Automated Microbial Genome Annotation

Report
MICROBIAL GENOME
ANNOTATION
Loren Hauser
Miriam Land
Yun-Juan Chang
Frank Larimer
Doug Hyatt
Cynthia Jeffries
1
NEB Educational Support
http://www.neb.com/nebecomm/course_support.asp?
2
Why study Computational Biology
and Bioinformatics?
 DNA sequencing output is growing faster than
Moore’s law!
 1 Illumina sequencing machine = 0.5 Tbp/week
 There are hundreds of these and thousands of
other sequencing machines around the world.
 New sequencing technology will conceivably
allow sequencing a human genome for less
than $1K in less than 1 day!
3
Why study Medical Bioinformatics?
 In the near future, most cancer diagnostics
will involved DNA or RNA sequencing!
 In the near future, every baby born in the
developed world will have their genome
sequenced. Protecting privacy and your
doctors ability to use that information are the
only real impediments!
 Hospitals are using DNA sequencing to track
antibiotic resistant bacterial infections.
4
DOE Undergraduate Research in
Microbial Genome Analysis and
Functional Genomics
http://www.jgi.doe.gov/education
5
Why Study Microbial Genomes?
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Large biological mass (50% of total)
photosynthetic (Prochlorococcus)
fix N2 gas to NH3 (Rhodopseudomonas)
NH3 to NO2 (Nitrosomonas)
bioremediation (Shewanella, Burkholderia)
pathogens, BW (Yersinia pestis - plague)
food production (Lactobacillus)
CH4 production (Methanosarcina)
H2 production (Rhodopseudomonas)
6
Example of Current Microbial
Genome Projects
 UC Davis – FDA funded 100K bacterial
genomes project associated with food.
 5 years = 20K per year / 200 days/year =
100 genomes/day!
7
Web Resources and Contact Information
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http://genome.ornl.gov/microbial/
http://www.jgi.doe.gov/
http://genome.jgi-psf.org/
http://www.jcvi.org/
http://www.ncbi.nlm.nih.gov/
http://www.sanger.ac.uk/
http://www.ebi.ac.uk/
ftp://ftp.lsd.ornl.gov/pub/JGI
 artemis ready files for each scaffold =
(feature table plus fasta sequence file)
 Contact:
 [email protected]; [email protected]
8
9
Evolution of Sequencing
Throughput
Sequencing Technology
Maxam and Gilbert
Manual Sanger
Automated Sanger (96 lanes/gel)
Automated Sanger (384 capilaries)
454 sequencing (new titanium)
Solexa (Illumina)
Solexa (Illumina)
PacBio realtime sequencing
Samples/run bp/sample runs/week
1
100
5
5
400
5
100
500
5
400
600
10
1,000,000
400
5
300,000,000
75
1
1,000,000,000
200
1
100,000,000
1000
10
bp/week
year
500 1977
10000 1985
250000 1995
2400000 2002
2E+09 2009
2.25E+10 2009
2.00E+12 ?2010
1E+12 ?2010
Sequenced Microbial Genomes
 ARCHAEAL GENOMES
 159 FINISHED; 218 IN PROGRESS
 BACTERIAL GENOMES
 3363 FINISHED; 11831 IN PROGRESS
 ENVIRONMENTAL COMMUNITIES
 > 50,000 samples (see MGRast)
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as of Sept 6, 2012
http://www.expasy.ch/alinks.html
http://www.genomesonline.org
http://metagenomics.anl.gov/
11
Published Genomes
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Nitrosomonas europaea - J.Bac. 185(9):2759-2773 (2003)
Prochlorococcus MED4 & MIT9313 - Nature 424:1042-1047 (2003)
Synechococcus WH8102 - Nature 424:1037-1042 (2003)
Rhodopseudomonas palustris - Nat. Biotech. 22(1):55-61 (2004)
Yersinia pseudotuberculosis - PNAS 101(22):13826-31 (2004)
Nitrobacter winogradskyi – Appl. Envir. Micro. 72(3):2050-63 (2006)
Nitrosococcus oceani - Appl. Envir. Micro. 72(9):6299-315 (2006)
Burkholderia xenovorans – PNAS 103(42):15280-7 (2006)
Thiomicrospira crunogena – PLoS Biology 4(12):e383 (2006)
Nitrosomonas eutropha C91 – Env. Micro. 9(12):2993-3007 (2007)
Sulfuromonas denitrificans – Appl. Envir. Micro. 74(4):1145-56 (2008)
Nitrosospira multiformis -- Appl. Envir. Micro. 74(11):3559-72 (2008)
Nitrobacter hamburgensis -- Appl. Envir. Micro. 74(9):2852-63 (2008)
Saccharophagus degradans – PLoS Genetics 4(5):e1000087 (2008)
R. palustris – 5 strain comparison – PNAS 105(47):18543-8 (2008)
L. rubarum and L. ferrodiazotrophum – Appl. Envir. Micro. (in press)
12
Basic Annotation Impacts
 Design of oligonucleotide arrays
 Design & prioritize protein expression
constructs
 Design & prioritize gene knockouts
 Assessment of overall metabolic capacity
 Database for proteomics
 Allows visualization of whole genome
13
Additional Analysis Impacts
 Revised functional assignments based on
domain fusions, functional clustering,
phylogenetic profile
 Regulatory motif discovery
 Operon and regulon discovery
 Regulatory and protein association
network discovery
14
Microbial
Annotation
Genome
Pipeline
Scaffolds
or
contigs
Simple
repeats
Prodigal
Complex
Repeats
Model
correction
tRNAs
Final Gene
List
InterPro
PRIAM
Blast
COGs
TMHMM
SignalP
rRNA,
Misc_RNAs
GC Content,
GC skew
Function call
Web
Pages
Feature
table
15
Prodigal (Prokaryotic Dynamic
Programming Genefinding Algorithm)
 Unsupervised: Automatically learns the statistical
properties of the genome.
