Transcript A Sample Slide - Institute for Environmental Genomics

H-044
Comprehensive Study on the Involvement of Shewanella oneidensis MR-1 c-Type Cytochromes in Anaerobic Respiration
Soumitra Barua1,2, Samantha Reed3, Dave Culley3, David Kennedy3, Margaret Romine3, Yunfeng Yang1, Jim Tiedje4, Jim Fredrickson3, Kenneth Nealson5, and Jizhong Zhou1,2
E-mail: [email protected]
1Environmental
Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; 2Institute for Environmental Genomics, The University of Oklahoma, Norman, OK 73072;
3Pacific Northwest National Laboratory, Richland, WA 99354; 4Center for Microbial Ecology Michigan State University, East Lansing, MI 48824; 5Department of Earth Sciences,
University of Southern California, Los Angeles, CA 90089
Approach
44 Genes are Predicted to Encode c-Type Cytochromes in MR-1, but only 41
are likely functional
Shewanella oneidensis MR-1, a facultative anaerobic g-proteobacterium, possesses
remarkably diverse respiratory capacity. Its complex electron-transport system allows the
coupling of metal reduction to bacterial energy generation and thus has a potential to be
applied in bioremediation of the DOE contaminated sites. However, many questions
underlying the anaerobic respiratory versatility of MR-1, remain poorly understood. To
better understand the electron transport system of this metal-reducing bacterium our
laboratory is investigating the c-type cytochromes of MR-1. Since c-type cytochromes are
essential for energy metabolism their mutation will directly affect the electron transport
network. Approximately 44 c-type cytochromes were identified in Shewanella genome
based on sequence analysis.
The recent determination of genome sequences from 10 additional Shewanella sp.
revealed mutations in 2 MR-1 genes encoding a cytochrome c and 1 that encodes the flavin
subunit of a split cytochrome c suggesting that ETS pathways in which they participate are
non-functioning in MR-1. Comparative sequence analysis revealed that the NrfB
pentaheme cytochrome (SO4570) was prematurely truncated at the C-terminus by 6
repeats of CAAGTGGTA. The same repeat results in loss of the downstream N-terminus
of the NrfC FeS binding protein (SO4569).
NrfA
(SO3980)
two step homologous
cross-over
Gene X
one step homologous
cross-over
Gene X
Gene Z
Gene Z
KmR
Gene Z
remove KmR
generate one mutant with Gene
Y replaced by ½ yeast bar code
Gene X
Gene Z
• Generate mutant with KmR replacing Gene
Y
• Generate mutant with yeast bar code
replacing Gene Y
A variety of approaches are ongoing or planned to
characterize these mutants in order to elucidate their
functional role in respiratory pathways in MR-1. These
approaches include:
•Growth comparisons to wild type cells in complex
or defined media supplemented with varied electron
donor and acceptor pairs
•Assays of reduction of various electron acceptors
•OmniLog Phenotypic microarray analysis of MR-1
and its cytc mutants
•DNA microarray analysis of the mutants
Growth of MR-1 and its cytc mutants at 3mM Nitrate
Growth of MR-1 & D so0970 at 30mM fumarate
0.45
0.45
0.4
0.4
0.35
0.35
0.3
0.3
MR-1-3mM Nitrate
0.25
∆so0610 (petC)-3mM Nit
∆so0845 (napB)-3mM Nit
0.2
∆so4047 (soxA)-3mM Nit
OD600
Gene Y
LB-MR-1-30mM Fumarate
0.25
Δso0970-Fum
0.2
∆so4360-3mM Nit
0.15
∆so4591 (cymA)-3mM Nit
0.15
MR-1-LB-30mM Lactate
0.1
0.1
0.05
0
Many MR-1 Predicted Cytochromes are also Present in Other Shewanella sp.
