PHYSIOLOGY AND METABOLISM

The cymA Gene, Encoding a Tetraheme c-Type Cytochrome, Is Required for Arsenate Respiration in Shewanella Species

Julie N. Murphy, Chad W. Saltikov
DOI: 10.1128/JB.01698-06

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Growth on arsenate (10 mM) and reduction to arsenite by Shewanella putrafaciens strain CN-32. ○, arrA null mutant; •, wild type; open bars, arsenite; dark bars, arsenate. The data points and error bars represent the means and standard deviations of triplicate cultures, respectively.

  • FIG. 2.
    FIG. 2.

    Phylogenetic analysis of CymA using distance analysis (neighbor joining). The analysis included CymAs of various Shewanella species and selected tetraheme cytochromes within the NirT/NapC family. Bootstrap values are labeled at nodes with ≥50% occurrences. Accession numbers are as follows: Shewanella oneidensis MR-1, NP_720107; Shewanella sp. strain ANA-3, ZP_00849215; Shewanella sp. strain MR-4, YP_735897; Shewanella sp. strain MR-7, ABI40840; Shewanella putrefaciens CN-32, ZP_00815799; Shewanella sp. strain W3-18-1, ZP_00905699; Shewanella sp. strain PV-4, ZP_00837170; Shewanella frigidimarina, YP_748973; Shewanella amazonensis, ZP_00584682; Shewanella baltica, ZP_00582221; Pseudomonas stutzeri, TP24038; Vibrio fischeri, YP_206147; Vibrio parahaemolyticus, Q53178; Paracoccus pantotrophus, Q56352; E. coli, P33226; Rhodobacter sphaeroides, AAB94872; Vibrio parahaemolyticus, NP_799637.

  • FIG. 3.
    FIG. 3.

    Anaerobic growth on arsenate or fumarate of wild-type and cymA null mutant (ΔcymA) (A) Shewanella putrafaciens strain CN-32 and CN-CYMA and (B) Shewanella sp. strain ANA-3 and AN-CYMA, respectively. The time course for growth was inferred from the optical density at 600 nm. •, ΔcymA grown on fumarate; ○, wild type grown on fumarate; ▴, ΔcymA grown on arsenate; ▵, wild-type grown on arsenate. The data points and error bars represent the means and standard deviations of triplicate cultures, respectively.

  • FIG. 4.
    FIG. 4.

    Restoration of growth on arsenate (10 mM) of Shewanella (A) CN-32 and (B) ANA-3 cymA null mutants (ΔcymA). The time course for growth was inferred from the optical density at 600 nm. •, wild-type strain; ○ ΔcymA null mutant with the vector (pBBR1MCS-2); ▵, ΔcymA null mutant with cymA on a plasmid; ⋄, heterologous complementation of AN-CYMA (ΔcymA) with pCNcymA. The data points and error bars represent the means and standard deviations of triplicate cultures, respectively.

  • FIG. 5.
    FIG. 5.

    Effects of different electron acceptors on cymA transcription in Shewanella putrefaciens strain CN-32 and Shewanella sp. strain ANA-3. CN-32 and ANA-3 were grown on different electron acceptors to the mid-exponential phase of growth (OD at 600 nm, 0.1). The nanogram genomic equivalent of mRNA for cymA was normalized to the total RNA used in the cDNA reaction. The data points and error bars represent the means and standard deviations of triplicate cultures, respectively.

  • FIG. 6.
    FIG. 6.

    The locations of heme motifs, cysteine-to-serine substitutions, and the predicted alpha helix membrane-anchoring domain (underlined) in CymA (A) and growth on arsenate of four cymA heme mutants containing single cysteine-to-serine substitutions (B). The time course for growth was inferred from the optical density at 600 nm. •, CN-32 plus vector; ▪, CN-CYMA plus vector; ⧫, pH 1(C46S); ×, pH 2(C78S); +, pH 3(C136S); ▴, pH 4(C173S). The data points and error bars represent the means and standard deviations of triplicate cultures, respectively.

Tables

  • TABLE 1.

