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Whole-genome Sequencing and Mining of Protease Coding Genes in Bacillus paralicheniformis MKU3, and its Degradomics in Feather Meal Medium

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Abstract

Bacillus paralicheniformis MKU3 produces commercially important keratinolytic proteases by utilizing chicken feather. To unravel the genetics of these degrading keratinolytic proteases in B. paralicheniformis MKU3, we sequenced the genome of this bacterium and studied the protease distribution and their characteristics using bioinformatics tools. Also, a proteomic analysis was performed to identify the consortium of proteases involved in feather hydrolysis. A total of 2,531,755 quality reads were obtained in whole genome sequencing with an approximate coverage fold of 105. The draft genome consists of 4,370,039 bp with 45 contigs. The draft genome codes for 4874 protein-coding genes. Furthermore, 109 genes coding for RNA, including 26 rRNA and 83 tRNA, were identified. Phylogenetic analysis of B. paralicheniformis MKU3 showed closest homolog to B. paralicheniformis F47. Genes coding for proteases belonging to five families were identified with the following proportions 37%, 36%, 9%, 14%, 2%, and 2% of serine-, metallo-, cysteine-, mixed-, and uncharacterized proteases, respectively. Metallo- and serine-protease represented more than 70% of the total proteases. Major protease families distributed in the genome were S8, S9, S33, M20, M50, C26, and C40. Most of the proteases showed significant similarity with the conserved domain database and also identified conserved catalytic sites and domains. SDS-PAGE and zymogram analysis of concentrated feather hydrolysis revealed the active proteases ranging from 10 to 250 kD in size. Proteomic analysis on the feather hydrolysis of B. paralicheniformis MKU3 identified two proteases belonging to serine proteases (S8) and other two as metalloproteases.

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Acknowledgements

UGC-NRCBS, DBT-IPLS, DST-PURSE programs of the School of Biological Sciences, MKU are gratefully acknowledged for providing the facility for genome sequencing and analysis. Authors thank Dr. U.S. Vishnu and Dr. J. Sankarasubramanian for their technical support. We also thank the Mass spectrometry facility of CCAMP/NCBS-TIFR.

Funding

This work was supported by research grant from the Department of Biotechnology, New Delhi, India (No. BT/PR4799/PID/6/642/2012).

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Authors and Affiliations

  1. Department of Genetics, School of Biological Sciences, Madurai Kamaraj University, Madurai, India

    Sangilimadan SanthaKalaikumari, Ramamoorthy Sivakumar & Jeyaprakash Rajendhran

  2. VIT Bhopal University, Bhopal, India

    Paramasamy Gunasekaran

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  1. Sangilimadan SanthaKalaikumari
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  2. Ramamoorthy Sivakumar
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  3. Paramasamy Gunasekaran
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  4. Jeyaprakash Rajendhran
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Contributions

SS performed the experiment, analyzed the data and drafted the manuscript. RS participated in editing the manuscript. JR designed, coordinated, supervised and reviewed the manuscript. PG also reviewed the manuscript. All the authors read and approved the final manuscript.

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Correspondence to Jeyaprakash Rajendhran.

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Electronic supplementary material

Below is the link to the electronic supplementary material.

List of proteases identified in the genome of B. paralicheniformis MKU3 identified from MEROPS database.

284_2020_2271_MOESM2_ESM.xlsx

List of proteases identified from B. paralicheniformis strains. Ten strains of B. paralicheniformis have been retrieved in this study and were analyzed for protease distribution.

284_2020_2271_MOESM3_ESM.xlsx

Extracellular proteases and proteins from B. paralicheniformis MKU3 listed in this file. These proteases and proteins were induced by chicken feather degradation by B. paralicheniformisMKU3.

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SanthaKalaikumari, S., Sivakumar, R., Gunasekaran, P. et al. Whole-genome Sequencing and Mining of Protease Coding Genes in Bacillus paralicheniformis MKU3, and its Degradomics in Feather Meal Medium. Curr Microbiol 78, 206–217 (2021). https://doi.org/10.1007/s00284-020-02271-1

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