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Regulation of biofilm formation by marT in Salmonella Typhimurium

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Abstract

In this study, we aimed at identifying the regulatory role of marT gene, known as the regulator of misL, on 15 different biofilm-related genes in S. Typhimurium 14028 strain. We also tested the strains for their ability to form biofilm and determined the adherence characteristics of the wild type and the mutant strains of the organism on Caco-2 and HEp-2 cells. For comparative analyses of the candidate genes, individual gene mutations were created via antibiotic gene cassette insertion into each gene of interest. marT gene was cloned behind an arabinose inducible BAD promoter in order to control marT expression. This recombinant plasmid was transfer into each of the 15 mutant strains to investigate the level of expression of each single gene in the presence and absence of marT induction. Besides determination of variations in biofilm formation by each mutant strain, the attachment characteristics of them onto Caco-2 and HEp-2 cell lines were also reported. As a result of attachments experiments on polystyrene surfaces, it was determined that the biofilm production capacity of each mutant strain decreased in a statistically significant manner (p < 0.05). QRT-PCR trials indicated that the marT gene regulates the expression of 14 genes, namely fimA, fimD, fimF, fimH, stjB, stjC, csgA, csgD, ompC, sthB, sthE, rmbA, fliZ and yaiC, in a positive manner. QRT-PCR studies were also revealed that the MarT protein positively regulates its own promoter. When the adherence characteristics of the mutant strains and the wild-type were investigated by using Caco-2 and HEp-2 cells, it was determined that the single gene mutations did have no effect on bacterial adhesion. In view of our mutational analyses and biofilm formation studies, it was concluded that fliZ, ompC, rmbA, stjB and stjC genes are related with biofilm formation in Salmonella, besides other cellular functions of them. Taken together, our data suggested that the regulatory role of MarT protein is not only restricted to the regulation of misL gene expression, but it rather acts as a general regulator on the biofilm-related genes in Salmonella.

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Acknowledgements

This work was supported by the grant 114Z871 from The Scientific and Technological Research Council of Turkey (TÜBİTAK). We would like to thank to Prof. Dr. İhsan Gürsel, Prof. Dr. Erkan Yılmaz, Prof. Dr. Sreeparna Banerjee and Professor Michael McClelland for their contributions.

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Authors and Affiliations

  1. Department of Biology, Middle East Technical University, Ankara, Turkey

    Zeynep Eran & Gülay Özcengiz

  2. Department of Biology, Ankara University, Ankara, Turkey

    Mustafa Akçelik

  3. Biotechnology Institute, Ankara University, Ankara, Turkey

    Betül Cansu Yazıcı & Nefise Akçelik

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  1. Zeynep Eran
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  2. Mustafa Akçelik
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  3. Betül Cansu Yazıcı
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  4. Gülay Özcengiz
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Contributions

ZE and BCY performed the experimental analyses. MA and GÖ guided the experiments and wrote the original draft. NA established the main idea of the manuscript, supervised the study, and edited the original draft.

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Correspondence to Nefise Akçelik.

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Supplementary file1 (DOCX 16 kb)

11033_2020_5573_MOESM2_ESM.png

Supplemental Fig. 1 PCR results verifying the entry of the kanamycin cassette into the targetgenes. The presence of 193bp amplicone was provedn the presence of the antibiotic cassette inthe target gene. (PNG 711 kb)

Supplemental Fig. 2 Light microscope image of Caco-2 monolayer (Leica, Type 090-135.001,X40). (PNG 372 kb)

Supplemental Fig. 3 Light microscope image of Caco-2 and HEp-2 monolayers (Leica, Type 090-135.001, X40). (PNG 227 kb)

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Eran, Z., Akçelik, M., Yazıcı, B.C. et al. Regulation of biofilm formation by marT in Salmonella Typhimurium. Mol Biol Rep 47, 5041–5050 (2020). https://doi.org/10.1007/s11033-020-05573-6

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  • DOI: https://doi.org/10.1007/s11033-020-05573-6

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