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Characterization of transcriptional activities at a divergent promoter of the type VI secretion system in enterohemorrhagic Escherichia coli O157:H7

  • Microbial Genetics, Genomics and Molecular Biology
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Abstract

The type VI secretion system (T6SS) is a novel secretion system found in many Gram-negative bacteria that plays a role in bacterial competition, virulence, and host immune evasion. The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 has a single functional T6SS gene cluster. In this study, we attempted to characterize the transcriptional pattern of the T6SS effector gene Z0264 in EDL933. Transcriptional analyses showed that Z0264 and other T6SS genes were transcribed in vitro in a growth-phase-dependent manner, but Z0264 was not secreted in the rich medium. Using adapter- and radioactivity-free transcription start site analysis, we identified an overlapping divergent promoter between Z0264 and Z0265. A β-galactosidase assay with truncated promoter regions showed that the divergent promoter is functional. In addition, we demonstrated the role of H-NS as a repressor in the transcription of Z0264. Notably, the cDNA PCR assay showed that the mRNA transcript from the Z0264 promoter did not include the entire main T6SS cluster, suggesting segmented gene expression by multiple promoters in the T6SS cluster. In conclusion, we identified a divergent promoter for Z0264 located in the T6SS cluster of EDL933 and characterized its in vitro transcriptional activity during growth. Our findings provide insights and a preliminary understanding of the regulatory mechanisms underlying T6SS transcription.

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References

  • Allsopp, L.P., Wood, T.E., Howard, S.A., Maggiorelli, F., Nolan, L.M., Wettstadt, S., and Filloux, A. 2017. Rsma and Amrz orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 114, 7707–7712.

    Article  CAS  Google Scholar 

  • Basler, M., Pilhofer, M., Henderson, G.P., Jensen, G.J., and Mekalanos, J.J. 2012. Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483, 182–186.

    Article  CAS  Google Scholar 

  • Bingle, L.E.H., Bailey, C.M., and Pallen, M.J. 2008. Type vi secretion: a beginner’s guide. Curr. Opin. Microbiol. 11, 3–8.

    Article  CAS  Google Scholar 

  • Brunet, Y.R., Bernard, C.S., Gavioli, M., Lloubès, R., and Cascales, E. 2011. An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster. PLoS Genet. 7, e1002205.

    Article  CAS  Google Scholar 

  • Bryan, A., Youngster, I., and McAdam, A.J. 2015. Shiga toxin producing Escherichia coli. Clin. Lab. Med. 35, 247–272.

    Article  Google Scholar 

  • Charity, J.C., Blalock, L.T., Costante-Hamm, M.M., Kasper, D.L., and Dove, S.L. 2009. Small molecule control of virulence gene expression in Francisella tularensis. PLoS Pathog. 5, e1000641.

    Article  Google Scholar 

  • Chen, Y., Wong, J., Sun, G.W., Liu, Y., Tan, G.Y.G., and Gan, Y.H. 2011. Regulation of type VI secretion system during Burkholderia pseudomallei infection. Infect. Immun. 79, 3064–3073.

    Article  CAS  Google Scholar 

  • Cianfanelli, F.R., Monlezun, L., and Coulthurst, S.J. 2016. Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends Microbiol. 24, 51–62.

    Article  CAS  Google Scholar 

  • Datsenko, K.A. and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645.

    Article  CAS  Google Scholar 

  • Dong, T.G. and Mekalanos, J.J. 2012. Characterization of the RpoN regulon reveals differential regulation of T6SS and new flagellar operons in Vibrio cholerae O37 strain V52. Nucleic Acids Res. 40, 7766–7775.

    Article  CAS  Google Scholar 

  • Griffin, P.M. and Tauxe, R.V. 1991. The epidemiology of infections caused by Escherichia coli O157: H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13, 60–98.

    Article  CAS  Google Scholar 

  • Jani, A.J. and Cotter, P.A. 2010. Type VI secretion: not just for pathogenesis anymore. Cell Host Microbe 8, 2–6.

    Article  CAS  Google Scholar 

  • Jurėnas, D. and Journet, L. 2021. Activity, delivery, and diversity of type VI secretion effectors. Mol. Microbiol. 115, 383–394.

    Article  Google Scholar 

  • Kaper, J.B. 1998. Enterohemorrhagic Echerichia coli. Curr. Opin. Microbiol. 1, 103–108.

    Article  CAS  Google Scholar 

  • Kaper, J.B., Nataro, J.P., and Mobley, H.L.T. 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2, 123–140.

    Article  CAS  Google Scholar 

  • Li, J., Yao, Y., Xu, H.H., Hao, L., Deng, Z., Rajakumar, K., and Ou, H.Y. 2015. SecReT6: a web-based resource for type VI secretion systems found in bacteria. Environ. Microbiol. 17, 2196–2202.

    Article  Google Scholar 

  • Lim, J.Y., Yoon, J., and Hovde, C.J. 2010. A brief overview of Escherichia coli O157:H7 and its plasmid O157. J. Microbiol. Biotechnol. 20, 5–14.

