identification of metal reductases and determination of ...€¦ · dwayne a. elias, heather m....
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Insoluble fraction
Matrix Number of Proteins
Cell Extract 972
Hydrophobic 515
Anion Exchange 360
Cation Exchange 392
Ni(II)-NTA 845
Common to all Matrices 143
Likely Candidates 41
Insoluble fraction
Matrix Number of Proteins
Cell Extract 326
Hydrophobic 122
Anion Exchange 164
Cation Exchange 198
Ni(II)-NTA 175
Common to all Matrices 26
Likely Candidates 11
Soluble fraction
Matrix Number of Proteins
Cell Extract 836
Anion Exchange ND
Cation Exchange ND
Ni(II)-NTA 657
Common to all Matrices N/A
Likely Candidates 71
Soluble fraction
Matrix Number of Proteins
Cell Extract 275
Anion Exchange 188
Cation Exchange 212
Ni(II)-NTA 196
Common to all Matrices 120
Likely Candidates 12
0
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Identification of Metal Reductases and Determination of their RelativeIdentification of Metal Reductases and Determination of their Relative
Abundance in Subsurface Sedimentary Systems using Proteomic AnalysisAbundance in Subsurface Sedimentary Systems using Proteomic AnalysisDwayne A. Elias, Heather M. Mottaz, Carrie D. Goddard, Alexander S. Beliaev, and Mary S. Lipton
AbstractHeavy metal and radionuclide contamination at Department of Energy (DOE)
sites nationwide constitute a major environmental problem. Of particular
interest are U and Tc, as well as Fe and Mn due to their potential direct and
indirect effects on contaminant biogeochemical behavior. For the past
decade bacteria that utilize metals as terminal electron acceptors have been
isolated and identified. These bacteria include members of three major
anaerobic groups; the denitrifying, sulfate- and Fe(III)- reducing bacteria.
The electron transfer pathways within these bacteria are still not well
understood. Moreover, this lack of information substantially impedes efforts
to increase in situ bioremediation efficiency. Hence, identification of metal
reductases, and determination of their similarity between these bacterial
groups is essential for understanding these mechanisms and assessing
bioremediative potential at DOE sites.
We have used cell fractionation techniques to resolve sub-cellular protein
fractions and quantify the purity of proteins within each enriched fraction.
Additionally, we have applied classical biochemical separations of fractions
to enrich for specific proteins responsible for metal reduction activity. The
application of advanced proteomics techniques allows for the identification of
all the proteins in the enriched fractions eliminating the need for purifying
each protein to homogeneity. We are utilizing a orthogonal purification
approaches in both series and parallel to created fractions containing
different complements of proteins. As each fraction exhibits the metal
reduction activity, the proteins common to all the fractions are the most likely
targets for further study by molecular biological techniques.
Accurate Mass and Time tag (AMT) Approach
Accurate mass observed: 1830.1985 DA
AMT is validated
Separation, analytical conditions
established
500 750 1,000 1,250 1,500m/z
916.0 920.5m/z
200 600 1,000 1,400 1,800m/z
y9
y8
b10
y7
b9b8
y6
y12
y10
y11b12
b6y5
b7
b11
y13b14b13
y4
Sequence determined from MS- MS
fragmentation pattern
Potential mass tag (PMT) identified
from D. radiodurans proteome
(APEEKAR) (GITINTAHVEYQTETR) (HYSHVDCPGHADYVK)
confirm sequence as
GITINTAHVEYQTETR
calculated mass = 1830.1987
Separate & measure using
Capillary LC- Ion Trap MS/MS
Separate & measure using
Capillary LC-FTICR MS
Mass tag = Protein marker
Application of AMT tags Development of AMT tags
Fe(III) reduction activity measurements
Concept of Experiments
Chemostat grown cells
French press lysis, centrifugation and ultracentrifugation
Soluble fraction Insoluble fraction
Anion, Cation, and Ni-
NTA column enrichment
Anion, Cation, Ni-NTA, and
Hydrophobic column enrichment
LTQ-FT MS analysis
Fractions with Fe(III)-NTA
reduction activity analyzed
•Activity of each fraction
was tested by a method
modified from that
described (Magnuson et
al., (2001) Biochem. J.
