OAK RIDGE NATIONAL LABORATORYU. S. DEPARTMENT OF ENERGY

E-mail: sbarua@ou.edu

Voice: (405) 325-3052

Comprehensive Study on the Involvement of Shewanella oneidensis MR-1 c-Type Cytochromes in Anaerobic Respiration

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

Soumitra Barua1,2, Samantha Reed3, Dave Culley3, David Kennedy3, Margaret Romine3, Yunfeng Yang1, Jim Tiedje4, Jim Fredrickson3, Kenneth Nealson5, and Jizhong Zhou1,2

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 c-type 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.

Abstract

H-044

Background

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 -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).

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.

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

NrfD (SO4568)

NrfA (SO3980)

NrfC (SO4569)

MK

NrfB (SO4570)

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.

Overview

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.

Acknowledgments

Media used: LB media Modified MR-1minimal media

Electron donor: Na-lactate: 30mMElectron acceptors: DMSO: 10mM(growth dynamics Na-fumarate: 30mMby BioscreenC Na-nitrate: 3mMin triplicates) Na-thioSO4: 3mM

TMAO: 20mMQualitative assay Fe-citrate: 10mM in colored metals: MnO2: 2.5mM BioscreenC

Cr(VI): 0.1mM

Growth of MR-1 and its cytc mutants at 3mM Nitrate

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 20 40 60 80

Time (hours)

OD

600

MR-1-3mM Nitrate

∆so0610 (petC)-3mM Nit

∆so0845 (napB)-3mM Nit

∆so4047 (soxA)-3mM Nit

∆so4360-3mM Nit

∆so4591 (cymA)-3mM Nit

MR-1-LB-30mM Lactate

Growth of MR-1 & D so0970 at 30mM fumarate

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 10 12 14

Time (hours)

OD

600 LB-MR-1-30mM Fumarate

Δso0970-Fum

Preliminary experiment 1

Anaerobic chromium [Cr(VI) 0.1mM] reduction of MR-1 & its cytc mutants

0

0.1

0.2

0.3

0.4

0.5

0.6

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0.8

0.9

0 5 10 15 20 25 30 35

Time (hours)

conc

entr

atio

n of

Cr(

VI)

at

OD

54

0 n

M

LB-0.1mMCr(VI)

LB-MR-1

Δso970(fcc)

Δso2361(ccoP)

Δso2363(ccoO)

Δso2727[cctA(STC)]

Δso4047(soxA)

Δso4360(mtrAD-like)

Anaerobic chromium [Cr(VI) 0.1mM] reduction of MR-1 & its cytc mutants

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0.1

0.2

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0 5 10 15 20 25 30 35

Time (hours)

conc

entr

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n of

Cr(

VI)

at

OD

54

0 n

M

LB-0.1mMCr(VI)

LB-MR-1

Δ1777(mtrA)

Δ1778(mtrC/omcB)

Δ1779(omcA)

Δ1780(mtrF)

Δ1782(mtrD)

Chromium reduction assay:

Preliminary experiment 2

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.

ORNL

one step homologous cross-over

• Generate mutant with KmR replacing Gene Y

• Generate mutant with yeast bar code replacing Gene Y

remove KmR

Gene YGene X Gene Z

KmRGene X Gene Z

Gene X Gene Z

generate one mutant with Gene Y replaced by ½ yeast bar code

two step homologous cross-over

PNNL

Gene YGene X Gene Z

Gene X Gene Z

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

Approach

Preliminary experiment 3

24 hrs growth of cytc mutants in MnO2

Dso1659 also showed similar mild growth effect in MnO2 as Dso0939 and Dso1421

Dm

trA

Dom

cA

Dm

trC/om

cB

Dm

trF

Dm

trD

Dcym

A

DifcA

-1

DpetC

Qualitative assay for MnO2 reduction:

• 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).Many MR-1 Predicted Cytochromes are also Present in Other Shewanella sp.

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 CN32 BALT FRIG AMAZ PV4 DENI ANA3 MR4 MR7 W318 heme #SO0264 monoheme c5 (ScyA) P P P P ? P P P P P 1SO0610 ubiquinol-cytochrome c reductase, cytochrome c1 (petC) P ? P P P P P P P P 1

SO2361 cytochrome c oxidase, cbb3-type, subunit III (ccoP) P P P P P P P P P P 2

SO2363 cytochrome c oxidase, cbb3-type, subunit II (ccoO) P P P P P P P P P P 1

SO3420 monoheme cytochrome c' P P P P P P P P P P 1

SO4606 diheme cytochrome c oxidase, subunit II (CyoA) P P P P P P P P P P 2

SO4666 cytochrome c (cytcB) P P P P P P P P P P 2

SO0845 diheme cytochrome c (NapB) P P P P P P P P P 2SO0970 fumarate reductase tetraheme cytochrome c (FccA) P P P P P P P P P 4

SO1777 periplasmic decaheme cytochrome c MtrA (mtrA) P P P P P P P P P 10SO1778 decaheme cytochrome c (MtrC) P P P P P P P P P 10SO2727 small tetraheme cytochrome c (CctA) P P P P P P P P P 4SO3980 cytochrome c552 nitrite reductase (NrfA) P ? P P P P P P P 5SO4047 SoxA-like diheme c P P P P P P P P P 2SO4048 diheme c4 P P P P P P P P P 2SO4570 pentaheme cytochrome c (NrfB), truncation P P P P P P P P P 5

SO4591 tetraheme cytochrome c (cymA) P P P P P P P P P 4

SO0939 split-soret diheme cytochrome c P P P P P P P P 2SO3141 nonaheme cytochrome c lipoprotein, degenerate P P P P P P P P 9SO1233 pentaheme cytochrome c (TorC) P P P P P P P 5SO1659 OmcA-like decaheme cytochrome c P P P P P P P 10SO1779 decaheme cytochrome c (omcA) P P P P P P P 10SO2178 cytochrome c551 peroxidase (ccpA) P P *** P P P P P 2SO0479 octaheme cytochrome c P P P P P P 8SO1780 outer membrane decaheme cytochrome c (MtrF) P P P P P P 10SO1782 periplasmic decaheme cytochrome c (MtrD) P P P P P P 10SO4142 monoheme cytochrome c P P P P P P 1SO4144 octaheme cytochrome c P P P P P P 8

SO4485 diheme cytochrome c P P P P P P 2SO2930 cytochrome c with carbohydrate binding domain ? P P P P P 2SO2931 cytochrome c lipoprotein ? P P P P P 2SO3623 split tetraheme flavocytochrome c (flavin subunit interrupted) P P P P P 4

SO4484 monoheme cytochrome c (Shp) P P P P P P 1SO1427 periplasmic decaheme cytochrome c (DmsC) P PP P P 10SO3056 split tetraheme flavocytochrome c P P P P 4SO1413 split tetraheme flavocytochrome c P P P ? 4SO1748 monoheme cytochrome c P P 1SO0714 periplasmic monoheme cytochrome c4 P 1SO0716 periplasmic monoheme cytochrome c (SorB) P 1SO0717 periplasmic monoheme cytochrome c4 P 1SO3300 split tetraheme flavocytochrome c ? PP 4SO1421 tetraheme flavocytochrome ? 4SO4360 MtrA-like decaheme cytochrome c 10SO4572 triheme cytochrome c ??? 3

Core

Absent in S. denitrificans

only

MR1 only

Increasingly rare in other

strains

•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.