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The MreA Metal-Binding Sites C40, H65, and C69 Play a Critical Role in the Metal Tolerance of Pseudomonas putida KT2440

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Abstract

Metal-binding proteins occur in the cytosol of most eubacteria. The hypothetical metal responsive protein MreA (PP-2969 gene; NreA) seems responsible for zinc, chromium, cadmium accumulation, and metal ion homeostasis. However, there is a lack of definitive evidence regarding the specific metal-binding sites of MreA protein. The present study aimed to identify putative metal-binding regions for MreA. In silico analysis revealed that amino acids C40, H65, and C69 (CHC region) seem critical for metal-protein interactions. We created site-directed mutants (SDM’s) of MreA for interacted amino acids to validate in silico results. The differential scanning fluorimetry (DSF) and atomic absorption spectroscopy (AAS) showed that SDM strains of MreA protein curtailed metal accumulation compared to the wild types indicating C40, H65, and C69 amino acids are critical for metal binding. Thus, we report potential implications for MreA-bioengineered strains of Pseudomonas putida KT2440 for metal ion homeostasis by alleviating metal toxicity in the biological environment.

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Acknowledgements

The authors dedicate this manuscript to Late Prof P. Maruthi Mohan. The authors thank Dr. Jens Klockgether, Clinical Research Group—OE 6711, Ped. Pneumology and Neonatology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany for providing the wild-type strain. The research work was supported by the Department of Science and technology [INSPIRE Faculty Award (DST) IFA12-LSPA-11to GR] and UGC-SAP DRS-II.

Funding

The research work was supported by the Department of Science and technology [INSPIRE Faculty Award (DST) IFA12-LSPA-11to GR] and [UGC-SAP DRS-II to SSS].

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Authors and Affiliations

  1. Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, Telangana, 500046, India

    Prakash-Babu Phanithi & Raghu Gogada

  2. Department of Biochemistry, Osmania University, Hyderabad, Telangana, 500 007, India

    Shanti Kumari Lunavat, Surya Satyanarayana Singh, Abdul Qadeer Mohammed, Rajesh Medisetty & Raghu Gogada

  3. Department of Biochemistry and Plant Physiology, MS Swaminathan School of Agriculture, Centurion University of Technology and Management, Paralakhemundi, Odisha, 761211, India

    Raghu Gogada

  4. Department of Biochemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh, 522510, India

    Venkata Prasuja Nakka

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  1. Shanti Kumari Lunavat
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Contributions

Conceptualization: [RG] and [SSS]; methodology: [RG] [SKL], [AQM], and [RM]; formal analysis and investigation: [SKL], [RG], [AQM], [RM], and [SSS]; writing—original draft preparation: [SKL], [RG], and [AQM]; writing—review and editing: [RG], [AQM], [VPN], [P-BP], [RM], and [SSS]; funding acquisition, resources, and supervision: [RG] and [SSS].

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Correspondence to Surya Satyanarayana Singh or Raghu Gogada.

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The animal immunization experiments were conducted following the approval from the University Ethical committee (383/01/a/CPCSEA) and comply with the laws of India.

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Lunavat, S.K., Singh, S.S., Mohammed, A.Q. et al. The MreA Metal-Binding Sites C40, H65, and C69 Play a Critical Role in the Metal Tolerance of Pseudomonas putida KT2440. Curr Microbiol 79, 142 (2022). https://doi.org/10.1007/s00284-022-02804-w

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