SO0264 monoheme c5 (ScyA)
SO0610 ubiquinol-cytochrome c reductase, cytochrome c1 (petC)
CN32 BALT FRIG AMAZ PV4 DENI ANA3 MR4 MR7 W318
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SO2363 cytochrome c oxidase, cbb3-type, subunit II (ccoO)
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SO3420 monoheme cytochrome c'
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SO4606 diheme cytochrome c oxidase, subunit II (CyoA)
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2
SO4666 cytochrome c (cytcB)
SO0845 diheme cytochrome c (NapB)
SO0970 fumarate reductase tetraheme cytochrome c (FccA)
SO1777 periplasmic decaheme cytochrome c MtrA (mtrA)
SO1778 decaheme cytochrome c (MtrC)
SO2727 small tetraheme cytochrome c (CctA)
SO3980 cytochrome c552 nitrite reductase (NrfA)
SO4047 SoxA-like diheme c
SO4048 diheme c4
SO4570 pentaheme cytochrome c (NrfB), truncation
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SO4591 tetraheme cytochrome c (cymA)
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SO0939 split-soret diheme cytochrome c
SO3141 nonaheme cytochrome c lipoprotein, degenerate
SO1233 pentaheme cytochrome c (TorC)
SO1659 OmcA-like decaheme cytochrome c
SO1779 decaheme cytochrome c (omcA)
SO2178 cytochrome c551 peroxidase (ccpA)
SO0479 octaheme cytochrome c
SO1780 outer membrane decaheme cytochrome c (MtrF)
SO1782 periplasmic decaheme cytochrome c (MtrD)
SO4142 monoheme cytochrome c
SO4144 octaheme cytochrome c
SO4485 diheme cytochrome c
SO2930 cytochrome c with carbohydrate binding domain
SO2931 cytochrome c lipoprotein
SO3623 split tetraheme flavocytochrome c (flavin subunit interrupted)
SO4484 monoheme cytochrome c (Shp)
SO1427 periplasmic decaheme cytochrome c (DmsC)
SO3056 split tetraheme flavocytochrome c
SO1413 split tetraheme flavocytochrome c
SO1748 monoheme cytochrome c
SO0714 periplasmic monoheme cytochrome c4
SO0716 periplasmic monoheme cytochrome c (SorB)
SO0717 periplasmic monoheme cytochrome c4
SO3300 split tetraheme flavocytochrome c
SO1421 tetraheme flavocytochrome
SO4360 MtrA-like decaheme cytochrome c
SO4572 triheme cytochrome c
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???
80
0
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Time (hours)
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14
Time (hours)
Overview
Preliminary experiment 2
Chromium reduction assay:
Anaerobic chromium [Cr(VI) 0.1mM]
reduction of MR-1 & its cytc mutants
Core
Absent in S.
denitrificans
only
Anaerobic chromium [Cr(VI) 0.1mM]
reduction of MR-1 & its cytc mutants
0.9
0.9
0.8
0.8
0.7
LB-0.1mMCr(VI)
0.6
LB-MR-1
Δso970(fcc)
0.5
Δso2361(ccoP)
Δso2363(ccoO)
0.4
Δso2727[cctA(STC)]
Δso4047(soxA)
0.3
Δso4360(mtrAD-like)
0.2
0.7
0.6
20
25
30
Δ1779(omcA)
Δ1780(mtrF)
Δ1782(mtrD)
0.2
0
15
Δ1778(mtrC/omcB)
0.3
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Δ1777(mtrA)
0.4
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LB-MR-1
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LB-0.1mMCr(VI)
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It is clear from this overview of the current data
available that many of these c-type cytochromes
participate in respiratory metabolism or detoxification
processes that have not yet been explored in detail. The
abundance of proteins induced by thiosulfate suggests that
additional S compounds warrant testing as potential
electron donor/acceptors. The co-localization of histine or
phenylalanine ammonia lysases with several of the split
flavin cytochromes (not included above) suggest that the
utilization of amino acids as electron donor/acceptors
should be evaluated.
The availability of these c-type cytochrome mutants and
of additional sequenced Shewanella strains provides and
excellent resource for comparative physiology studies and
will greatly facilitate our goal of characterizing respiratory
networks in Shewanella sp.
