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Significance of Glutamate Racemase for the Viability and Cell Wall Integrity of Streptococcus iniae

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Abstract

Streptococcus iniae is a pathogenic and zoonotic bacterium responsible for human diseases and mortality of many fish species. Recently, this bacterium has demonstrated an increasing trend for antibiotics resistance, which has warranted a search for new approaches to tackle its infection. Glutamate racemase (MurI) is a ubiquitous enzyme of the peptidoglycan synthesis pathway that plays an important role in the cell wall integrity maintenance; however, the significance of this enzyme differs in different species. In this study, we knocked out the MurI gene in S. iniae in order to elucidate the role of glutamate racemase in maintaining cell wall integrity in this bacterial species. We also cloned, expressed, and purified MurI and determined its biochemical characteristics. Biochemical analysis revealed that the MurI gene in S. iniae encodes a functional enzyme with a molecular weight of 30 kDa, temperature optimum at 35°C, and pH optimum at 8.5. Metal ions, such as Cu2+, Mn2+, Co2+ and Zn2+, inhibited the enzyme activity. MurI was found to be essential for the viability and cell wall integrity of S. iniae. The optimal growth of the MurI-deficient S. iniae mutant can be achieved only by adding a high concentration of D-glutamate to the medium. Membrane permeability assay of the mutant showed an increasing extent of the cell wall damage with time upon D-glutamate starvation. Moreover, the mutant lost its virulence when incubated in fish blood. Our results demonstrated that the MurI knockout leads to the generation of S. iniae auxotroph with damaged cell walls.

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Abbreviations

CFU:

colony-forming unit

GlcNAc:

N-acetyl-glucosamine

INT:

iodonitrotetrazolium chloride

LB:

Luria—Bertani (medium)

MurNAc:

N-acetylmuramic acid

TSYE:

tryptone soy yeast extract

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Funding

This work was supported by the Natural Science Foundation of Hebei Province (C2019205044), Outstanding Youth Foundation of Department of Education of Hebei Province (YQ2014026), Research Fund of Hebei Normal University (L2016Z03), State Key Laboratory of Pathogen and Biosecurity (Academy of Military Medical Science) (SKLPBS1529), and Science and Technology Research Project of Hebei Normal University (ZD2018070).

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Authors and Affiliations

  1. College of Life Science, Hebei Normal University, 050024, Shijiazhuang, China

    M. Muhammad, J. Bai, J. Ju, B. Zhao & D. Liu

  2. Department of Biochemistry, Kano University of Science and Technology, Wudil, Nigeria

    M. Muhammad

  3. Department of Biochemistry, Bayero University Kano, Kano, Nigeria

    A. J. Alhassan

  4. Department of Medical Laboratory Science, Bayero University Kano, Kano, Nigeria

    H. Sule

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  1. M. Muhammad
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  2. J. Bai
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  3. A. J. Alhassan
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  4. H. Sule
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  5. J. Ju
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  6. B. Zhao
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  7. D. Liu
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Corresponding authors

Correspondence to B. Zhao or D. Liu.

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Ethical approval. All experimental animal procedures were carried out strictly according to the recommendations from the Guide for the Care and Use of the Laboratory Animals of Hebei Province of China. Animal experiment protocols were approved by the Animal Monitoring Committee of Hebei Normal University.

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Conflict of interest. The authors declare no conflict of interest.

Published in Russian in Biokhimiya, 2020, Vol. 85, No. 2, pp. 287-296

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Muhammad, M., Bai, J., Alhassan, A.J. et al. Significance of Glutamate Racemase for the Viability and Cell Wall Integrity of Streptococcus iniae. Biochemistry Moscow 85, 248–256 (2020). https://doi.org/10.1134/S0006297920020121

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