Electronic supplementary material
Comparative proteomic analysis reveals mechanistic insights into
Pseudomonas putida F1 growth on benzoate and citrate
Manolis Mandalakis,†,‡ Nicolai Panikov,†,§ Shujia Dai,† Somak Ray,† Barry L. Karger*,†
† Barnett Institute, Northeastern University, Boston, MA 02115
‡ Present Address: Department of Chemistry, University of Crete, Heraklion, Greece, GR71409
§ Present Address: Harvard School of Public Health, Department of Immunology and
Infectious Diseases, Boston, MA 02115
* To whom correspondence should be addressed. B.L. Karger: E-mail: b.karger@neu.edu,
Phone: 617.373.2867, FAX: 617.373.8795
1
Computational analysis of shotgun proteomics data
The proteomic data were processed using Proteome Discoverer (version 1.3; Thermo
Fisher Scientific, Waltham, MA) and a local MASCOT server (version 2.3; Matrix Science,
London, UK). The MS/MS spectra were searched against the protein database of P. putida
F1, consisting of SwissProt and its TrEMBL supplement (5,245 protein entries by June 2012;
http://www.uniprot.org/). A precursor ion tolerance of 10 ppm and a fragment ion tolerance
of 0.8 Da (CID) or 0.05 Da (HCD) were specified. Trypsin with a maximum of two missed
cleavages was selected; carbamidomethylation of cysteine, TMT labeling on both lysine and
N-terminal amine of peptides were set as fixed modifications. Finally, a target-decoy
approach (Elias and Gygi 2007) was applied to maintain the false discovery rate (FDR) below
1% for peptide identification. Proteins with at least two unique peptides were regarded as
confident identifications, while all single-peptide hits were excluded from further analysis.
Relative quantitation of proteins was achieved by pairwise comparison of TMT
reporter ion intensities among samples using DanteR software (version 0.1.1; Pacific
Northwest National Laboratory, Richland, WA; http://omics.pnl.gov). Protein ratios were
calculated as the geometric mean of the respective peptide ratios with their statistical
significance assessed by ANOVA (Roxas and Li 2008). Peptides showing co-isolation
interference higher than 25% were discarded from this analysis, and quantitation was applied
only for proteins showing at least two peptide ratios. Furthermore, an empirical Bayesian
analysis (EBA) reported by Margolin et al. (2009) and Wojcechowskyj et al. (2011) was
applied to derive differentially expressed proteins. In essence, the log-ratio distribution of
quantified proteins was modeled by a mixture of three independent component distributions.
The central part of the data or the “null model” with no differential expression was assumed
to be a Gaussian distribution, while the positive and negative tails, which correspond to the
differentially expressed proteins, were modeled by a Generalized Pareto distribution. The
2
parameters describing the distributions were derived by applying the method of maximum
likelihood fitting, and the resulting values were used to estimate the complete marginal
density of the distribution. The Bayesian posterior probability for each protein not belonging
to the “null model” was estimated from the null and marginal density.
Based on the empirical Bayesian framework, the posterior probability of a protein
showing differential abundance was defined by a local false discovery rate (FDR) threshold.
The local FDR for threshold K is the sum of all the null probabilities for ratios that would be
denoted as discoveries (i.e. that have a posterior probability of being non-null that is higher
than K). By using EBA, the thresholds of ratios for differentially expressed proteins were set
at a corresponding local FDR of 5% (i.e. fold-change < 0.68 or > 1.47). An additional
threshold for the ANOVA p-values was set at <0.01 to keep only those proteins showing
statistically significant changes.
The lists of up- and down-regulated proteins were divided into functional categories
by performing an extensive search of several on-line protein function databases (KEGG
Pathway Database, InterPro, UniProt). Protein lists were also subjected to Gene Ontology
(GO) enrichment analysis using DAVID functional classification tool (Database for
Annotation, Visualization and Integrated Discovery, http://david.abcc.ncifcrf.gov/) (Huang et
al. 2009a, 2009b).
