PHYSIOLOGY AND METABOLISM

Production of 3-Nitrosoindole Derivatives by Escherichia coli during Anaerobic Growth

Young-Man Kwon, Bernard Weiss
DOI: 10.1128/JB.00586-09

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Pellets of bacterial cultures grown anaerobically in the presence of 100 mM nitrate. The photograph shows the lower portions of tubes containing centrifuged saturated cultures. The strains used were CC106, a tna mutant (tube A); BW1824, a tna + transductant of CC106 (tube B); RV (tube C); and BW2018, a tnaA mutant of RV (tube D).

  • FIG. 2.
    FIG. 2.

    Visible spectra of CHCl3-extracted cell pellets. Equal amounts of saturated cultures were used (see Materials and Methods). The strains used were RV (tnaA +) (solid line) and BW2018 (tnaA mutant) (dashed line).

  • FIG. 3.
    FIG. 3.

    Formation of 3-nitrosoindole and derivatives (based on data from references 2, 29, 30, and 43).

  • FIG. 4.
    FIG. 4.

    TLC of chromophores in a CHCl3 extract of strain RV. The unstained chromatogram was photographed in ambient light. Spot O is at the origin.

  • FIG. 5.
    FIG. 5.

    Susceptibility of DNA repair mutants to the lethal effects of indole production (tnaA + genotype) in the presence of nitrate. For each strain tested, multiple tubes were inoculated with 2 × 105 bacteria per ml and incubated anaerobically for the indicated times, and then the preparations were plated on LB medium to determine the concentration of viable cells. The strains (and relevant genotypes) were as follows: KD1092 (wild type [wt]), BW1177 (nfi), BW2021 (tnaA), and BW2022 (tnaA nfi) (A); BW2023 (recA) and BW2024 (recA tnaA) (B); and BW1996 (uvrA) and BW1997 (uvrA tnaA) (C).

  • FIG. 6.
    FIG. 6.

    Other indole derivatives (see Discussion).

Tables

  • TABLE 1.

    Bacterial strains used

    StrainRelevant genotypeaSource or referenceb
    BW386 recA56 srlD300::Tn10 Laboratory collection
    BW1177KD1092 trpA58 nfi-1::cat 41
    BW1824CC106 tna+ dgoA::Tn10dkan This study
    BW1996KD1092 uvrA277::Tn10 P1(N3055) × KD1092c
    BW1997KD1092 uvrA277::Tn10 ΔtnaA739::kan P1(N3055) × BW2021c
    BW2018RV ΔtnaA739::kan P1(JW3686-7) × RV
    BW2019RV tnaB::mini-Tn5-lac-tet/1 P1(MT113) × RV
    BW2020RV narG205::Tn10 P1(RK5268) × RV
    BW2021KD1092 ΔtnaA739::kan P1(JW3686-7) × KD1092
    BW2022KD1092 ΔtnaA739::kan nfi-1::cat P1(JW3686-7) × BW1175
    BW2023KD1092 recA56 srlD300::Tn10 P1(BW386) × KD1092c
    BW2024KD1092 ΔtnaA739::kan recA56 srlD300::Tn10 P1(BW386) × BW2021c
    CC106 tnaA Δ(gpt-lac)5 (F′ lacZCC106)d 14
    JCB387RV ΔnirB 15
    JW3686-7ΔtnaA739::kan 3
    KD1092 trpA58 16
    MT113 tnaB::mini-Tn5-lac-tet/1 4
    N3055 uvrA277::Tn10 CGSCe
    RK5268 narG205::Tn10 48
    RVΔlac tna+ 15

    • ↵ a All strains are derivatives of E. coli K-12 and are F− λ− unless stated otherwise. cat, kan, and tet are genes specifying resistance to chloramphenicol, kanamycin, and tetracycline, respectively.

    • ↵ b Transductions with phage P1 are described as follows: P1(donor) × recipient.

    • ↵ c Tetracycline-resistant transductants were tested for UV sensitivity (recA).

    • ↵ d The tnaA mutant genotype was found in this study (see Results).

    • ↵ e CGSC, Coli Genetic Stock Center, Yale University (http://cgsc.biology.yale.edu ).

  • TABLE 2.

    Requirements for production of the red chromophores

    ConditionsRed pellet
    Completea +
        −tnaA −
        −tnaB −
        −narG −
        −nirB +
        +O2 b −
        −NO3− −
        20 mM NO3 − +
        −NO3 − + 20 mM NO2 − −
        +0.1 M 3-(N-morpholino)propanesulfonic acid/NaOH buffer (pH 7.8)−
    Tryptophan-free mediumc −
        +tryptophan (0.5 mM)+
        +indole (0.5 mM)+

    • ↵ a Cultures were grown to saturation at 37°C in sealed, filled tubes using an initial concentration of 100 to 200 cells/ml. After 20 h the cultures were centrifuged, and the pellets were observed. The complete conditions for chromophore production included a tna+ strain (RV) grown anaerobically in glycerol-fumarate medium containing 100 mM NaNO3. Additional strains used were BW2018 (tnaA), BW2019 (tnaB), BW2020 (narG), and JCB387 (nirB).

    • ↵ b The culture was aerated by shaking in an open flask.

    • ↵ c Vitamin assay Casamino Acids (Difco) was substituted for tryptone and yeast extract in the growth medium.

  • TABLE 3.

    Identification of indole derivatives by mass spectrometry

    Postulated derivativem/z predictedm/z foundTLC spota
    Indoxyl red247.08659247.08612A
    Indole trimer364.14444364.14379B
    Indole red262.09749262.09706C

    • ↵ a Spot from which the compound was isolated (see Fig. 4).

  • TABLE 4.

    Mutation frequencies of tnaA+ and tnaA mutant strains grown anaerobically in the presence of nitrate

    Selected traitaMutation frequencyb
    BW1177 (nfi)BW2022 (nfi tnaA)
    Strr 2.6 × 10−9 7.1 × 10−9
    AUr 7.6 × 10−7 9.9 × 10−7
    Valr 1.0 × 10−7 1.6 × 10−7
    Trp+ <2.0 × 10−9 <2.0 × 10−9

    • ↵ a Strr, streptomycin resistance; AUr, resistance to 6-azauracil; Valr, valine resistance; Trp+, tryptophan independence.

    • ↵ b Each value is the median for at least five independent cultures that were grown as described in Materials and Methods.

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KEYWORDS

Escherichia coli K12
Indoles

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