359:147-152.) as shown
using purified MtrC and
OmcA from S. oneidensis
MR-1.
•Protein concentration was
measured using the BCA
micro plate method.
pH 6.8
pH 7.0
pH 7.5
blank
No Fe(III)-NTA added
MtrC/OmcA pi,pH 6.8, 7.0, 7.5
MtrA MtrC OmcA MtrC/OmcA
MtrA/MtrC MtrA/OmcA MtrC/OmcA
Our lab has developed a procedure for the high-throughput detection of
heme-containing peptides using a search for a mass of 615.1694 Da that
accounts for the formal charge of the c-type heme. This is valid for peptides
700-7000 Da with a 2% false positive rate. (F. Yang et al. (2005) J.
Proteome Res. In press).
S. oneidensis MR-1 Dsv. desulfuricans G20 G. metallireducens andG. sulfurreducens
MS/MS fragmentation
spectra of heme-
containing peptides (A)
K. GADSCLMC*HK.K
and (B)
R.MSWNGGHHDNADV
ACASC*HQVHVAK.D
from tryptic digest of
MtrA. *indicated the
heme attachment site.
0
0.5
1
1.5
2
2.5
pro
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(m
g/m
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100
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250
Acti
vit
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mo
l/m
in,
/mg
pro
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)
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3.5
4
4.5
0 2 4 6 8 10 12 14 16 18
Elution time (min)
pro
tein
(m
g/m
l)
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50
100
150
200
250
Act
ivity (
mol/m
in, /
mg p
rote
in)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25 30
Elution time (min)
pro
tein
(m
g/m
l)
0
50
100
150
200
250
Act
ivit
y (µ
mol/m
in, /m
g p
rote
in)
Insoluble fraction
Anion exchange
Insoluble fraction
Hydrophobic
Insoluble fraction
Ni(II)-NTA
Protein concentration
Total activity
Specific Activity
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25Elution time (min)
pro
tein
(m
g/m
l)
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50
100
150
200
250
Acti
vit
y (
mol/
min
, /m
g p
rote
in
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 10 12 14 16
Elution Time (min)
pro
tein
(m
g/m
l)
-20
0
20
40
60
80
100
120
140
Acti
vit
y (
mol/
min
, /m
g p
rote
in)
0
0.2
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1
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1.6
1.8
pro
tein
(m
g/m
l)
0
5
10
15
20
25
30
35
Acti
vit
y (µ
mo
l/m
in,
/mg
pro
tein
)
G. sulfurreducensInsoluble fractionMatrix Number of Proteins
Cell Extract 414
Hydrophobic 228
Anion Exchange 207
Cation Exchange 234
Ni(II)-NTA 227
Common to all Matrices 87
Likely Candidates 38
G. metallireducens
Insoluble fractionMatrix Number of Proteins
Cell Extract 237
Hydrophobic 76
Anion Exchange 43
Cation Exchange 51
Ni(II)-NTA 64
Common to all Matrices 9
Soluble fractionMatrix Number of Proteins
Cell Extract 560
Anion Exchange 229
Cation Exchange 174
Ni(II)-NTA 154
Common to all Matrices 14
•Protein detection was valid if 2 peptides unique to the parent
protein were present in all 3 MS analyses and passed strict
filters.
•Proteins from all column fractions of each matrix were then
pooled and queried against other matrix results to find proteins
in common for insoluble or soluble subcellular fractions.