 Indifferent to GC Content: Prodigal performs well
irrespective of the GC content of the organism.
 Draft: Prodigal can train on multiple sequences
then analyze individual draft sequences.
 Open Source: Prodigal is freely available under
the GPL.
 Reference: Hyatt D, Chen GL, Locascio PF, Land
ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic
gene recognition and translation initiation site
identification. BMC Bioinformatics. 2010 Mar
8;11(1):119. (Highly Accessed)
16
G+C Frame Plot Training
 Takes all ORFs above a specified length in
the genome.
 Examines the G+C bias in each frame
position of these ORFs.
 Does a dynamic programming algorithm
using G+C frame bias as its coding
scoring function to predict genes.
 Takes those predicted genes and gathers
dicodon usage statistics.
17
Gene Prediction
 Dicodon usage coding score
 Length factor added to coding score (GCcontent-dependent)
 Coding/noncoding thresholds sharpened (starts
downstream of starts with higher coding get
penalized by the difference).
 Dynamic programming to put genes together.
 Bonuses for operon distances, larger bonus for
-1/-4 overlaps.
 Same strand overlap allowed (up to 60 bases).
 Opposite strand -->3'r 5'f<- allowed (up to 250
bases)
18
Start Site Scoring
Shine Dalgarno Motif
 Examines initially predicted genes and gathers
statistics on the starts (RBS motifs, ATG vs GTG
vs TTG frequency)
 Moves starts based on these discoveries.
 Gathers statistics on the new set of starts and
repeats this process until convergence (5-10
iterations).
 RBS motifs based on AGGAGG sequence, 3-6
base motifs, with one mismatch allowed in 5
base or longer motifs (e.g. GGTGG, or AGCAG).
 Does a final dynamic programming with the
start scoring function.
19
Start Site Scoring
Other Motifs
 If Shine-Dalgarno scoring is strong, use it –
this accounts for ~85% of genomes.
 If Shine-Dalgarno scoring is weak, look for
other motifs
 If a strong scoring motif is found, use it
(example GGTG in A. pernix)
 If no strong scoring motif is found, use
highest score of all found motifs (example –
Crenarchaea, Tc and Tl start sites are the
same, but internal operon genes use weak
Shine-Dalgarno motifs)
20
Annotated Gene Prediction
21
Prodigal Scoring
22
Gene Prediction Problems –
Pseudogenes
23
Pseudogenes – Internal deletion
24
Pseudogenes – Premature stop codon
25
Pseudogenes – N-terminal deletion
26
Pseudogenes – Transposon insertion
27
Pseudogenes – Multiple frameshifts
28
Pseudogenes – Premature Stop and
Frameshift
29
Pseudogenes – Dead Start Codon
30
31
GENE PAGE
32
33
34
35
ORGANISM’S (PSYC) COGS LIST
Contig
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Contig1
Gene
1
3
4
6
8
10
11
14
15
16
17
19
20
23
24
25
26
27
28
29
29
30
31
33
34
35
36
37
38
39
40
41
42
43
45
46
47
51
52
53
57
58
59
59
60
61
62
64
65
66
67
68
69
70
71
74
76
77
78
78
Num Prot
-------------------------------------------------------------
Group
I
E
S
K
R
L
COG
COG1211
COG0137
COG1376
COG0583
COG0628
COG0593
J
G
COG0172
COG0021
L
L
G
R
P
P
COG0551
COG0507
COG0057
COG1451
COG0168
COG0569
M
COG2885
M
R
R
R
COG1538
COG1538
COG2274
COG1566
C
R
Q
P
S
R
P
R
COG2010
COG3019
COG2132
COG3667
COG3544
COG0491
COG2217
COG1826
S
G
O
J
J
M
S
O
O
C
O
O
F
R
I
R
M
S
P
P
E
L
O
COG1937
COG2814
