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

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M. Muhammad^1,2, J. Bai¹, A. J. Alhassan³, H. Sule⁴, J. Ju¹, B. Zhao^1,a*, and D. Liu^1,b*

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

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

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

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

^* To whom correspondence should be addressed.

Received June 14, 2019; Revised September 19, 2019; Accepted October 7, 2019

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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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KEY WORDS: Streptococcus iniae, glutamate racemase, gene knockout, cell wall integrity, biochemical characterizations

DOI: 10.1134/S0006297920020121

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