Time (hours)
Time (hours)
Acknowledgments
Preliminary experiment 3
Qualitative assay for MnO2 reduction:
Increasingly
rare in other
strains
24 hrs growth of cytc mutants in MnO2
MR1
only
Dso1659 also showed similar mild growth effect in MnO2 as Dso0939 and Dso1421
DcymA
P
60
DmtrD
P
40
DmtrF
P
P
20
heme #
1
1
SO2361 cytochrome c oxidase, cbb3-type, subunit III (ccoP)
P
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0
0
By comparing genotypes to physiology/biochemistry of the Shewanella strains whose genome has been sequenced, we can better derive
predictions of gene function. For example, the absence of most MR-1 type cytochromes in S. denitrificans suggests that anaerobic respiration
in this organism will differ vastly from that in MR-1. This bacterium is able to denitrify, but conducts this process using a set of proteins that
are distinct from those in MR-1 (but shared with other Shewanella sp. in this group). The putative SO0714-SO0717 complex is shared only by
S. baltica suggesting that it will be possible to identify a mode of growth shared only with MR-1. The occurrence of intact versions of
cytochrome containing complexes in other genomes provides a means to explore functions that have been lost in MR-1.
Protein
0.05
DifcA-1
OAK RIDGE NATIONAL LABORATORY
U. S. DEPARTMENT OF ENERGY
Gene X
DpetC
SO3141 is degenerate, requiring 4 frameshifts to reconstruct the proper reading frames to
produce the expected intact nonaheme cytochrome c. Orthologs to this outer membrane
lipoprotein are present in all sequenced Shewanella strains except S. frigidimarina and S.
denitrificans. A 3rd defective function is the result of interruption of the flavin subunit
(SO3624) of the enzyme complex including cytochrome c (SO3623) by ISSod3_10. Intact
versions of this locus occur in 6 other Shewanella strains.
One additional putative cytochrome c is encoded by SO1748, a predicted outer
membrane monoheme cytochrome. In summary, we have identified 41 genes that are
predicted to encode c-type cytochromes that participate in functioning electron transport
pathways.
Gene Z
DomcA
NrfD
(SO4568)
Gene Y
DmtrA
MK
NrfB
(SO4570)
Gene X
ORNL
LB media
Modified MR-1minimal media
Electron donor:
Na-lactate: 30mM
Electron acceptors: DMSO: 10mM
(growth dynamics Na-fumarate: 30mM
by BioscreenC
Na-nitrate: 3mM
in triplicates)
Na-thioSO4: 3mM
TMAO: 20mM
Qualitative assay
Fe-citrate: 10mM
in colored metals:
MnO2: 2.5mM
BioscreenC
Cr(VI): 0.1mM
DmtrC/omcB
NrfC
(SO4569)
Components of conventional nitrite ETS chain are
defective in MR-1, but present in all other sequenced
Shewanella strains except S. baltica and S. denitrificans
PNNL
Media used:
concentration of Cr(VI) at OD540 nM
Background
Targeted genomic deletions of all but 4 (SO0264, SO1233, SO2178 and SO3056) of the predicted intact c-Type
cytochromes have been successfully constructed by either homologous cross-over with host-encoded recombinases
(PNNL) or with introduced phage cre-lox recombinases (ORNL). Each mutant was tagged with a unique bar code to
facilitate tracking individual strains in planned competitive growth studies. These mutants are intended as a resource to
facilitate characterization of respiratory pathways in MR-1.