Insoluble fraction
Anion exchange
Insoluble fraction
Hydrophobic
Insoluble fraction
Ni(II)-NTA
Insoluble fraction
Ni(II)-NTA
Working closely with other groups, a number
of candidate proteins in S. oneidensis MR-1
have been verified:1. MS proteomic data generated with the
Shewanella federation have shown the
importance of MtrABC and OmcA.
2. K.O. mutants of MtrABC and OmcA generated
through the EMSL Grand Challenge show
decreased Fe(III)-reduction in all but MtrA; (see
Y. Gorby presentation and J. Fredrickson poster).
1. Purified MtrC and OmcA reduce Fe(III)-NTA and
HFO in vivo but not MtrA; (L. Shi et al, submitted
J. Biol. Chem.).
• Subcellular fractions of S. oneidensis MR-1,
Dsv. desulfuricans G20, G. metallireducens
and G. sulfurreducens have been enriched
by multiple matrices and the proteins from
fractions displaying Fe(III)-reduction activity
identified as putatively being involved in
metal-reduction in each of these organisms.
• The combination of reductase enrichment with
high-throughput, comprehensive MS analysis
yields more information without lengthy
purification of each protein and the possible
loss of activity during purification.
• These amino acid sequences will be used to
reannotate genes in these organisms against
newer sequencing efforts, thus improving their
functional categorization.
• These sequences will also be used together
with our heme-containing peptide detection
methods to identify organisms from sediment
extracts of DOE sites including FRC, Rifle and
Hanford.
AcknowledgementsAcknowledgements
This research was supported
by the Natural and
Accelerated Bioremediation
Research Program (NABiR),
Office of Biological and
Environmental Research,
U.S. Department of Energy
(DOE). Pacific Northwest
National Laboratory, a
multiprogram national
laboratory, is operated by
Battelle for the U.S.
Department of Energy under
Contract DE-AC05-
76RL01830.
Mass spectrometryMass spectrometry
LjiljanaLjiljana PasaPasa TolicTolic
KeqiKeqi Tang Tang
SuzanaSuzana MartinovicMartinovic
David AndersonDavid Anderson
ChristopheChristophe Masselon Masselon
Deanna AuberryDeanna Auberry
Ron MooreRon Moore
Harold UdsethHarold Udseth
Aleksey Aleksey TolmachevTolmachev
RuiRui Zhang Zhang
WeijunWeijun QianQian
BogdanBogdan BogdanovBogdanov
SeongheeSeonghee AhnAhn
Andre Andre VilkovVilkov
JeongkwonJeongkwon Kim Kim
MishaMisha GorshkovGorshkov
Eugene Eugene NikoleavNikoleav
Data processing, software developmentData processing, software development
and statistics and statistics
Gordon AndersonGordon Anderson NikolaNikola TolicTolic
Mary PowersMary Powers Dave ClarkDave Clark
Kerry SteeleKerry Steele Dave PriorDave Prior
HiediHiedi Sofia Sofia Gary Gary KiebelKiebel
Ken AuberryKen Auberry Eric Eric StrittmatterStrittmatter
Bill Cannon Bill Cannon Matt Monroe Matt Monroe
SeparationsSeparations
YufengYufeng ShenShen RuiRui Zhao Zhao
KostasKostas PetritisPetritis David SimpsonDavid Simpson
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
1260.6 1370.0
1024.6
1261.7876.4
907.31054.9
985.3870.3
1487.3
1192.91305.31135.1
982.6
831.01119.8
1651.41536.8617.4 742.0
1800.1672.6 1572.5
518.2 1798.5 1928.8553.3818.5 1888.6
1613.5 1758.6 1949.6436.2341.1
y62+
QVHVA-NH3
Heme+a4
y7
b193+
b8