COG0435
COG0261
COG0211
COG2834
COG4399
COG1138
COG0526
COG0526
COG3088
COG4235
COG0563
COG1949
COG1502
COG0790
COG1519
COG1385
COG1840
COG1178
COG3842
COG0188
COG0625
R
T
COG0515
COG0515
Gene Name
COG Des c ription
Sc ore
E-Value
Category
4-diphos phoc
Is pD
y tidy l-2-methy l-D-erithritol
86
9.00E-19
s y nthas
Lipid
e metabolis m
Argininos uc
ArgG
c inate s y nthas e 565 1.00E-162 Amino ac id trans
Unc harac teriz
ErfK
ed protein c ons erv
61
ed 4.00E-11
in bac teria
Func tion unk now
Trans c riptional
Ly s R
regulator
123
2.00E-29 Trans c ription
Predic ted permeas
PerM
e
145
6.00E-36 General func tion
ATPas e inv
DnaA
olved in DNA replic
104
ation
8.00E-24
initiation DNA replic ation
No COG
Sery l-tRNASerS
s y nthetas e
557 1.00E-160 Trans lation ribos
Trans k etolas
Tk
e
tA
992
0 Carbohy drate tra
No COG
Zn-finger domain
TopA as s oc iated with
38 topois
9.00E-04
omeras
DNA
e ty
replic
pe I ation
ATP-dependent
Rec D
ex oDNAs e (ex
61
onuc
3.00E-10
leas e V) DNA
alphareplic
s ubunit
ation
- h
Gly c eraldehy
GapA
de-3-phos phate 344
dehy drogenas
7.00E-96e/ery
Carbohy
thros
drate
e-4-pho
tra
Predic ted metal-dependent
COG1451
hy
104
drolas
3.00E-24
e
General func tion
Trk -ty pe K+
Trk
trans
G
port s y s tems
181
membrane
1.00E-46
c omponents
Inorganic ion tra
K+ trans port
Trk
sA
y s tems NAD-binding
125
4.00E-30
c omponent
Inorganic ion tra
No COG
Outer membrane
OmpA protein and 113
related
1.00E-26
peptidogly
Cell
c an-as
envelope
s oc iate
bio
No COG
Outer membrane
TolC
protein
114
2.00E-26 Cell envelope bio
Outer membrane
TolC
protein
114
2.00E-26 Cell envelope bio
ABC-ty pe bac
SunT
terioc in/lantibiotic
410ex
1.00E-115
porters c ontain
Defens
an
e mec
N-termi
han
Multidrug res
EmrA
is tanc e efflux pump
82
7.00E-17 Defens e mec han
No COG
No COG
Cy toc hrome
Cc
ccmonoA
and diheme
38
v
3.00E-04
ariants
Energy produc tio
Predic ted metal-binding
COG3019
protein
136
1.00E-33 General func tion
Putative multic
SufIopper ox idas es
271
9.00E-74 Sec ondary meta
Unc harac teriz
Pc ed
oB protein involv
148
ed in 6.00E-37
c opper res
Inorganic
is tanc e ion tra
Unc harac teriz
COG3544
ed protein c ons erv
43
ed 8.00E-06
in bac teria
Func tion unk now
Zn-dependent
GloB
hy drolas es inc luding
100
2.00E-22
gly ox y lasGeneral
es
func tion
Cation trans
Zport
ntA ATPas e
754
0 Inorganic ion tra
Sec -independent
TatA
protein s ec retion
59
4.00E-11
pathway c
Intrac
omponents
ellular traffi
No COG
Unc harac teriz
COG1937
ed protein c ons erv
49
ed 5.00E-08
in bac teria
Func tion unk now
Arabinos e efflux
AraJ permeas e
57
2.00E-09 Carbohy drate tra
Predic ted glutathione
ECM4
S-trans507
feras
1.00E-145
e
Pos ttrans lationa
Ribos omal RplU
protein L21
126
3.00E-31 Trans lation ribos
Ribos omal RpmA
protein L27
119
4.00E-29 Trans lation ribos
Outer membrane
LolA
lipoprotein-s
122
orting
3.00E-29
protein
Cell envelope bio
Unc harac teriz
COG4399
ed protein c ons erv
41
ed 2.00E-04
in bac teria
Func tion unk now
Cy toc hrome
Cc
cmF
biogenes is fac589
tor
1.00E-169 Pos ttrans lationa
Thiol-dis ulfide
Trx is
A omeras e and thioredox
49
2.00E-07
ins
Pos ttrans lationa
Thiol-dis ulfide
Trx is
A omeras e and thioredox
49
2.00E-07
ins
Pos ttrans lationa
Unc harac teriz
Cc ed
mH
protein involv
157
ed in 4.00E-40