    Article  CAS  Google Scholar 

  • Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25, 402–408.

    Article  CAS  Google Scholar 

  • Miller, J. 1972. Assay of β-galactosidase. In Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.

    Google Scholar 

  • Monjarás Feria, J. and Valvano, M.A. 2020. An overview of antieukaryotic T6SS effectors. Front. Cell. Infect. Microbiol. 10, 584751.

    Article  Google Scholar 

  • Mougous, J.D., Cuff, M.E., Raunser, S., Shen, A., Zhou, M., Gifford, C.A., Goodman, A.L., Joachimiak, G., Ordoñez, C.L., Lory, S., et al. 2006. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526–1530.

    Article  CAS  Google Scholar 

  • Münch, R., Hiller, K., Grote, A., Scheer, M., Klein, J., Schobert, M., and Jahn, D. 2005. Virtual footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes. Bioinformatics 21, 4187–4189.

    Article  Google Scholar 

  • Navarro-Garcia, F., Ruiz-Perez, F., Cataldi, Á., and Larzábal, M. 2019. Type VI secretion system in pathogenic Escherichia coli: structure, role in virulence, and acquisition. Front. Microbiol. 10, 1965.

    Article  Google Scholar 

  • Pukatzki, S., Ma, A.T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W.C., Heidelberg, J.F., and Mekalanos, J.J. 2006. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the dictyostelium host model system. Proc. Natl. Acad. Sci. USA 103, 1528–1533.

    Article  CAS  Google Scholar 

  • Reese, M.G. 2001. Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput. Chem. 26, 51–56.

    Article  CAS  Google Scholar 

  • Sana, T.G., Soscia, C., Tonglet, C.M., Garvis, S., and Bleves, S. 2013. Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa. PLoS ONE 8, e76030.

    Article  CAS  Google Scholar 

  • Si, M., Wang, Y., Zhang, B., Zhao, C., Kang, Y., Bai, H., Wei, D., Zhu, L., Zhang, L., Dong, T.G., et al. 2017. The type VI secretion system engages a redox-regulated dual-functional heme transporter for zinc acquisition. Cell Rep. 20, 949–959.

    Article  CAS  Google Scholar 

  • Silverman, J.M., Brunet, Y.R., Cascales, E., and Mougous, J.D. 2012. Structure and regulation of the type VI secretion system. Annu. Rev. Microbiol. 66, 453–472.

    Article  CAS  Google Scholar 

  • Simons, R.W., Houman, F., and Kleckner, N. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53, 85–96.

    Article  CAS  Google Scholar 

  • Solovyev, V. and Salamov, A. 2011. Automatic annotation of microbial genomes and metagenomic sequences. In Li, R.W. (ed.), Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies, pp. 61–78. Nova Science Publishers, Hauppauge, New York, USA.

    Google Scholar 

  • Wan, B., Zhang, Q., Ni, J., Li, S., Wen, D., Li, J., Xiao, H., He, P., Ou, H.Y., Tao, J., et al. 2017. Type VI secretion system contributes to enterohemorrhagic Escherichia coli virulence by secreting catalase against host reactive oxygen species (ROS). PLoS Pathog. 13, e1006246.

    Article  Google Scholar 

  • Wang, C., Lee, J., Deng, Y., Tao, F., and Zhang, L.H. 2012. ARF-TSS: an alternative method for identification of transcription start site in bacteria. BioTechniques 52, 1–3.

    Article  Google Scholar 

  • Zhang, Y., Gao, H., Osei-Adjei, G., Zhang, Y., Yang, W., Yang, H., Yin, Z., Huang, X., and Zhou, D. 2017. Transcriptional regulation of the type VI secretion system 1 genes by quorum sensing and ToxR in Vibrio parahaemolyticus. Front. Microbiol. 8, 2005.

    Article  Google Scholar 

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Acknowledgements

This study was supported by a grant from National Research Foundation (NRF-2018R1D1A1B07051455) to S.K.K. and a grant (NRF-2017R1A2B4013056) to J.W.Y., Republic of Korea.

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Authors and Affiliations

  1. College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon, 24341, Korea

    Se Kye Kim, Jun Bong Lee & Jang Won Yoon

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  1. Se Kye Kim
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  2. Jun Bong Lee
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Contributions

S.K.K. and J.W.Y. conceptualized and designed the study.

S.K.K. and J.B.L. carried out the experiments and analyzed the data. S.K.K. wrote the paper with the help of J.W.Y.

Corresponding author

Correspondence to Jang Won Yoon.

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The authors have no potential conflicts of interest to disclose.

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Kim, S.K., Lee, J.B. & Yoon, J.W. Characterization of transcriptional activities at a divergent promoter of the type VI secretion system in enterohemorrhagic Escherichia coli O157:H7. J Microbiol. 60, 928–934 (2022). https://doi.org/10.1007/s12275-022-2109-9

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  • DOI: https://doi.org/10.1007/s12275-022-2109-9

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