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Microbial Genomics:
Bizarre Bacteria, Exotic Environments,
and How They Interact
Naomi Ward
The Institute for Genomic Research (TIGR)
What is TIGR?
• independent, not-for-profit research institute
• vast majority of work supported by federal
grants and contracts (NIH, NSF, USDA, DOE)
•1995: first whole genome sequence of a freeliving organism (Haemophilus influenzae)
• conducts genomic and post-genomic research
on microbes, plants, parasites, and humans
• www.tigr.org
Archaeoglobus
fulgidus
Bacillus
subtilis
Mycoplasma
pneumoniae
Synechocystis
sp.
Haemophilus
influenzae
Mycoplasma
genitalium
Rickettsia
prowazekii
Methanobacterium
thermoautotrophicum
Helicobacter
pylori
Chlamydia
trachomatis
Mycobacteriu
m
tuberculosis
Aquifex
aeolicus
Saccharomyces
Borrelia Treponema
pallidum
cerevisiae burgdorferi
Methanococcus
jannaschii
Archaea
Eubacteria
Eucaryote
Escherichia
coli
Pyrococcus
horikoshii
Neisseria
meningitidis
Neisseria
meningitidis
Aeropyrum
pernix
Deinococcus
Chlamydia radiodurans Xylella
fastidiosa
pneumoniae
Thermotog
a maritima
Vibrio
Campylobactercholerae
jejuni
Pseudomonas
Helicobacter
aeruginosa
pylori
Chlamydia
pneumoniae
Chlamydia
trachomatis
GENOMICS
ECOLOGY
TAXONOMY
Research areas/questions:
1. What underlies the ecological “success” of
cosmopolitan taxa?
Ubiquitous distribution
Related to genomic repertoire?
2. What role does the normal flora play in
biology of the host and ecosystem function?
Nutrition
Reproduction
Susceptibility to disease
Ecosystem resilience
How has 16S ribosomal RNA gene sequence
analysis helped bacterial taxonomy?
“We, as primates evolutionarily committed to vision as
a principal sense, comprehend this blooming and
buzzing confusion by ordering and classifying,
separating and comparing. ”
Stephen Jay Gould
Morphology = a natural taxonomy for bacteria
How has 16S ribosomal RNA gene sequence
analysis helped bacterial taxonomy?
Morphology + biochemistry + molecular chronometer
= a more natural taxonomy for bacteria
How has 16S ribosomal RNA gene sequence analysis
helped microbial ecology?
Genomic
DNA
extraction
PCR
amplification
of 16S rDNA
Cloning
P. aeruginosa
ATTCGATCCG...
B. cereus
M. luteus
B. subtilis
P. stutzeri
Sequencing
and analysis
Why do we care about uncultured bacteria?
Because they represent >99% of all bacteria
(Great Plate Count Anomaly)
Cultivation-dependent method
isolates
16S rRNA gene sequencing
Culturing methods
Microbial diversity
structure
Molecular microbial “ecology”
Cultivation-independent method
Development of large
database
Notable uncultured bacteria
80% of the human intestinal flora
EPR and Galapagos Rift 2002 (25th anniversary)
Riftia tube biofilm: what is
the bacterial composition,
and does it attract and
facilitate attachment of
larval tubeworms?
WGS (Whole Genome Shotgun) Sequencing strategy:
vector
vector
primer
insert
Genomic DNA
Paired reads
repeat
repeat
WGS (Whole Genome Shotgun) Sequencing strategy:
Closed (finished)
genome
sequence
Annotating the genome sequence:
Classification of proteins into families
with conserved function
Sequence similarity
Domain structure
Biochemical annotations
Evolutionary relatedness
Identification with profile HMMs
Bacterial genomes <1Mb:
Mycoplasma spp.
Ureaplasma urealyticum
Phytoplasma asteris
Mesoplasma florum
Wigglesworthia glossinidia
Buchnera aphidicola
Blochmannia floridanus
Tropheryma whipplei
Borrelia garinii
Bacterial genomes >5.5Mb:
Escherichia coli (some), Ralstonia solanacearum
Photorhabdus luminescens, Bacillus anthracis
Burkholderia mallei, pseudomallei
Nocardia farcinica
Pseudomonas aeruginosa, putida, syringae
Nostoc sp., Bacteroides thetaiotaomicron
Photobacterium profundum
Sinorhizobium meliloti
Rhodopirellula baltica, Mesorhizobium loti
Streptomyces coelicolor, avermitilis
Bradyrhizobium japonicum
1
2
3
4
5
6
7
8
9
10
11 Mb
Cryptosporidium spp.