(b22-H2O)3+
b223+
b233+
(b24+H2O)3+
y162+
y172+
y182+
b192+
b202+
b212+
b222+
y8
b242+
y11
ACASC*HQVHV
y13-H2O
1418.7
B
Heme+
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
m/z
616.2
617.2
1064.3
719.0614.2 1046.2918.2
1349.31246.31133.3 1551.1821.1768.2692.7 1396.4936.2884.1604.9 1484.9548.2387.1346.3285.1 484.2
y72+
y7-Heme
y8-Heme-H2O
y8-Heme (M+H)+
(M+H)+-H2O
y4y5
y6
y7
b8
y8
b9y3-Heme
A
Reference Protein Description
SO3988 aerobic respiration control protein ArcA
SO4320 agglutination protein
SO1490 alcohol dehydrogenase II
SO1429 anaerobic dimethyl sulfoxide reductase A subunit
SO2756 antioxidant AhpC/Tsa familySO1637 bacterial surface antigen
SO1112 bacterioferritin subunit 1
SO2882 conserved hypothetical protein
SO3765 conserved hypothetical protein
SO1824 conserved hypothetical protein
SO1518 conserved hypothetical proteinSO1377 conserved hypothetical protein
SO3906 conserved hypothetical protein
SO4134 conserved hypothetical protein
SO2593 conserved hypothetical protein
SO3842 conserved hypothetical proteinSO2264 cysteine desulfurase
SO4666 cytochrome c
SO2361 cytochrome c oxidase cbb3-type, subunit III
SO3286 cytochrome d ubiquinol oxidase subunit I
SO1778 decaheme cytochrome c MtrC
SO1779 decaheme cytochrome c OmcASO0970 fumarate reductase flavoprotein subunit precursor
SO4384 hypothetical protein
SO3733 hypothetical protein
SO1521 iron-sulfur cluster-binding protein
SO1519 iron-sulfur cluster-binding proteinSO4349 ketol-acid reductoisomerase
SO1295 major outer membrane lipoprotein putative
SO3740 NAD(P) transhydrogenase alpha subunit
SO1019 NADH dehydrogenase I C/D subunits
SO1103 NADH:ubiquinone oxidoreductase
SO0902 NADH:ubiquinone oxidoreductase SO4321 OmpA family protein
SO3545 OmpA family protein
SO3896 outer membrane porin putative
SO3904 outer membrane protein TolC
SO4591 tetraheme cytochrome cSO2907 TonB-dependent receptor domain protein
SO2427 TonB-dependent receptor putative
SO0610 ubiquinol-cytochrome c reductase cytochrome c1
Reference Protein Description
SO4054 5,10-methylenetetrahydrofolate reductase
SO3988 aerobic respiration control protein ArcA
SO0958 alkyl hydroperoxide reductase C subunit
SO2882 conserved hypothetical protein
SO3842 conserved hypothetical protein
SO2264 cysteine desulfurase
SO1140 dihydrodipicolinate reductase
SO1937 ferric uptake regulation protein
SO0970 fumarate reductase
SO3855 malate oxidoreductase
SO1776 outer membrane protein precursor MtrB
SO2935 oxidoreductase
Reference Protein Description
GSU1644 ABC transporter, ATP-binding protein
GSU3401 periplasmic amino acid-binding protein
GSU1013 chemotaxis MotB protein, putative
GSU0384 conserved hypothetical protein
GSU0095 conserved hypothetical protein TIGR00103
GSU3259 cytochrome c family protein
GSU3259 cytochrome c family protein
GSU0274 cytochrome c family protein
GSU2811 cytochrome c Hsc (hsc)
GSU2813 cytochrome c551 peroxidase (ccpA-2)
GSU0509 Fe(III) reductase, alpha subunit (sfrA)
GSU0510 Fe(III) reductase, beta subunit (sfrB)
GSU1932 hypothetical protein
GSU2090 hypothetical protein
GSU2496 hypothetical protein
GSU1467 iron-sulfur cluster-binding protein
GSU1468 keto/oxoacid ferredoxin oxidoreductase, a s.u.
GSU1469 keto/oxoacid ferredoxin oxidoreductase, b s.u.
