cell density at
the location is 105 cells per ml and
accept the figure of 5% belonging to
A. macleodii, in 200 liters we will
have about 5x108 cells of A.
macleodii (about the population that
you have in a common 100 ml
laboratory culture)

21. End sequencing of ca 8000 fosmids
• 15968 individual reads
• 424 (2.6%) could be affiliated to A.
macleodii DE (>98% nuc identity over
300 nucs), average 99.04%
Purificación
López-García

22. Alteromonas macleodii DE
1x106 2x106 3x106 4x106
57,2
GC% 44,9
33,4
0,16
GC%
0
skew
-0,16
Microaerobic Metal Flagellins LPS O-Chain Peripheral
extracellular Phage Nitrate respiration
respiration resistance Hg resistance Giant protein LPS O-chain
polysaccharide CRISPR
Hydrogenases
(pEPS)
eFosmids
6 Mb
Not AD1
synthenic AD2
rRNAs
tRNAs
100%
95%
AD 1
80%
100%
English
95%
Channel
673
80%
100%
Balearic 95%
Sea 45
80%
100%
Black 95%
Sea 11
80%

23. 4216 Genes annotated in 182 fosmids of A.
macleodii sequenced.
Similarity #ORFs
100 1032 (24.47%)
98-100 1837 (43.57%)
BlastN
90-98 405 (9.60%)
80-90 132 (3.13%)
70-80 25 (0.59%)
<70 781 (18.52%)
80-100 4
60-80 33
BlastP
40-60 147 Regulatory and outer layer related genes increase
20-40 149
<20 454

24. 16S 23S

25. Peripheral extracellular
polysaccharide (pEPS)
Fosmid eAmacleodii
AD1000-21-D04
2,600,664 99,98 2,789,943
Amacleodii DE
98,04 60 85 98,65 98,45
Amacleodii AD1
Fosmid eAmacleodii 100 100
AD1000-5-F10
Fosmid eAmacleodii 99,99
AD1000-42-B04
Fosmid eAmacleodii
AD1000-9-F08
Phenylalanyl tRNA
synthetase α/β subunit
Phosphodiesterase.
Msc protein Mannose ybaK/ebsC p.
(MscS family) O-antigen polymerase Hydrolase
guanylyltransferase
Fosmid eAmacleodii Fructosamine
EPS biosynthesis
AD1000-21-D04 Kinase
Eps P/D
99,98
Amacleodii DE
69 94 85
Amacleodii AD1
Ribosomal L20,35 Transcription
Phenylalanyl tRNA
repair Sensor histide
synthetase α/β subunit
kinase
β-hexoaminidase
EPS Cluster
Polysaccharide related p. Glycosyl transferase
tRNA synthetase Hypothetical p.
Periplasmic Transferases p.
Ribosomal proteins Integrase, IS element

26. Different exposed structures e.g. exopolysaccharide

27. Fosmids containing
Ribosomal Operon II
Intervening Sequence
tRNA
5S
Fosmid eAmacleodii
16S 23S
AD1000-207-B8
99,46 98,83 100
Amacleodii DE
99,06 98,31 99,18
Amacleodii AD1
Fosmid eAmacleodii
100 100 100 ATP-dependent
Methyl-accepting AD1000-231-F10 helicase HrpB
chemotaxis p. Methyl-accepting RNA
CheY/A/W/D
Synonymous 2
chemotaxis p. Ligase
Non-synonymous 0 0
Fosmid eAmacleodii Chemotaxis 4 Na+/H+ Peptidyl- Membrane
AD1000-207-B8
methylesterase 1 1
antiporter dipeptidase Dcp transglycolase
99,78 98,77 99,98 99,23 99,96
Amacleodii DE
423.708 520.976
99,7 99,21 98,01 98 99,38 99,67
Amacleodii AD1
99,9 Aconitate Glycosyltransferase
hydratase Lipopolysaccharide
Fosmid eAmacleodii heptosyltransferase
AD1000-231-F10 Synonymous 3
Non-synonymous 2
7 34
2 22
5
4
8 42
0 15
43 22 22 20
17 2 2 1
32
2
8 1 1
2 0 1
Bacterial type II secretion
0 10 1
1 0 2
system protein E:GAF
GGDEF Transcriptional
Cryptic haloacid
Permease DMT Repressor LacI Twin-arginine translocation Branched-chain amino
dehalogenase
Hydrolase/ Oxidoreductase pathway signal Acid aminotransferase
phosphatase
Two-component system Iron-containing alcohol Phosphoglyceromutase
Sensor kinase deshydrogenase
Trehalose Cluster
1 2 3 4
Ribosomal proteins Trehalose Cluster
Pseudoalteromonas atlantica T6c Transferase proteins
tRNA tblast
TblastX
46 51 56 62 67 73 78 83 89 94
Chemotaxis related proteins x% ID
Pseudoalteromonas tunicata D2 rRNA 1 transcriptional regulator
Transposase/Integrase 2 trehalose permease
Shewanella frigidimarina NCIMB400 Hypotetical protein 3 TonB-dependent OM receptor
Glycosyl transferase 4 Trehalase

