Technical CommentsHEALTH AND MEDICINE

Comment on “A commensal strain of Staphylococcus epidermidis protects against skin neoplasia” by Nakatsuji et al.

See allHide authors and affiliations

Science Advances  11 Sep 2019:
Vol. 5, no. 9, eaaw3915
DOI: 10.1126/sciadv.aaw3915
  • Fig. 1 Same genetic control of toxicity and mutagenicity effects of 6-HAP and S. epidermidis MO34 extract.

    (A) Schematic representation of the genetic control of 6-HAP metabolism in E. coli and yeast (S. cerevisiae). Genes responsible for the detoxification pathways are indicated in blue, and genes responsible for activation are in red. E. coli genes are listed first, yeast genes are in capital letters after the slash. Note that the Moco-dependent defense against 6-HAP is not present in yeast. Ade, adenine; (6-HAP)R, 6-HAP-riboside; (6-HAP)MP, 6-HAP-riboside monophosphate; (6-HAP)DP, 6-HAP-riboside diphosphate; d(6-HAP)DP, 6-HAP-deoxyriboside diphosphate; (6-HAP)TP, 6-HAP-riboside triphosphate; d(6-HAP)TP, 6-HAP-deoxyriboside triphosphate; d(6-HAP)MP, 6-HAP-deoxyriboside monophosphate. (B and C) Hypersensitivity of E. coli Δmoa mutants to 6-HAP or to extract of S. epidermidis strains MO34 or 1457. The compounds to be tested were spotted on the disc at the center of the plate. The upper row shows the growth on minimal M9 plates; the lower row shows the ability of compounds spotted in the center to induce Rifr mutations. Ext., extract; NIC, Natland International Corporation. (D) 6-HAP by itself does not produce any toxic or mutagenic effects because mutations blocking 6-HAP conversion to the ribonucleotide monophosphate level prevent both 6-HAP–induced toxicity and mutagenicity as seen in (C). (E) The extract of S. epidermidis strain MO34 is mutagenic in the ham1 yeast strain unable to deactivate d(6-HAP)TP. Canavanine resistance (Canr) is measured (see Materials and Methods). Medians with 95% confidence intervals are shown. Double asterisks indicate mutant frequencies significantly higher than for the spontaneous sample (U test, P < 0.003). Note the 10-fold difference in the 6-HAP (MPB, MP Biomedicals) concentration used for the WT strain and ham1 mutant.

  • Fig. 2 6-HAP does not inhibit DNA synthesis in vitro.

    (A) Primer-template design. (B) DNA polymerase primer extension reactions in the presence of 6-HAP or with control solvent, DMSO. 6-HAP (1 mM) did not prevent incorporation of dATP opposite template T, even when in 100- to 10,000-fold excess. The reaction was with the Exo Klenow fragment of E. coli Pol I, 0.25 U per reaction, for 10 min at 37°C. See Materials and Methods for a full description and fig. S1 for additional corroborating results.

  • Fig. 3 Mutagenic specificity of MO34 is identical to that of 6-HAP.

    (A) An essentially identical linear dose-response curve can be obtained for the frequency of Cycr E. coli mutants as a function of 6-HAP or MO34 extract concentration. This requires 250-fold less of pure 6-HAP than of MO34 extract, likely representing the relative 6-HAP content of the extract. Presented are median mutant frequencies obtained from four separate experiments with 95% confidence intervals. (B and C) The extract from the S. epidermidis strain MO34 induces the same proportion of transition mutations as authentic 6-HAP (B) and with the same frequency (C).

  • Table 1 DNA sequence changes in spontaneous, MO34-, and 6-HAP–induced d-cycloserine–resistant cycA mutants.

    Mutation typeSpontaneous
    No. (%)
    MO34 (1 μg/ml)
    No. (%)
    6-HAP (4 ng/ml)
    No. (%)
    Transitions7 (11%)64 (97%)*65 (99%)*
    G·C→A·T5 (7.9%)19 (29%)34 (52%)
    A·T→G·C2 (3.2%)45 (68%)31 (47%)
    Transversions7 (11%)00
    G·C→T·A3 (4.8%)00
    G·C→C·G1 (1.6%)00
    A·T→C·G3 (4.8%)00
    Deletions 1 nt9 (14%)01 (1%)
    Deletions >1 nt15 (24%)2 (3%)0
    Duplications9 (14%)00
    IS elements16 (25%)00
    Total mutations6366**66**

    *Significantly different from the spontaneous value (Fisher’s exact test, P < 10−4).

    **Two Cycr clones contained two mutations in the cycA gene.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw3915/DC1

      Fig. S1. HAP in a form of nucleobase does not interfere with DNA synthesis.

      Fig. S2. Distribution of sequenced d-cycloserine–resistance mutations along the cycA gene.

      Fig. S3. Confirmation of chemical identity of preparations of 6-HAP used in the study.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. HAP in a form of nucleobase does not interfere with DNA synthesis.
      • Fig. S2. Distribution of sequenced D-cycloserine–resistance mutations along the cycA gene.
      • Fig. S3. Confirmation of chemical identity of preparations of 6-HAP used in the study.

      Download PDF

      Files in this Data Supplement:

    Navigate This Article