References
Elias JE, Gygi SP (2007) Target-decoy search strategy for increased confidence in large-scale
protein identifications by mass spectrometry. Nat Methods 4:207–214
Roxas BA, Li Q (2008) Significance analysis of microarray for relative quantitation of
LC/MS data in proteomics. BMC Bioinformatics 9:187
3
Margolin AA, Ong SE, Schenone M, Gould R, Schreiber SL, Carr SA, Golub TR (2009)
Empirical Bayes analysis of quantitative proteomics experiments. PLoS ONE 4:e7454
Wojcechowskyj
JA,
Lee
JY,
Seeholzer
SH,
Doms
RW
(2011)
Quantitative
phosphoproteomics of CXCL12 (SDF-1) signaling. PLoS ONE 6:e24918
Huang DW, Sherman BT, Lempicki RA (2009a) Systematic and integrative analysis of large
gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57
Huang DW, Sherman BT, Lempicki RA (2009b) Bioinformatics enrichment tools: paths
toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–
13
4
Table S1. List of preferentially expressed proteins in benzoate-grown cells.
Pput_1871
Uniprot
accession
A5W1L0
3-oxoadipate CoA-transferase alpha subunit
MW
[kDa]
24.3
No of unique
peptides
11
69
Counts of
TMT127
7
Counts of
TMT129
0
Counts of
TMT127/TMT129
0
Fold-change
(TMT127/TMT129)
∞
Benzoate Catabolism
Pput_1870
A5W1K9
3-oxoadipate CoA-transferase beta subunit
22.5
6
48
8
0
0
∞
3
Benzoate Catabolism
Pput_2053
A5W233
Muconate cycloisomerase
-
40.2
26
80
75
18
14
30.29
2.7E-07
4
5
Benzoate Catabolism
Pput_4346
A5W8K9
Benzoate Catabolism
Pput_4342
A5W8K5
Beta-ketoadipyl CoA thiolase
41.8
26
86
41
18
14
29.47
3.4E-07
4-carboxymuconolactone decarboxylase
14.8
6
49
14
5
5
26.90
6
Benzoate Catabolism
Pput_2055
3.1E-05
A5W235
Catechol 1,2-dioxygenase
34.2
24
70
56
26
20
26.10
7
Benzoate Catabolism
2.0E-07
Pput_2054
A5W234
Muconolactone delta-isomerase
11.1
5
40
22
16
11
20.70
8
2.7E-05
Benzoate Catabolism
Pput_2551
A5W3H6
1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase
27.1
18
78
51
23
18
13.76
6.1E-07
9
Benzoate Catabolism
Pput_4343
A5W8K6
3-oxoadipate enol-lactonase
28.6
17
88
32
18
17
11.73
2.6E-06
10
Benzoate Catabolism
Pput_2554
A5W3H9
Benzoate 1,2-dioxygenase, alpha subunit
51.5
44
93
262
92
72
8.71
1.2E-14
11
Benzoate Catabolism
Pput_2552
A5W3H7
Oxidoreductase FAD/NAD(P)-binding domain protein
36.4
24
77
70
26
25
8.37
3.6E-06
12
Benzoate Catabolism
Pput_4344
A5W8K7
3-carboxy-cis,cis-muconate cycloisomerase
48.2
27
82
35
22
20
7.62
4.5E-05
13
Benzoate Catabolism
Pput_2553
A5W3H8
Benzoate 1,2-dioxygenase, beta subunit
19.1
17
63
75
32
28
7.20
7.0E-08
14
Benzoate Catabolism
Pput_2549
A5W3H4
Catechol 1,2-dioxygenase
33.4
21
87
44
18
15
4.92
2.4E-03
15
Ribosomes
Pput_0513
A5VXS3
50S ribosomal protein L17
14.4
12
65
43
40
37
2.62
2.0E-03
16
Ribosomes
Pput_0476
A5VXN6
50S ribosomal protein L11
14.9
13
73
50
52
46
2.34
2.4E-04
17
Ribosomes
Pput_0482
A5VXP2
30S ribosomal protein S12
13.7
10
67
45
51
40
2.09
5.5E-03
18
Ribosomes