bios y nthes
Pos
is ttrans
of c -ty
lationa
pe c y t
Cy toc hrome
COG4235
c biogenes is fac103
tor
3.00E-23 Pos ttrans lationa
Adeny late k
Adk
inas e and related168
k inas
3.00E-43
es
Nuc leotide trans
Oligoribonuc
Orn
leas e (3'->5' ex oribonuc
293
7.00E-81
leas e)
RNA proc es s ing
Phos phatidy
Cls
ls erine/phos phatidy
230
lgly
1.00E-61
c erophos Lipid
phate/c
metabolis
ardiolipin
m
FOG: TPR COG0790
repeat SEL1 s ubfamily
56
4.00E-09 General func tion
3-deox y -D-manno-oc
KdtA
tulos onic
330
-ac id2.00E-91
trans feras
Cell
e
envelope bio
Unc harac teriz
COG1385
ed protein c ons120
erved 1.00E-28
in bac teria
Func tion unk now
ABC-ty pe Fe3+
AfuA trans port s y s
164
tem periplas
1.00E-41
mic
Inorganic
c omponent
ion tra
ABC-ty pe Fe3+
ThiP trans port s y s
287
tem permeas
1.00E-78
e Inorganic
c omponent
ion tra
ABC-ty pe s
PotA
permidine/putres c
318
ine trans
5.00E-88
port s y
Amino
s tems ac
AT
id
Pas
trans
e
Ty pe IIA topois
Gy rA
omeras e (DNA
941
gy ras e/topo 0
II DNA
topois
replic
omeras
ation
e I
GlutathioneGs
S-trans
t
feras e
94
7.00E-21 Pos ttrans lationa
No COG
Serine/threonine
SPS1protein k inas e
83
2.00E-17 General func tion
Serine/threonine
SPS1protein k inas e
83
2.00E-17 General func tion
36
Taxonomic Distribution of Top
KEGG BLAST Hits
37
Frequency distance distributions
Salgado et al.
PNAS (2000)
97:6652
Fig. 2
38
Frequency distance distributions
Salgado et al.
PNAS (2000)
97:6652
Fig. 3b
39
Branched Chain Amino Acid
Transporter family
Organism
Nostoc punctiforme
Trichodesmium erythraeum
Helicobacter pylori J99
Helicobacter pylori 26695
Campylobacter jejuni subsp. jejuni NCTC 11168
Geobacter metallidurans
Desulfovibrio desulfuricans
Escherichia coli K12
Escherichia coli O157:H7 EDL933
Buchnera sp. APS
Pseudomonas aeruginosa, PAO1
Pseudomonas fluorescens
Pseudomonas syringae
Psychrobacter
Vibrio cholerae O1 biovar eltor str. N16961
Yersinia pestis, CO92
Yersinia pseudotuberculosis
Haemophilus influenzae Rd KW20
Pasteurella multocida subsp. multocida str. Pm70
Xylella fastidiosa (3 strains)
Azotobacter vinlandii
Psychrobacter
Burkholderia fungorum
Burkholderia mallei
Burkhoderia pseudomallei
Ralstonia metallidurans
Ralstonia eutropha
Nitrosomonas europaea
Neisseria meningitidis MC58
Neisseria meningitidis Z2491
Caulobacter crescentus
Mesorhizobium loti
Agrobacerium tumefaciens
Bradyrhizobium japonicum
Brucella melitenis
Brucella suis
Sinorhizobium meliloti
Rickettsia conorii
Rickettsia prowazekii
Rhodobacter sphaerodes
Rhodospirillum rubrum
Rhodopseudomonas palustris
Cyano
Cyano
epsilon
epsilon
epsilon
delta
delta
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
gamma
beta
beta
beta
beta
beta
beta
beta
beta
alpha
alpha
alpha
alpha
alpha
alpha
alpha
alpha
alpha
alpha
alpha
alpha
JGI
JGI
COG
COG
COG
JGI
JGI
COG
COG
COG
COG
JGI
JGI
JGI
COG
COG
JGI
COG
COG
COG, JGI
JGI
JGI
JGI
JGI
JGI
JGI
COG
COG
COG
COG
COG
COG
JGI
JGI
JGI
ATPase
ATPase
Permease PBP
COG0410 COG0411 COG0559
COG0683
3
3
6
4
2
1
3
6
0
0
0
0
0
0
0
0
1
1
2
2
1
1
2
1
2
2
4
4
1
1
2
2
1
1
2
2
0
0
0
0
3
3
7
4
3
3
5
3
7
4
8
5
0
0
1
0
0
0
1
0
2
2
4
2
2
2
4
2
0
0
0
0
0
0
0
0
0
0
0
0
3
4
8
2
0
0
0
0
22
20
34
29
6
6
11
8
7
7
13
10
9
8
16
12
18
19
36
28
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7
7
16
10
7
7