Guillardia theta
Nanoarchaeum
equitans
CcBv
Mimivirus
Encephalitozoon
cuniculi
Methanosarcina
acetivorans
Ashbya gossypii
Bacteria
Archaea
Eukarya
Viruses
GENOMICS
Silicibacter
pomeroyi
ECOLOGY
TAXONOMY
The genome of Silicibacter pomeroyi DSS-3:
a key organism in the marine food web
1974 - Pomeroy recognized that
marine bacteria are an active
component of the oceanic food web
Roseobacters have a key
role in marine and
atmospheric sulfur cycles
"Demethylation"
Pathway
"Cleavage"
Pathway
DMSP
CH3
CH3 S CH2 CH2 COO-
MeSH
DMS
CH3 SH
SH-
SO4-2
CH3 S CH3
methionine
protein
Roseobacters are
ubiquitous
moo
Cultured
Uncultured
Open ocean
Coastal ocean
Sediments
Marine plant- or
animal-associated
Sea ice
Biofilms
Most ecologically important marine organisms
are oligotrophs
Roseobacters are the exception
% of Clones
0
5
10
15
20
25
30
SAR11
Roseobacter
SAR116
SAR86
Taxa with culturable
members
Marine Actinobacteria
Marine Picophytoplankton
SAR202
SAR324
Aphotic Zone
SAR406/Marine Group A
Giovannoni and Rappé 2000
What did we learn from the S. pomeroyi genome?
Genes involved in proposed pathways for organic sulfur
transformation in marine roseobacters
* Gene identified in S. pomeroyi genome
1. DMSP transport protein
2. DMSP lyase
3. DMSP methyltransferase
4. MMPA b-lyase or generic C-S lyase
5. MMPA methyltransferase
6. DMSO reductase/DMS oxidase
7. Acrylase
8. Acrylase-CoA ligase
9. Cystathionine g-synthase [metB]
10. Inorganic sulfur metabolism
Strict heterotroph vs supplemental lithotrophy?
- Two coxSML operons in genome (oxidation of CO to CO2)
- CO oxidation at concentrations typical of surface sea water
experimentally confirmed
Coastal
[CO]
Open ocean
[CO]
Consumption of CO in the headspace of triplicate cultures
Moran et al., 2004 Nature
Strict heterotroph vs supplemental lithotrophy?
- Gene cluster for oxidation of reduced inorganic sulfur (sox)
- Presence of thiosulfate experimentally confirmed to enhance biomass
production by 45% relative to cultures receiving no sulfur
Growth in acetate medium with (closed symbols) or without (open
symbols) 10mM thiosulfate
Moran et al., 2004 Nature
Metagenomics allows access to the full genomic
potential of uncultivated microbes
Cultivation-dependent methods
Genome sequencing
isolates
16S rRNA gene sequencing
Culturing
methods
Development of
large database
Microbial diversity
Development of
large database
function
structure
Metagenome sequencing
Molecular microbial “ecology” (16S-based)
Cultivation-independent methods
Is the supplemental lithoheterotrophy
of S. pomeroyi representative of uncultured
relatives?
Analyzed gene stoichiometry in Sargasso Sea environmental
shotgun library (Venter et al.., 2004):
Moran et al., 2004 Nature
Suggests lithoheterotrophy involving CO and inorganic S are
distributed beyond this group
Confirmation that supplemental
lithoheterotrophy is a more widespread lifestyle
Phylogenetic tree showing
the relationship of COoxidizing isolates (with a
"JT-" prefix) to select
cultured and
environmental sequences
Tolli et al., 2006 AEM
Why sequence a genome?
S. pomeroyi: a marine “opportunitroph” and lithoheterotroph
CO2
Transporters for
peptide uptake
amino acid efflux
CO oxidation
CO
Marine DOM
in surface water
{
{
photodegradation
TRAP and glycine betaine
transporters
Mannitol
Glycerol
Other algal osmolytes
Citrate
Malonate
Glyoxylate
Phytoplankton
breakdown
GENOMICS
Hyphomonas
neptunium
(dimorphic
prosthecate
bacteria
DPB)
ECOLOGY
TAXONOMY
Other markers:
Hyphomonas belongs to
the order
Caulobacterales?
23S rRNA
HSP70 proteins
30 concatenated ribosomal proteins
16S rRNA: the conventional wisdom
EF-Tu proteins
Hyphomonas belongs to the order
Rhodobacterales
Why sequence a genome?
Whole-genome analysis confirms
the Hyphomonas-Caulobacter relationship
Overall gene complement
Cell cycle regulation
Outer membrane complements
Hn shares more
genes with Cc
than with Sp
“We taxonomists and natural historians are said
to be the fuddy-duddies and accountants of
science--maintainers of the lists and guardians
of the storehouses...
The physicist Lord Rutherford, at the turn of the
century, called us glorified stamp collectors…”
Stephen Jay Gould
Multiple genome sequences are necessary to define
the Streptococcus agalactiae (Group B strep) “pan-genome”
Tettelin et al., 2005 PNAS
Background, The “V” project
(Genomic tools for studying the ecology
of the human vaginal microflora)
Women can be clustered into “supergroups” representing
specific vaginal community types
Rank abundance of vaginal microbial community supergroups in
African-American (red) and Caucasian (blue) women
Preliminary data from Larry Forney
Thanks to:
NSF
NIH-NIAID
USDA
NOAA OE
RAs, Team Leaders, and Directors of TIGR
Seqcore/JTC
Jonathan Badger, Kevin Penn, Sunni Page,
Tommie Schneider