Pput_0720
A5VYC4
50S ribosomal protein L21
11.6
11
78
70
64
57
1.91
2.0E-03
19
Ribosomes
Pput_0488
A5VXP8
50S ribosomal protein L4
21.8
14
82
77
74
72
1.82
8.1E-04
20
Ribosomes
Pput_0487
A5VXP7
50S ribosomal protein L3
22.6
24
82
121
122
113
1.75
1.2E-04
21
Ribosomes
Pput_0503
A5VXR3
50S ribosomal protein L18
12.6
13
72
51
56
50
1.69
9.3E-03
22
Ribosomes
Pput_0483
A5VXP3
30S ribosomal protein S7
17.6
23
84
87
92
80
1.68
1.6E-03
23
Ribosomes
Pput_0511
A5VXS1
30S ribosomal protein S4
23.0
24
81
112
113
106
1.49
1.9E-03
24
Oxidoreductases
Pput_3089
A5W507
Pyrrolo-quinoline quinone
69.1
49
79
121
96
92
4.40
2.4E-19
25
Oxidoreductases
Pput_3161
A5W579
4-hydroxyphenylpyruvate dioxygenase
70.4
18
42
19
15
15
4.23
3.0E-06
26
Oxidoreductases
Pput_4748
A5W9Q8
Azurin
16.1
10
68
33
29
29
2.45
3.3E-04
27
Oxidoreductases
Pput_3083
A5W501
Aldehyde dehydrogenase
54.9
27
73
22
19
18
2.29
1.4E-05
28
Oxidoreductases
Pput_0584
A5VXZ2
Aldehyde dehydrogenase
55.2
39
84
56
53
46
1.95
3.7E-05
29
Oxidoreductases
Pput_3606
A5W6H0
Fatty acid oxidation complex subunit alpha [Includes: Enoyl-CoA
77.3 hydratase/Delta(3)-cis-Delta(2)-trans-enoyl-CoA
37
62
41
isomerase/3-hydroxybutyryl-CoA
39
37
epimerase
1.62
9.9E-04
30
Oxidoreductases
Pput_1281
A5VZY2
NADH:flavin oxidoreductase/NADH oxidase
39.8
14
57
9
12
9
1.61
6.0E-03
31
Oxidoreductases
Pput_3522
A5W688
Acyl-CoA dehydrogenase domain protein
40.5
18
64
19
19
18
1.48
4.2E-03
32
Central metabolism
Pput_1428
A5W0C6
Acetyl-coenzyme A synthetase
76.7
35
59
52
45
43
2.85
7.4E-15
33
Central metabolism (citrate cycle)
Pput_5256
A5WB64
Acetyl-CoA carboxylase carboxyltransferase subunit alpha 51.9
26
68
33
30
30
2.62
7.4E-11
34
Central metabolism
Pput_5255
A5WB63
Oxaloacetate decarboxylase alpha subunit
38
77
57
51
50
2.59
5.4E-11
35
Central metabolism
Pput_4462
A5W8X2
D-isomer specific 2-hydroxyacid dehydrogenase, NAD-binding
34.9
protein
19
70
22
20
19
2.09
7.8E-04
36
Central metabolism (citrate cycle)
Pput_1663
A5W109
Succinate dehydrogenase subunit A
63.4
42
74
134
124
121
1.58
9.6E-07
37
Central metabolism (citrate cycle)
Pput_1660
A5W106
Citrate synthase
47.6
33
80
89
85
82
1.55
1.5E-03
No
Protein Function
Locus Name
1
Benzoate Catabolism
2
Protein description
65.6
% Coverage
5
p-value
-
Table S1. (continued)
156.8
4
3
2
0
0
∞
-
56.7
63
97
620
627
569
2.00
7.0E-36
A5WA35
ATP-dependent protease ATPase subunit HslU (Unfoldase HslU)
50.0
41
73
103
90
84
1.85
1.1E-04
A5W9A3
Chaperone protein DnaK (HSP70) (Heat shock 70 kDa protein)
65.5
(Heat shock
78protein 70)
85
375
370
311
1.69
1.7E-07
Pput_3256
A5W5H2
Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ Mal
20.5
allergen
16
98
25
18
18
1.60
5.5E-05
Transporters (Benzoate)
Pput_4340
A5W8K3
Outer membrane porin
8
20
9
8
8
10.78
5.6E-06
44
Transporters (osmoregulation)
Pput_0242
A5VX06
L-cystine-binding protein / Diaminopimelate-binding protein 28.7