GSU1470 keto/oxoacid ferredoxin oxidoreductase, g s.u.
GSU0644 KH domain protein
GSU0872 lipoprotein, putative
GSU2940 lipoprotein, putative
GSU0343 NADH dehydrogenase I, F subunit (nouF)
GSU0344 NADH dehydrogenase I, G subunit, putative
GSU0346 NADH dehydrogenase I, I subunit (nuoI-1)
GSU0350 NADH dehydrogenase I, M subunit (nuoM-1)
GSU1700 NADP-dependent malic enzyme (maeB)
GSU0024 OmpA domain protein
GSU0360 OmpA domain protein
GSU1609 outer membrane efflux protein
GSU0814 outer membrane efflux protein, putative
GSU2939 outer membrane porin FmdC, putative
GSU0073 outer membrane protein, putative
GSU2268 outer membrane protein, putative
GSU2267 outer membrane protein, putative
GSU2305 peptidoglycan-associated lipoprotein
GSU0097 pyruvate ferredoxin/flavodoxin oxidoreductase
GSU3291 V-type H(+)-translocating pyrophosphatase
Reference Protein Description
gi|23473865|ref|ZP_00129160.1| Anaerobic selenocysteine dehydrogenase
gi|23475856|ref|ZP_00131126.1| Bacterioferritin (cytochrome b1)
gi|23475073|ref|ZP_00130363.1| Coenzyme F420-reducing hydrogenase
gi|23475394|ref|ZP_00130681.1| Dissimilatory sulfite reductase (desulfoviridin)
gi|23475395|ref|ZP_00130682.1| Dissimilatory sulfite reductase (desulfoviridin)
gi|23473599|ref|ZP_00128895.1| FAD/FMN-containing dehydrogenases
gi|23475074|ref|ZP_00130364.1| Heterodisulfide reductase, subunit A
gi|23475071|ref|ZP_00130361.1| Membrane protein involved in colicin uptake
gi|23474623|ref|ZP_00129916.1| Ni,Fe-hydrogenase I
gi|23473161|ref|ZP_00128457.1| Proline dehydrogenase
gi|23473601|ref|ZP_00128897.1| Pyruvate:ferredoxin oxidoreductase
Reference Protein Description
GSU3289 conserved hypothetical protein
GSU2504 cytochrome c family protein
GSU1397 cytochrome c family protein, putative
GSU2811 cytochrome c Hsc (hsc)
GSU0509 Fe(III) reductase, alpha subunit (sfrA)
GSU1177 fumarate reductase, flavoprotein subunit (frdA)
GSU1178 fumarate reductase, iron-sulfur protein (frdB)
GSU2496 hypothetical protein
GSU1468 keto/oxoacid ferredoxin oxidoreductase, a s.u.
GSU1469 keto/oxoacid ferredoxin oxidoreductase, b s.u.
GSU1470 keto/oxoacid ferredoxin oxidoreductase, g s.u.