OD 600
Under anaerobic conditions, Shewanella oneidensis MR-1 utilizes a wide range
of electron acceptors for respiration such as fumarate, nitrate, nitrite,
dimethylsulfoxide (DMSO), thiosulfate, trimethylamine oxide (TMAO), and So,
as well as Fe(III) oxides, Mn(IV) oxides, Cr(VI), Tc(VII), U(VI), and V(V). This
diverse respiratory capability is due in part to the presence of an abundance of ctype cytochrome genes. Since c-type cytochrome proteins are essential for energy
metabolism their mutation will directly affect the electron transport network. To
investigate their involvement in anaerobic respiration of S. oneidensis, targeted
deletions of 37 out of 41 predicted intact c-type cytochrome encoding genes have
been generated by either homologous cross-over using host-encoded recombinases
(PNNL) or by introduced phage cre-loxP recombinases (ORNL). Growth studies
indicate significant effects of these mutants with different electron acceptors
compared to the wild type. Decreased growth of 5 mutants on 3 mM nitrate
suggests their involvement in nitrate regulation. Ten different mutants showed
defects in the reduction of Mn(IV) relative to WT MR-1 suggesting a complex
network of electron transfer reactions. These mutants were also evaluated
anaerobically for their Cr(VI) reduction capability. Initial test results suggest that
11 mutants were partially defective in Cr(VI) reduction providing new clues of
function for several uncharacterized cytochromes. Mutants in the high affinity
cbb3 cytochrome oxidase components exhibit a defect both aerobically and
anaerobically with TMAO suggesting a role for this complex in both suboxic and
anaerobic respiratory processes. Whole-genome expression gDNA microarray
analyses, competitive growth studies, and fuel cell studies are underway to
explore functions of this multicomponent, branched electron transport system.
• SO4483-SO4485 are induced by nitrate, TMAO, and DMSO relative to
fumarate (Beliaev 2005) and by uranium (Bencheikh-Latmani 2005)
suggesting a possible protective role in anaerobic respiratory processes.
• Mutants lacking SO1427 and CymA are unable to grow on DMSO.
However, the cluster of genes encoding these genes is induced by
thiosulfate and not DMSO suggesting that a different sulfur-containing
substrate may be a more favorable e- acceptor. A second DMSO-like
cluster is also present, but conditions that promote its expression have
yet to be defined. Note that the predicted localization of the terminal
reductases are lipoproteins are hence may be involved in electron
transfer to insoluble sulfur-containing materials.
• The cytochrome bc1 complex transfers electrons from ubiquinol to the
cbb3-type cytochrome oxidase. Mutants lacking SO0610 are defective
in reduction of MnO2 and chromate.
• Fumarate reduction is abolished in the FccA mutant demonstrating that
this periplasmic localized protein is the sole fumarate reductase. The
CymA mutant is also unable to grow on fumarate as expected.
• Mutants lacking SO0479 are deficient in manganese oxide reduction.
This novel cytochrome has its own cytochrome assembly proteins.
This locus is adjacent to a Nos-like copper transporter suggesting that
one or more members of this complex contain a copper center.
• The cbb3-type cytochrome oxidase complex is used under conditions of
low oxygen tension. Mutations in CcoO (SO2363) result in reduced
growth on both TMAO and nitrate suggesting that this complex is
necessary for removal of residual oxygen during respiration of these
substrates.
• ScyA (SO0264) is proposed to be the donor to the cbb3-type
cytochrome oxidase because it is the only abundant high potential
soluble cytochrome under aerobic conditions (Meyer 2004).
Preliminary experiment 1
concentration of Cr(VI) at OD540 nM
Abstract
Voice: (405) 325-3052
This research was funded by grants from the U.S.
Department of Energy Genomics: GTL program
through Shewanella Federation. Oak Ridge
National Laboratory is managed by the University
of Tennessee-Battelle LLC for the Department of
Energy under contract DOE-AC05-00OR22725.
•Beliaev, A.S., et. al., J. Bacteriol. 2005. 187(20):7138-7145.
•Bencheikh-Latmani, R., et. al., Appl. Environ. Microbiol. 2005.
71(11):7453-7460.
•Hedderich, R., et. al., FEMS Microbiol Rev. 1999. 22:353–381.
•Marietou A, et. al., FEMS Microbiol Lett. 2005. 248(2):217-225.
•Meyer, T.E., et. al., OMICS. 2004. 8(1):57-77.
•Mowat, C.G.,et. al., Nat. Struct. Mol. Biol. 2004. 11(10):1023-1024
•Schwalb, C., et. al., Biochem. 2003. 42(31):9491-9497.