28
80
53
52
48
1.75
3.0E-03
45
Transporters
Pput_1565
A5W0R1
Sulfate ABC transporter, periplasmic sulfate-binding protein 36.7
13
51
16
17
16
1.67
8.6E-04
46
Transporters (osmoregulation)
Pput_1112
A5VZG3
L-glutamate-binding protein / L-aspartate-binding protein
32
84
100
100
92
1.55
7.9E-04
47
Transporters (osmoregulation)
Pput_0316
A5VX79
Substrate-binding region of ABC-type glycine betaine transport
34.5
system
29
83
38
38
36
1.48
3.6E-03
48
Unknown function
Pput_0823
A5VYM6
Putative uncharacterized protein
7.6
3
32
4
4
4
24.27
5.5E-04
49
Unknown function
Pput_4972
A5WAD2
Putative uncharacterized protein
15.5
4
33
7
6
6
2.48
1.3E-03
50
Unknown function
Pput_2249
A5W2M8
Putative uncharacterized protein
17.1
9
54
13
13
13
1.47
1.3E-04
51
Arylsulfatase
Pput_2406
A5W335
Sulfatase
59.0
30
72
35
25
22
10.80
1.3E-08
52
Signaling/Regulation (or general function)
Pput_1445
A5W0E3
Arc domain protein DNA binding domain protein
12.0
6
63
9
7
7
2.66
4.1E-04
53
General function
Pput_1812
A5W1F5
Alpha/beta hydrolase fold protein
30.3
14
81
23
19
17
2.05
6.4E-03
38
Stress-Response
Pput_1102
A5VZF3
39
Stress response
Pput_4363
A5W8M6
ATP dependent helicase, Lhr family
60 kDa chaperonin (GroEL protein) (Protein Cpn60)
40
Stress response
Pput_4875
41
Stress-Response
Pput_4593
42
Stress response (oxidative stress)
43
45.9
33.2
6
Table S2. List of preferentially expressed proteins in citrate-grown cells.
Central_Metabolism
Locus
Name
Pput_2593
Uniprot
accession
A5W3L8
MW
[kDa]
23.2
No of unique
peptides
4
29
Counts of
TMT127
0
Counts of
TMT129
4
Counts of
TMT129/TMT127
0
Fold-change
(TMT129/TMT127)
∞
2
Central_Metabolism
Pput_4173
A5W837
Lipid-A-disaccharide synthase
41.2
4
21
0
3
0
∞
3
Central_Metabolism
Pput_3549
A5W6B4
tRNA--hydroxylase
-
23.1
2
13
0
2
0
∞
4
Central_Metabolism
Pput_0592
A5VY00
-
Alpha/beta hydrolase fold protein
39.5
2
4
0
3
0
∞
5
Central metabolism
Pput_2563
-
A5W3I8
Succinate semialdehyde dehydrogenase
49.2
10
28
5
8
5
8.00
1.1E-03
6
Central metabolism
7
Central metabolism
Pput_0598
A5VY06
Biotin carboxyl carrier protein
16.2
10
63
18
12
8
7.94
5.3E-07
Pput_1026
A5VZ77
Glycine cleavage system H protein 1
13.9
2
23
3
4
3
2.49
8
6.2E-03
Central_Metabolism
Pput_4919
A5WA79
tRNA sulfurtransferase
54.9
21
55
20
24
19
1.99
2.6E-03
9
Central_Metabolism
Pput_1735
A5W181
NADH dehydrogenase subunit M
55.6
3
5
4
5
4
1.98
7.9E-03
10
Central metabolism
Pput_0363
A5VXC6
Pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase
55.4
25
54
45
58
40
1.93
2.8E-03
11
Central metabolism
Pput_4406
A5W8R8
Cytochrome b
46.0
3
5
3
3
3
1.92
6.1E-03
12
Central metabolism
Pput_1483
A5W0I0
N-acetylneuraminate synthase
42.5
24
71
36
38
34
1.88
1.0E-03
13
Central metabolism
Pput_2393
A5W322
Thiamine pyrophosphate enzyme domain protein TPP-binding
60.0
protein
25
63
8
14
8
1.86
4.9E-05
14
Central_Metabolism
Pput_0984
A5VZ35
Fumarase
48.5
22