    Bacterial strains and plasmids used in this study

    Strain or plasmidGenotype or markers, characteristics, and usesSource or reference
    E. coli strains
        TOPO Top 10E. coli host for cloning; F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupGInvitrogen
        UQ950E. coli DH5α λ(pir) host for cloning; F− Δ(argF-lac)169 φ80dlacZ58(ΔM15) glnV44(AS) rfbD1 gyrA96(NalR) recA1 endA1 spoT1 thi-1 hsdR17 deoR λpir+28
        WM3064Donor strain for conjugation; thrB1004 pro thi rpsL hsdS lacZΔM15 RP4-1360 Δ(araBAD)567 ΔdapA1341::[erm pir(wt)]28
    Shewanella strains
        Shewanella sp. ANA-3Isolated from an As-treated wooden pier piling in a brackish estuary (Eel Pond, Woods Hole, MA)27
        AN-CYMAShewanella sp. strain ANA-3; ΔcymA; does not respire As(V) or fumarateThis study
        S. putrefaciens CN-32Isolated from anaerobic subsurface core sample, New Mexico8, 34
        CN-CYMAS. putrefaciens CN-32; ΔcymA; does not respire As(V)This study
        Shewanella sp. W3-18-1Isolated from Pacific Ocean marine sediments at 630 m19
    Plasmids/vectors
        pCR4-TOPO4-kb cloning vector; lacZ-ccdB: Kmr AmprInvitrogen
        pSMV109.1-kb mobilizable suicide vector; oriR6K mobRP4 sacB Kmr Gmr28
        ANpΔcymA1.2-kb fusion PCR fragment containing ΔcymA from ANA-3 cloned into the SpeI site of pSMV10; used to make the AN-CymA ΔcymA strain.This study
        CNpΔcymA2-kb fusion PCR fragment containing ΔcymA from S. putrefaciens cloned into the SpeI site of pSMV10; used to make the CN-CymA ΔcymA strain.This study
        pBBR1MCS-25.1-kb broad-host-range plasmid; Kmr lacZ14
        pANcymAANA-3 cymA PCR fragment, including the promoter region, cloned into the SpeI site of pBBR1MCS-2This study
        pCNcymAS. putrefaciens cymA PCR fragment cloned into the SpeI sites of pBBR1MCS-2This study
        pH 1(C46S)pCNcymA plasmid with SXXCH of heme 1This study
        pH 2(C78S)pCNcymA plasmid with CXXSH of heme 2This study
        pH 3(C136S)pCNcymA plasmid with SXXCH of heme 3This study
        pH 4(C173S)pCNcymA plasmid with SXXCH of heme 4This study
  • TABLE 2.

    Growth characteristics of AN-CYMA and CN-CYMA on known terminal electron acceptors

    SubstrateGrowtha
    ANA-3CN-32MR-1b
    ΔcymAWild typeΔcymAWild typeΔcymAWild type
    Arsenate−+−+−−
    Fumarate−+++−+
    DMSO−−NDND−+
    Nitrate++++−+
    TMAO++−−++
    Thiosulfate++++++
    Oxygen++++++
    Fe(III)−+−+−+
    Mn(IV)−+−+−+

    • ↵a Plus indicates growth on or reduction [Fe(III) and Mn(IV)] of the substrate similar to the wild type. Minus indicates lack of growth on the corresponding substrate. Lactate (20 mM) served as the electron donor and carbon source. Terminal electron acceptors were included in the anaerobic medium at 10 mM. ND, not determined.

    • ↵b Sources, references 20 and 32.

  • TABLE 3.

    Physiological effects of S. putrefaciens CN-32 with cymA heme variants on the reduction of arsenatea

    StrainTime (h)Arsenate (mM)Arsenite (mM)
    All09.9 ± 0.220.23 ± 0.32
    CN-32 + pBBR1MCS-212.50.7 ± 0.119.7 ± 0.00
    50.509.7 ± 0.06
    CN-CYMA + pBBR1MCS-212.59.7 ± 0.140.12 ± 0.16
    50.51.4 ± 0.218.6 ± 0.01
    CN-CYMA + pH 1(C46S)12.59.2 ± 0.301.0 ± 0.27
    27.5010.2 ± 0.50
    50.509.8 ± 0.26
    CN-CYMA + pH 2(C78S)12.510.1 ± 0.120.10 ± 0.12
    50.51.5 ± 0.218.1 ± 0.13
    CN-CYMA + pH 3(C136S)12.59.8 ± 0.030.18 ± 0.16
    50.52.5 ± 0.367.2 ± 0.11
    CN-CYMA + pH 4(C173S)12.59.9 ± 0.220.07 ± 0.05
    50.51.4 ± 1.989.9 ± 0.61

    • ↵a The cultures represented in Fig. 6B were sampled at the indicated times, and the filtrates were analyzed by high-performance liquid chromatography for arsenate and arsenite. The data and errors represent the means and standard deviations of triplicate cultures, respectively.

Additional Files

  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Table S1, four-digit hexadecimal code describing the SNP genotype of P. aeruginosa strains
      Excel document, 22K
    • Supplemental file 2 - Table S2, primer sequences and description of �cargo� genes of PAGI-2 and PAGI-3
      Word document, 80K.
    • Supplemental file 3 - Fig. S2, overview of defined island types and subtypes
      Word document, 460K.
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