GSU1909 ketol-acid reductoisomerase (ilvC)
GSU2263 oxidoreductase, Gfo/Idh/MocA family
GSU2731 polyheme membrane-associated cytochrome c (ferA)
GSU0097 pyruvate ferredoxin/flavodoxin oxidoreductase
GSU3294 rubredoxin-oxygen oxidoreductase, putative
GSU2814 rubrerythrin
GSU2612 rubrerythrin/rubredoxin protein, putative
GSU3246 thioredoxin peroxidase
GSU3453 uroporphyrinogen decarboxylase (hemE)
Soluble fractionMatrix Number of Proteins
Cell Extract 398
Anion Exchange 119
Cation Exchange 137
Ni(II)-NTA 360
Common to all Matrices 91
Likely Candidates 20
Contact information:
Reference Protein Description
gi|23474600|reCOG0059: Ketol-acid reductoisomerase
gi|23475184|reCOG0062: Uncharacterized conserved protein
gi|23475800|reCOG0141: Histidinol dehydrogenase
gi|23473865|reCOG0243: Anaerobic selenocysteine dehydrogenases
gi|23473249|reCOG0247: Fe-S oxidoreductase
gi|23473599|reCOG0277: FAD/FMN-containing dehydrogenases
gi|23474001|reCOG0370: Fe2+ transport system protein B
gi|23474626|reCOG0374: Ni,Fe-hydrogenase I large subunit
gi|23476244|reCOG0378: Ni2+-binding GTPase, hydrogenase expression
gi|23475512|reCOG0479: fumarate reductase, Fe-S protein
gi|23474201|reCOG0492: Thioredoxin reductase
gi|23474074|reCOG0535: Predicted Fe-S oxidoreductases
gi|23475197|reCOG0569: K+ transport systems, NAD-binding component
gi|23474087|reCOG0607: Rhodanese-related sulfurtransferase
gi|23473167|reCOG1014: Pyruvate:ferredoxin oxidoreductase, alpha subunit
gi|23475406|reCOG0731: Fe-S oxidoreductases
gi|23473995|reCOG0735: Fe2+/Zn2+ uptake regulation proteins
gi|23473199|reCOG0739: Membrane proteins related to metalloendopeptidases
gi|23475776|reCOG0778: Nitroreductase
gi|23473738|reCOG0822: NifU homolog involved in Fe-S cluster formation
gi|23473165|reCOG1014: Pyruvate:ferredoxin oxidoreductase, b subunit
gi|23473601|reCOG1014: Pyruvate:ferredoxin oxidoreductase, g subunit
gi|23474512|reCOG1034: NADH:ubiquinone oxidoreductase
gi|23475493|reCOG1035: Coenzyme F420-reducing hydrogenase, beta subunit
gi|23475494|reCOG1145: Ferredoxin
gi|23475077|reCOG1146: Ferredoxin
gi|23475074|reCOG1148: Heterodisulfide reductaseand related polyferredoxins
gi|23473631|reCOG1271: Cytochrome bd-type quinol oxidase, subunit 1
gi|23473400|reCOG1454: Alcohol dehydrogenase, class IV
gi|23473386|reCOG1529: Aerobic-type carbon monoxide dehydrogenase
gi|23473320|reCOG1538: Outer membrane protein
gi|23474371|reCOG1538: Outer membrane protein
gi|23475532|reCOG1538: Outer membrane protein
gi|23474624|reCOG1740: Ni,Fe-hydrogenase I small subunit
gi|23475073|reCOG1908: Coenzyme F420-reducing hydrogenase, delta subunit
gi|23473162|reCOG1979: Fe-dependent alcohol oxidoreductases/dehydrogenase
gi|23473640|reCOG2033: Desulfoferrodoxin
gi|23475490|reCOG2048: Heterodisulfide reductase, subunit B
gi|23475856|reCOG2193: Bacterioferritin (cytochrome b1)
gi|23474929|reCOG2249: Putative NADPH-quinone reductase
gi|23473856|reCOG2414: Aldehyde:ferredoxin oxidoreductase
gi|23474145|reCOG2414: Aldehyde:ferredoxin oxidoreductase
gi|23473649|reCOG2885: Outer membrane protein
gi|23475536|reCOG3672: Predicted periplasmic protein
gi|23474732|reCOG3681: Uncharacterized conserved protein
gi|23474430|reCOG4231: Indolepyruvate ferredoxin oxidoreductase, a and b s.u.
gi|23475858|reCOG4624: Iron only hydrogenase large subunit
gi|23473580|reCOG4656: Predicted NADH:ubiquinone oxidoreductase, s.u. RnfC
gi|23475449|reCOG4659: Predicted NADH:ubiquinone oxidoreductase, s.u. RnfG
gi|23475818|reCOG4775: Outer membrane protein/protective antigen OMA87