58
26
28
25
1.82
1.2E-04
15
Central_Metabolism
Pput_0364
A5VXC7
Pyruvate dehydrogenase E1 component
99.3
69
73
152
158
147
1.78
2.5E-11
16
Central_Metabolism
Pput_5247
A5WB55
Aspartate ammonia-lyase
51.5
15
52
17
18
17
1.53
1.4E-04
17
Chemotaxis
Pput_1535
A5W0N2
Putative CheW protein
17.8
10
89
11
17
11
6.41
1.7E-04
18
Chemotaxis
Pput_0796
A5VYK0
Histidine kinase
25.2
4
23
4
4
3
3.62
2.9E-03
19
Chemotaxis
Pput_3612
A5W6H6
Putative CheW protein
34.5
9
31
8
8
8
2.11
4.1E-03
20
Chemotaxis
Pput_0623
A5VY31
Methyl-accepting chemotaxis sensory transducer
69.8
26
58
22
27
22
1.81
2.2E-05
21
Chemotaxis
Pput_1457
A5W0F5
Flagellar brake protein YcgR (Cyclic di-GMP binding protein 28.4
YcgR)
17
74
17
18
16
1.74
1.0E-04
22
Chemotaxis
Pput_1489
A5W0I6
Flagellin domain protein
29.7
19
67
73
77
67
1.71
7.3E-03
23
Chemotaxis
Pput_1507
A5W0K4
Flagellar basal body-associated protein FliL
17.8
4
39
5
5
4
1.70
8.9E-03
24
Chemotaxis
Pput_0804
A5VYK8
Methyl-accepting chemotaxis sensory transducer
48.8
10
27
5
7
5
1.68
8.1E-03
25
Chemotaxis
Pput_1461
A5W0F9
Putative CheW protein
34.5
13
57
13
13
13
1.54
5.9E-05
26
Transport, porins
Pput_0283
A5VX47
TonB-dependent siderophore receptor
76.5
3
3
2
2
2
2.99
5.4E-03
27
Transport, porins
Pput_1235
A5VZT6
Outer membrane porin
46.1
25
69
62
68
61
2.81
3.9E-08
28
Transport, porins
Pput_0249
A5VX13
Outer membrane porin
48.3
25
59
66
69
63
2.79
1.0E-08
29
Transport, porins
Pput_1128
A5VZH9
OmpA/MotB domain protein
33.9
6
21
5
5
5
2.24
6.1E-03
30
Transport, porins
Pput_4302
A5W8G6
Outer membrane porin
46.6
32
64
55
64
54
1.76
3.0E-04
31
Transport, Protein translocation
Pput_0474
A5VXN4
Protein translocase subunit secE/sec61 gamma
13.3
3
17
6
6
6
1.95
3.3E-03
32
Transport, Protein translocation
Pput_0864
A5VYR6
Protein translocase subunit yajC
11.9
5
42
9
10
8
1.62
3.2E-03
33
pH Regulation
Pput_1868
A5W1K7
Na(+)/H(+) antiporter nhaA 2 (Sodium/proton antiporter nhaA47.7
2)
6
10
6
7
6
5.18
7.4E-03
34
pH Regulation
Pput_0115
A5VWN0
Carbonic anhydrase
17
92
35
36
35
1.49
8.6E-04
No
Protein Function
1
Protein description
Butyryl-CoA:acetate CoA transferase
26.0
% Coverage
7
p-value
-
Table S2. (continued)
35
Metabolic regulation (oxidants detoxification)
Pput_1122
A5VZH3
Glutaredoxin-like protein
17.1
5
38
15
15
15
1.69
5.7E-03
36
Metabolic regulation
Pput_5212
Endoribonuclease L-PSP
13.5
12
76
25
26
24
1.65
9.1E-03
37
Unknown function
Pput_1996
A5WB20
A5W1X6
Putative uncharacterized protein
8.9
3
43
0
2
0
∞
-
38
Unknown function
Pput_1536
A5W0N3
Putative uncharacterized protein
14.7
2
17
2
3
2
5.81
1.6E-03
39
Unknown function
Pput_3448
A5W614
Putative uncharacterized protein
8.8
7
46
270
306
267
4.90
3.3E-118
40
Unknown function
Pput_4305
A5W8G9
Putative uncharacterized protein
52.8
3
8
5
5
5
3.47
7.6E-03
41
Unknown function
Pput_1215
A5VZR6
Putative uncharacterized protein
21.5
16
74
28
32
26
3.36
2.0E-08