15
9
27
26
50
59
7
7
12
13
6
6
12
11
5
5
12
8
0
0
1
0
0
0
1
0
6
6
12
5
6
7
13
9
20
20
40
38
40
Probable Ancient Gene (Liv Operon)
41
Branched Chain Amino Acid Transporter
family – Rhodopseudomonas palustris
Target ID
Description
Putative Ligand
RPA0985
putative branched-chain amino acid transport
system substrate-binding protein
branched chain AAs
RPA4029
RPA4648
possible branched-chain amino acid ABC transport
system substrate-binding protein
possible ABC transporter binding protein
component
branched chain AAs
Thermal Shift Assay
Binding Ligand
4-Hydroxybenzoate,
Benzoate, Salicylate,
Benzaldehyde
4-Hydroxybenzoate, pCoumarate
Δ Tm °C for 1000 uM
Ligand OR (100uM ) Ligand
Tm(°C)
No
Ligand
29.0,13.5, 2.5, 2.0
56.5
17.0, 2.0
58.6
spermidine/putrescine
p-Coumarate
2.0
55.5
RPA1250
amide-urea binding protein
branched chain AAs
Urea
5.0
63.0
RPA1789
putative branched-chain amino acid transport
system substrate-binding protein
branched chain AAs
p-Coumarate
7.0
67.0
branched chain AAs
Urea
6.0
59.5
branched chain AAs
Ala, Gly,Ser, Met, Leu,
Cys
11.5, 6.5, 4.5, 2.5, 2.0, 2.0
77.5
nitrate/taurine
Malate
4.0
52.5
amino acids, prefers
polar aas
Met, Cys, His
10.0, 6.5, 3.5
63.0
13.0, (6.0, 2.0 )
61.5
6.0, 3.0, 3.0, 2.0, 2.0
52.0
RPA3669
RPA3810
RPA2043
RPA2628
RPA0668
RPA1741
RPA2193
RPA3486
RPA2499
putative urea short-chain amide or branched-chain
amino acid uptake ABC transporter periplasmic
solute-binding protein precursor
putative periplasmic binding protein of ABC
transporter
putative ABC transporter, periplasmic substratebinding protein
polar amino acid ABC transport substrate-binding
protein, aapJ-2 (aapJ-2)
putative ABC transporter subunit, substrate-binding
component
possible branched-chain amino acid transport
system substrate-binding protein
putative ABC transporter, perplasmic binding
protein, branched chain amino acids
putative branched-chain amino acid transport
system substrate-binding protein
possible ABC transporter, periplasmic protein
branched chain AAs
branched chain AAs
4-Hydroxybenzoate,
Salicylate,
Benzaldehyde
Met, Leu, Malate, Gly,
Pro
branched chain AAs
Glutarate
5.0
64.5
branched chain AAs
Glutarate
3.0
44.5
nitrate/taurine or
aliphatic sulfonates
Asn
7.0
53.5
42
Example of Lateral Transfer
43
Transporter Gene Loss
in Yersina Pestis
 36 Genes involved in transport from YPSE
are nonfunctional in YPES
 13 lost due to frameshifts
 11 lost due to deletions
 6 lost due to IS element insertions
 4 (2 pair) lost due to recombination
causing deletions and frameshifts
 2 lost due to premature stop codons
44
45
Nostoc punctiforme
Signal Transduction Histidine Kinases
46
Nostoc punctiforme
Signal Transduction Histidine Kinases
47
Nostoc punctiforme
Signal Transduction Histidine Kinases
Gene #
R1448
R1449
R1550
R1597
R1685
R1759
R1760
R1778
R1798
R1868
R2035
R2209
R2262
R2263
R2268
R2271
R2272
R2375
R2408
R2421
R2485
R2901
R2903
R2909
R3010
R3052
aa#
374
444
595
1042
1559
706
595
451
1098
713
1080
430
657
740
709
504
1801
1211
928
421
530
629
1116
103
210
475
COG
COG0642
COG0642
COG0642
COG4191
COG0642
COG4251
COG0642
COG5002
COG0642
COG0642
COG0642
COG0642
COG0642
COG0642
COG0642
COG4191
COG3899
COG5278
COG4585
COG4585
COG0642
COG0642
COG4251
COG4251
COG0642
COG2205
N-term.
(TM)
N-term.