28. Fosmids containing
Ribosomal Operon III
Sensory BOX GGDEF
Bacterio-phytochrome Lactate Cluster
Fosmid eAmacleodii 5S 23S 16S
AD1000-249-G9 4 3 2 1
Fosmid eAmacleodii 68 85 98 99 98 82 91
AD1000-357-C11 99 99 99 4 3 2 1
85 98 98 82 80 98
Amacleodii DE 4
99 68 99 98 98 98,2 81 96 85 97
Amacleodii AD1 4
Fosmid eAmacleodii 99 100 99 99 98 ChrR
Pseudouridine
AD1000-112-F00 synthase D
Adenine Lipoprotein ComL
Recombination GGDEF domain TRP repeat
phosphoribosyltransferase
Adenylate kinase RecR p. DNA polymerase III CDP-diacylglycerol--serine Caseinolyti RNA polymerase
Transcriptional regulator O-phosphatidyltransferase
Chaperone HtpG subunits gamma and tau c peptidase sigma 70 factor
B
A. macleodii ATCC 4 3 2 1
Vibrio parahemolyticus 16 4 3 2 1
Psychromonas ingrahamii 37 3 2
rRNA Lactate Cluster
tRNA
Integrase, IS element 1 Hyphothetical protein
2 Lactate Responsive regulator
Glycosyl transferase 3 Lactate permease
4 Lactate dehydrogenase
Hypothetical p.

29. Different degradative capabilities eAmacleodii AD1000-
207-B8 can use trehalose while DE cannot (checked
phenotypically)

30. Blastn
Blastp Sulfotransferase
Poliysaccharide UDP galactopyranose Motor
P-ring Acyl CoA Flagellar M-ring Flagellar
biosynthesis p. switch p.
FlaA precursor DegT/J reductase FlaL Sensor biosynthesis p. Chemotaxis
P-loop
CheW/R FliC aminotransferase kinase FliM FlgH Cluster A/Z/Y
FlaM/N FlgB/C/D/E FlgJ/K/L Flagellin FlaK FlaM CheW
F/G/H Maf-1 FlaR FliC FliK FliO FliA
FliE
AD1000-39-B06
Amacleodii AD1/
98,9% 99~100%
Fosmid eAmacleodii ~72%
AD1000-9-A03 96% 82% ~60%
52% ~65% 80% 96% 99~100%
Amacleodii DE/ AD3/ AD4
NeuB:Antifreeze- Acylneuraminate
FliC. like p. cytidylyltransferase
FliD Methionyl-tRNA
formyltransferase
FliC Amacleodii AD1/
AD1000-39-B06
72% 99% 98% 99%
Fosmid eAmacleodii
FlgL AD1000-9-A03
66% 81% 85% 99%
FlgK
Amacleodii DE/ AD3/ AD4
FlgE
FlgD
FlgF FlgG FliC FlaK FlaM
FlgG FlaL
Sensor
Flagellum Cluster Glycosyl transferase
Extracellular Hypothetical p.
Regulatory Transferases p.
Integrase, IS element
Periplasmic
Phage related
Cytoplasmic tRNA

31. Vibrio parahaemolyticus
FlaL
FlaM
FlaK

32. Synonymous 241
FlaK
Non-synonymous 49
81%
Regulatory domain
DNA binding domain

33. Synonymous 128
FlaL
Non-synonymous 44
85%
(sensor of two-component system)
FlaL
Sensory box
TM1
Variable region
in AD1000-39-B06

34. Flagellum cluster Variability
Extracellular>Regulation>>cytoplasmic
Acetyl CoA Diguanalate Transglycosilase
cyclase STL Smr Domain ABC Transporter
carboxylase
containing p. RTX Toxin
DNA pol Cell cycle p. Kef-type K+ FGAM Thrombospondin type 3
subunit α MesJ transport system Synthetase repeat:OmpA/MotB
Synonymous 151 181 38 69 110 155 31 32 12 16 28 10 15 75 75 31 33
Non-synymous 28 19 6 3 16 23 9 0 6 4 4 10 3 15 18 6 2 Mer B,A,R
eAmacleodii
AD1000-7-B08 A. macleodii DE
98% 98%
60% 78% 83% 83% 1.483.121 99% 1.518.371
A. macleodii DE
HlyD Sterol 3.203.823
HKLuxR UmuC desaturase Mer B,A,R
FliD FliC FlgL FlgK FlgJ FlgL FlgG FlgE FlgC FlgA FlgN
Transposase
FlaG FlgH FlgF FlgD FlgB
FlgM
CheR/V
GI 9

35. Different exposed receptors, flagelins and their
glycosilation BUT ALSO
behaviour at the level of flagellum synthesis i.e. the
lineage represented by the sequenced strain and that
represented by fosmid eAmacleodii AD1000-39-B06
could have different responses to environmental signals
that either induce or block synthesis of the flagellum i.e.
produce flagella under different conditions.