42
Unknown function
Pput_3595
A5W6F9
Putative uncharacterized protein
20.8
5
46
4
4
4
2.26
4.9E-03
43
Unknown function
Pput_2585
A5W3L0
Putative uncharacterized protein
96.2
6
10
5
2
2
1.92
7.0E-03
44
Unknown function
Pput_3839
A5W753
Putative uncharacterized protein
33.5
14
48
17
16
16
1.82
4.0E-03
45
Unknown function
Pput_5262
Putative uncharacterized protein
15.5
2
11
4
4
4
1.75
7.5E-03
46
Unknown function
Pput_1477
A5WB70
A5W0H4
Putative uncharacterized protein
98.4
26
39
22
25
21
1.56
6.4E-04
8
Table S3. Kinetic model describing bacterial growth in the activated and balanced batch
culture
State variable
Cell mass, x
Concentration of the
C-source, s
Respiratory CO2 (p)
production
Dissolved oxygen (O)
Monod model
Simplified Monod model
dx
s
= ( m - a)x, m = mm
dt
Ks + s
[1]
ds
mx
=dt
Y
[2]
dp
= Yp/x m x + ax
dt
[3]
dO
= K L (O0 - O) - Yo/x ( m x + ax)
dt
[4]
dx ìïï mm x if s > 0 growth phase
=í
dt ïîï -ax if s = 0 starvation
ds ïìï -mm x / Y if
=í
dt ïïî 0
if
dp ïìï Yp / x mm x / Y
=í
dt ïïî ax
s> 0
[2a]
s= 0
if
s> 0
if
s= 0
ìï -Y m x if
dO
= KL (O0 - O) - ïí o / x m
ïïî -Yo / x ax
if
dt
[1a]
[3a]
s> 0
s= 0
[4a]
Symbols: μ specific growth rate, SGR; μm, maximal SGR achieved under unrestricted growth
conditions; a, specific turnover/death rate (could be considered as self-digestion of cellular
polymers accounting maintenance requirements); Y, Yp/x and Yo/x are stoichiometric coefficients
quantifying respectively cell mass yield per unit mass of consumed C-substrate, CO2 yield per
mass unit of biomass and oxygen needed to produce unit of biomass; KL, oxygen mass-transfer
rate between gas and liquid phases
9
Total proteins:1875
-Log10 p-value
15
46 preferentially expressed
proteins in citrate-grown cells
53 preferentially expressed
proteins in benzoate-grown cells
10
5
-Log 10 p-value > 2
0
-2
-1
0
Log2 Fold-Change
1
2
Figure S1. Volcano plot illustrating the fold-change versus p-values of P. putida F1 proteins
expressed in benzoate or citrate grown cells. (Fold change is the ratio of protein levels in the
benzoate versus citrate-grown cells). The horizontal dotted lines represent the p-value cut-off
thresholds (p-value <0.01), while the vertical dotted lines represent the fold-change cut-off
thresholds (fold change >1.47) derived from the empirical Bayesian analysis.
10
Incubation time: 0.4 h
3.5E+05
Benzoate
Incubation time: 1.2 h
Incubation time: 1.5 h
3.0E+05
Absorbance (275 nm)
Incubation time: 2.3 h
2.5E+05
Injection
peak
Peak1
cis,cis -muconate
2.0E+05
Peak2
catechol
1.5E+05
1.0E+05
5.0E+04
0.0E+00
0
1
2
3
4
5
6
7
8
9
10
11
12
Chromatographic time (min)
Figure S2. HPLC chromatograms (at 275 nm) of P. putida F1 culture supernatants over the
course of benzoate degradation. The gradual accumulation of cis,cis-muconate (Peak 1) and
catechol (Peak 2) intermediates with decreasing levels of benzoate are shown.
11