RRR
1
1
unk. (2)
Other domain PAS/PAC GAF(PHY)
1 Chase/1 Hpt
unk. (2)
unk.(1)
unk. (3)
1
HAMP +
TM
1
4 (3)
3(1)
2 (1)
1
1
1
5
1
1
1
1
2
1
1 Prt. Kin.
1 Chase
1 Cache
(sp)
unk. (1)
unk. (4)
unk.
1
1
unk. (1)
1
1
1
3
1
1
2 (1)
1
HisKA HATPase
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1?
1
1
1
HisKA_3
1
HisKA_3
1
1
1
1
1
1
1
0
0.5
1
0
1
1
C-term.
RRR
Operon structure
K1448/K1449
K1448/K1449
2
1
1
1
1
1
2
RRR1757/RRR1758/K1759/K1760
RRR1757/RRR1758/K1759/K1760
K1778/WHTH1779
K-R1798/K-F1799/LuxR-F1800
K-R2035/cNMP-F2036
2262-9
2262-9
2262-9
K2271/PK&K2272
K2271/PK&K2272
3
LuxR2407/K2408
LuxR2420/K2421
1
K2901/RRR2902/K2903
K2901/RRR2902/K2903
48
Nostoc punctiforme
Signal Transduction Histidine Kinases
169
12
3
154
2
1
12
3
3
6
3
1
23
15
66
46
61
59
21
64
82
3
1
1
1
2
5
8
TM
RRR
predicted genes total
pseudogenes
genes with sensors but no kinase domain (do these work with the genes with no sensor domains - not in the same operon)
functional Signal Transduction Histidine Kinases
with 2 kinase domains (fused genes? or a 1 gene cascade?)
with an Adenylate Cyclase domain
with a Ser/Thr Protein Kinase domain, a COG3899 domain, 1 or more GAF domains, and possibly other domains
with Hpt domain
with CBS domains
with Chase domains
with Cache domains
with an Amino acid transporter as a sensor? domain
with a N-terminal RRR domain
with only a N-terminal RRR as a sensor? domain
with 1 or more RRR domains (86 RRR domains)
with 1 or more C-terminal RRR domains (ie. Hybrid kinases)
with 1 or more PAS/PAC domains (147 PAS/PAC domains total)
with 1 or more GAF or Phytochrome domains (96 total - 38 phytochromes)
with HAMP domains (34 total)
with unknown N-terminal sensor domains
with multiple N-terminal sensor domains
with no sensor domain (do these work with the genes with no kinase domains - not in the same operon)
with large C-terminal unknown domain
with N-terminal RRR & WHTH (fused genes?)
cNMP binding sensor domain
with HisKA_2 type dimerization/autophosphorylation domains
with HisKA_3 type dimerization/autophosphorylation domains
putative operons with common (bidirectional) promoter
Transmembrane alpha helical domain
response regulator receiver domain (Phospho accepting Asp containing domain)
49
Nostoc punctiforme
Regulatory Proteins
570 Regulatory Proteins
Comments/Pseudogenes
201 Transcription/Elongation/Termination Factors
14
9
0
0
2
2
1
Sigma Factors
Cyanobacterial Sigma Factors
Sigma-54 (RpoN)
Sigma 32 (RpoH)
Sigma 28 (Flagella/Sporulation)
Sigma-24 (RpoE/FecI) (ECF subfamily)
Unknown Sigma factor (ECF subfamily)
17
1
8
1
1
5
1
Anti/Anti-Anti Sigma Factors
Anti-Sigma regulatory factor (Ser/Thr protein kinase and phosphatase)
Anti-Sigma-factor antagonist (STAS) domain protein
Anti-Sigma-factor antagonist (STAS) and sugar transfersase
Predicted transmembrane transcriptional regulator (anti-sigma factor)
Putative Anti-Sigma regulatory factor (Ser/Thr protein kinase)
Sigma 54 modulation protein/ribosomal protein S30EA
3
1
1
1
Termination/Antitermination Factors
NusA antitermination factor
NusB antitermination factor
NusG antitermination factor
2 sets of pseudogenes: pNPAR018 truncated by transposase; pNPAR022, 3, 4 ar
1 set of pseudogenes: NpR2325/6
S1 RNA binding domain:KH domain / RNA binding
0 Elongation Factors
0 GreA/GreB family elongation factors
167
3
1
2
1
1
6
1
1
1
5
1
5
Transcription factors
Ferric uptake regulator (FUR) family
Negative regulator of class I heat shock protein
phage shock protein A, PspA
Phosphate uptake regulator, PhoU
Plasmid maintenance system antidote protein
Predicted transcriptional regulator
SOS-response transcriptional repressor, LexA
Putative transcriptional acitvator, Baf
Transcriptional Regulator, AbrB family
Transcriptional Regulator, AraC family
Transcriptional Regulator, AraC family with Methyltransferase activity
Two Component Transcriptional Regulator, AraC family
4 different COGs
50