36. Constant-Diversity
Nature Reviews Microbiology ,
November 2009

37. San Francisco Bay salt pond
POM Alteromonas macleodii and Co
CO2 + H2O
300 major
organic
compounds
Problem: Solution:
only the transporters will to have many
occupy the 3 Mpb of one
individual cell
DNA ? lineages sharing
the feast
(3132494 pb)
Problem: what about clonal sweeps? Solution:
phages
One lineage will be more efficient in consuming the
limiting nutrient

38. Red Queen and
Constant-Diversity

39. What does natural selection act upon – genes,
individuals, groups of individuals (such as a kin group),
or the species as a whole?
• The unit of selection in the prokaryotic world
might be a complex consortium that includes several
clonal cellular lineages sharing a core genome but
differing in an adaptive gene complements that
allows the population a much more efficient
exploitation of available resources. These pan-clonal
populations are maintained equalized by phage
predation in an analogous way as the immune
system in a mammal maintains in check tumors. If
this is so, phage populations should be considered
as belonging to the same selection unit.

40. Alicante (Spain), Evolutionary
University Miguel Hernández Genomics Group
Ana Belén Rohit Ghai
Post-doc. Post-doc.
Inma García
Pre-doc. Carol Mizuno
Aitor Gonzaga Pre-doc.
Pre-doc. Sandra Rocamora
Pre-doc.

41. Thank you very much!
Alicante (Spain),
University Miguel Hernández
Aitor Gonzaga
Ana-Belén
Martín-Cuadrado
Rohit Ghai

43. Evapora. Carbonate Gypsum Halite
Ratio Domain Intermediate Domain Domain No BACTERIOME
36-70 g/l life *Santa pola saltern :
(Vi/Vo) 70-140 g/l 140-220 g/l 220-290 g/l >290 g/l DNA Bulk DNA
0.22 microns sp37, sp33, sp32, Time-series
5 microns
extraction pyrosequencing
1041 Mpb
(400pb)
*Torrevieja saltern: 145 Mpb
Diatoms Particles / small Archaea
Haloarchaea eukaryotes (& Bacteria) * Santa pola saltern:
Cyanobacteria Metagenomic
Precipitation
sp37 clones
Mg & K
Green Algae Sulphur Phototrophic Bacteria fosmids library
Artenia Salina (35 Kb) *Torrevieja saltern: 5952 clones
Dunaliella
GI 1 GI 2 GI 3 GI 4
100
Haloquadratum walsbyi chromosome
% id
90
(3132494 bp)
Nu. SNP
80
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Lengt (bp)
100 90 80 70 60 50 40 30 %sim. eHw-1 H. walsbyi DSM 16790
Cell surface glycoprotein
Cell surface adhesin 0 0
Putative S-layer protein
Dentin-sialophosphoprotrein 10 10
2'3'-cyclic-nucleotide diesterase 20 20
IS element C23
Ig-like domain 240631 279189 0 10000 20000 30000 0 10000 20000 30000 40000 50000 60000 70000
Protease eHw-4 H. walsbyi C23
ABC-type cobalamin/iron(III)
eHw-1
0 0
10 10
HBSQ001 Hmu2 20 20
239281 312087
0 10000 20000 30000 40000 0 10000 20000 30000
eHw-4 eHw-5 eHw-559
0 0
10 10
eHw-7
20 20
0 10000 20000 30000 0 10000 20000 30000
eHw-559 eHw-9 eHw-6
0 0
eHw-5 10 10
20 20
0 10000 20000 30000 0 10000 20000 30000
eHw-12
eHw-7 eHw-12
0 0
eHw-6
10 10
20 20
eHw-9 0
0 10000 20000 30000 10000 20000 30000

44. OBJETIVE
HOW MANY DIFFERENT
Haloquadratum walsbyi
clones exists?
Core-genome
how
different Pan-genome “In situ”, from the
do they they are? of H. walsbyi metagenome
vary along
time?
are equally Accesory-genome
distributed ?

45. What we have?: Sequences
Two genomes Haloquadratum walsbyi strains sequenced
(in theory) HBSQ001 C23
4 SAGs of H. + (2000)
Sanger
(2011)
454 pyro.
walsbyi 3132494 bp 3148034 bp
Metagenomic collections of Santa Pola saltern :
DNA Bulk DNA pyrosequencing (FLX, 400pb)
5 m 0.22 m extraction
sp37 (2008)
sp33 (2010)
sp32 (2007)
prokariots time-series (2005) 1041 Mpb
+
Torrevieja (Alicante) Saltern (2010) 145 Mbp
Fosmids libraries. 33 Sequenced fosmids Analysis of
2947
fosmid ends
DNA Metagenomic fosmids library (35 Kb)
5 m 0.22 m extraction
sp (2002) 2000 clones
sp37 (2008) 5952 clones
23 fosmids (2006) (Sanger)
prokariots
Torrevieja saltern: 2880 clones 10 fosmids GI1 (2009) FLX