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. 2020 Mar 11:42:12.
doi: 10.1186/s41021-020-00149-z. eCollection 2020.

Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms

Masanobu Kawanishi  1 , Chiaki Shimohara  1 , Yoshimitsu Oda  1 , Yuuta Hisatomi  1 , Yuta Tsunematsu  2 , Michio Sato  2 , Yuichiro Hirayama  2 , Noriyuki Miyoshi  3 , Yuji Iwashita  4 , Yuko Yoshikawa  3   5 , Haruhiko Sugimura  4 , Michihiro Mutoh  6 , Hideki Ishikawa  7 , Keiji Wakabayashi  3 , Takashi Yagi  1 , Kenji Watanabe  2
Affiliations

Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms

Masanobu Kawanishi et al. Genes Environ. .

Abstract

Introduction: Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb +) microorganisms possess a 54-kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan.

Findings: The analysis revealed that two of six strains possessed a clb gene cluster. These clb + strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium.

Conclusion: These results support the observations that the clb gene cluster is widely distributed in nature and clb + E. coli having genotoxic potencies is not rare among microorganisms.

Keywords: Colibactin; Genotoxicity; Genotyping.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Typical gel images of amplicons from genomic DNA of a clb+ and a clb− strain. Genomic DNA of JCM5263 (clb+) and JCM20114 (clb−) were analyzed. The clb genes and expected sizes of their amplicons (bp) in PCR are as follows: clbA, 613; clbB, 555; clbC, 503, clbD, 431; clbF, 465; clbG, 599; clbH, 693; clbI, 643; clbJ, 544; clbK, 690; clbL, 401; clbM, 592; clbN, 581; clbO, 438; clbP, 464; clbQ, 430
Fig. 2
Fig. 2
The read coverage of the clb genes in genomic DNA of E. coli strains determined by Illumina MiSeq. The color represents the read coverage of the indicated clb gene in the indicated strains
Fig. 3
Fig. 3
Micronuclei formation in CHO AA8 cells infected with clb+E. coli. Relative cell growth and mean values of MN frequencies at least 1000 cells are shown. In the graph, MOI = 0 represents the vehicle control (treatment with IM). Horizontal red lines in MN graphs indicate MN frequencies two fold higher than those of each vehicle control. N.A. indicates data are not available due to the high cytotoxicity
Fig. 4
Fig. 4
Dose-dependent induction of micronuclei in CHO AA8 cells infected with clb+E. coli. Mean ± SD values of at least three independent experiments are shown. MOI = 0 represents the vehicle control. * indicates p < 0.05 and ** indicates p < 0.01 (versus that of MOI = 0) according to the t-test
Fig. 5
Fig. 5
Induction of SOS response (umuC gene) by E. coli extracts in umu test. Relative LacZ activity to the clb− strain JCM1649T. Mean values of duplicated determinations are shown. 4-NQO as a positive control of DNA damaging agent (incubated for 3 h at 37 °C)
See this image and copyright information in PMC

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