Burkholderia xenovorans
Regulatory Proteins
946 Regulatory Proteins
Comments
704 Transcription/Elongation/Termination Factors
22
4
2
2
1
12
1
Sigma Factors
Sigma 70 (RpoD)
Sigma-54 (RpoN)
Sigma 32 (RpoH)
Sigma 28 (Flagella/Sporulation)
Sigma-24 (RpoE/FecI) (ECF subfamily)
Unknown Sigma factor (ECF subfamily)
13
1
1
1
2
4
1
1
1
1
Anti/Anti-Anti Sigma Factors
Anti Sigma-E protein, RseA, Burkholderiaceae specific
Anti-Sigma regulatory factor (Ser/Thr protein kinase and phosphatase)
Anti-Sigma(ECF) factor, ChrR
Anti-Sigma-factor antagonist (STAS) domain protein
Predicted transmembrane transcriptional regulator (anti-sigma factor)
Putative Anti-Sigma regulatory factor (Ser/Thr protein kinase)
Putative Anti-Sigma-28 factor, FlgM
Putative Sigma E regulatory protein, MucB/RseB
Sigma-54 modulation protein
6
1
2
1
1
1
Termination/Antitermination Factors
transcription termination factor Rho
Response regulator receiver (CheY) and ANTAR domain protein
NusA antitermination factor
NusB antitermination factor
NusG antitermination factor
also called ribosomal protein S30AE
Cold-shock DNA-binding domain(related to S1 RNA binding domain)
ANTAR = RNA binding, anti-termination
S1 RNA binding domain:KH domain / RNA binding
3 Elongation Factors
3 GreA/GreB family elongation factors
660
7
1
2
1
1
Transcription factors
Cold-shock DNA-binding domain protein
Possible Ferric uptake regulator (FUR) family
Ferric uptake regulator (FUR) family
Negative regulator of class I heat shock protein
Negative transcriptional regulator
51
Regulatory Protein
Identification Scheme
Number Category Product Description
COG1
COG2
InterPro
COG0840
COG0840
Pfam
Smart
MCPsignal
MA
MCPsignal and Tar
MCPsignal
MCPsignal and Cache
MCPsignal
MCPsignal
MA and TarH
MA
MA
MA and GAF
MA and GAF
5
5
5
5
5
5
5
5
5
Chemotaxis Signal
Possible
Transduction
Bacterial chemotaxis sensory transducer
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer, TarH (aspartate) sensor
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer, Pas/Pac sensor
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer, Cache sensor
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer, GAF sensor
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer, Phytochrome sensor
Chemotaxis Signal
Bacterial
Transduction
chemotaxis sensory transducer, Phytochrome sensor
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Chemotaxis Signal
CheW Transduction
protein
COG0835
CheW
Chemotaxis Signal
CheW Transduction
protein
IPR002545
Chemotaxis Signal
Two component
Transduction
CheW protein
IPR002545 and IPR001789
Chemotaxis Signal
Possible
Transduction
CheA Signal Transduction Histidine Kinases (STHK), weak homolog,
COG0643
no good domain identification
Chemotaxis Signal
Possible
Transduction
CheA Signal Transduction Histidine Kinases (STHK)
COG0643
HATPase_c
Chemotaxis Signal
CheA Signal
Transduction
Transduction Histidine Kinases (STHK)
COG0643
IPR008207 and IPR003594
Chemotaxis Signal
CheA Signal
Transduction
Transduction Histidine Kinases (STHK)
IPR002545 and IPR003594 and IPR004105
Chemotaxis Signal
CheB methylesterase
Transduction
COG2201
CheB_methylest
Chemotaxis Signal
CheB methylesterase
Transduction
IPR000673 and IPR001789 CheB_methylest
Chemotaxis Signal
Two component
Transduction
CheB methylesterase
COG2201
IPR001789
CheB_methylest
Chemotaxis Signal
MCP methyltransferase,
Transduction
CheR-type
COG1352
CheR
Chemotaxis Signal
MCP methyltransferase,
Transduction
CheR-type
IPR000780
CheR
Chemotaxis Signal
MCP methyltransferase,
Transduction
CheR-type with PAS/PAC sensor
COG1352
CheR
Chemotaxis Signal
MCP methyltransferase/methylesterase,
Transduction
CheR/CheB with PAS/PAC sensor
COG1352
CheR and CheB_methylest
Chemotaxis Signal
CheC,Transduction
inhibitor of MCP methylation
COG1776
Chemotaxis Signal
CheD,Transduction
stimulates methylation of MCP proteins
COG1871
TIGR
IPR004089
COG0840
COG0840
COG0840
COG0840
COG0840
IPR001294
IPR001294 and IPR004089
sensory_box
CheW
CheW
CheW
HATPase_c
HPT and CheW
MeTrc
MeTrc
MeTrc
MeTrc
sensory_box
sensory_box
52
Summary of automated transporter
annotation --- Zymomonas
317
Transporter Proteins
69
82
116
2
2
14
29
1
2
3
4
5
8
9
Channels/Pores
Electrochemical Potential-driven transporters
Primary Active Transporters
Group Translocators
Transport Electron Carriers
Accessory Factors Involved in Transport
Incompletely Characterized Transport Systems
23
46
73
9
103
2
13
2
1
1
14
12
17
1.A
1.B
2.A
2.C
3.A
3.B
3.D
4.A
5.A
5.B
8.A
9.A
9.B
alpha-type channels
beta barrel porins
Porters (uniporters, symporters, antiporters)
Ion-gradient-driven energizers
P-P-bond-hydrolysis-driven transporters
Decarboxylation-driven transporters
Oxidoreduction-driven transporters
Phosphotransfer-driven group translocators
Transmembrane 2-Electron Transfer Carriers
Transmembrane 1-Electron Transfer Carriers
Auxiliary transport proteins
Recognized transporters of unknown biochemical mechanism
Putative uncharacterized transport proteins
53
Zymomonas transporters
complete listing
GROUP
Porters
Porters
2.A.53
sulfate transporter or Xanthine/uracil/vitamin C transporter
carbonic anhydrase, sulfate transporter SulP family
2 proteins
or0489
2.A.53
or1027
2.A.53
GROUP
Porters
Porters
Porters
Porters
Porters
Porters
Porters
Porters
2.A.6
putative lipooligosaccharide nodulation factor exporter, NolGHI, RND superfamily
hydrophobe/amphiphile efflux-1 HAE1, RND superfamily
acriflavin resistance protein, RND superfamily
efflux transporter, RND family, MFP subunit
acriflavin resistance protein, RND superfamily
acriflavin resistance protein, RND superfamily
hopanoid biosynthesis associated RND transporter like protein HpnN
export membrane protein SecD, RND superfamily
8 proteins
or0146
or0252
or0704
or1290
or1378
or1379
or1439
or1719
GROUP
Porters
Porters
Porters
2.A.64
twin-arginine translocation protein TatC
twin-arginine translocation protein TatB
twin-arginine translocation protein TatA
3 proteins
or1107
2.A.64.1.1
or1108
2.A.64.1.1
or1109
2.A.64.1.1
GROUP
Porters
Porters
Porters
Porters
Porters
2.A.66
multi antimicrobial extrusion protein MatE
polysaccharide biosynthesis protein
polysaccharide biosynthesis protein
polysaccharide biosynthesis protein
virulence factor MviN family
5 proteins
or0190
or0202
or1191
or1303
or1478
GROUP
Porters
Porters
2.A.69
predicted transporter, putative auxin efflux carrier component
predicted transporter, putative auxin efflux carrier component
2 proteins
or0625
2.A.69.2./1
or0626
2.A.69.2./1
2.A.6.3/2
2.A.6.2
2.A.6.2
2.A.6.2/3.A.1.122/8.A.1
2.A.6.2
2.A.6.2
2.A.6.5/7
2.A.6.4.1
2.A.66.1
2.A.66.2
2.A.66.2
2.A.66.2
2.A.66.4.1
54
Transcriptome Analysis Pipeline:
RNA sequences to GRN
Collect
RNAseq
data
Map
reads to
genomes
Predict
operons
In silico
Compare operon
determinations
(genome coordinates)
Improve
algorithm
Determine
orthologs with
OrthoMCL
Determine
orthologous
operons
Calculate
reads/bp
Display
frequency
plot
Cluster analysis
of gene
expression
changes
Determine
operons from
frequency plot
Align
orthologous
promoters
Determine TISs
with 5’ RACE.
Determine
TFBS from
alignments
Cluster analysis
from gene
expression arrays
GRN genetic
regulatory
network
Predict
TFBS
In silico
Dynamic range and sensitivity
New gene, wrong start, riboswitch
Small Regulatory RNA ???
Differential gene expression
Operon with Internal Promoter
60
Long Term Vision
 Develop TPing SOPs, and an automated
analysis pipeline.
 Initially produce TPs and preliminary GRNs for
all important DOE microbial genomes (i.e.
BESC), and eventually all DOE microbial
genomes.
 Incorporate the TP analysis pipeline into ORNL’s
automated microbial annotation pipeline, and
eventually into IMG and GenBank files.
 Add additional experimental methods to improve
the GRN determinations.

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