Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2023

Molecular characterisation of the putative de novo biotin biosynthesis genes bioA and bioB from Burkholderia pseudomallei (#141)

Ashley Johnson 1 , Justine Bendo 1 , Charlene Kahler 1 , Aleksandra Debowski 1 2 , Philip Ireland 3 , Keith Stubbs 2 , Mitali Sarkar-Tyson 1
  1. Marshall Centre for Infectious Disease, School of Biomedical Sciences, The University of Western Australia, Crawley, WA, Australia
  2. School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia
  3. Defence Science and Technology Laboratory, Porton Down, Salisbury, United Kingdom

Burkholderia pseudomallei, the aetiologic agent of melioidosis, is a facultative intracellular and inherently antibiotic-resistant pathogen. Melioidosis is estimated to cause 89,000 deaths annually1. This highlights the urgent requirement for development of novel antibiotics to effectively treat this severe disease.

Biotin plays a critical role in intermediary metabolism as an essential cofactor of biotin-dependent enzymes2. Unlike bacteria, humans are unable to synthesise biotin de novo, instead requiring dietary intake3. Thus, biotin biosynthesis presents a niche target for development of novel therapeutics against bacterial pathogens, and has consequently drawn significant interest in the field of drug discovery. The genes involved in its biosynthesis have been well-characterised in some bacteria. However, this pathway is yet to be fully explored in B. pseudomallei.

The bioA and bioB genes of B. pseudomallei K96243, assigned through JCVI metagenomics analysis using curated Hidden Markov Model-based protein families, are demonstrated in this study to be highly likely involved in de novo biotin biosynthesis4.

Through bioinformatics and computational characterisation, the bpsl0367 (bioA) and bpsl0364 (bioB) genes of B. pseudomallei were predicted to encode BioA, 7,8-diamino-pelargonic acid (DAPA) synthase, and BioB, biotin synthase, respectively. Deletion mutants BpsΔbioA and BpsΔbioB, and complement strains BpsΔbioA/bioA and B.psΔbioB/bioB were generated and assessed for biotin auxotrophy. For the BpsΔbioA and BpsΔbioB mutant strains, a growth defect was observed in minimal media lacking free biotin as compared to the wild-type strain.

The phenotype was confirmed to be a result of deletion of the bioA and bioB genes through restoration of growth to wild-type levels, as seen with BpsΔbioA/bioA and BpsΔbioB/bioB complement strains, and when the BpsΔbioA and B.psΔbioB mutants were supplemented with exogenous biotin. This work suggests that the bioA and bioB mutants of B. pseudomallei K96243 are biotin auxotrophs, and thus involved in biotin metabolism. Further characterisation through molecular and biochemical methods will allow us to confirm the predicted enzymatic activity of the proteins, and assess their potential as targets for novel antibiotic development.

  1. Limmathurotsakul, D., Golding, N., Dance, D.A., Messina, J.P., Pigott, D.M., Moyes, C.L., Rolim, D.B., Bertherat, E., Day, N.P., Peacock, S.J. and Hay, S.I., 2016. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nature microbiology, 1(1), pp.1-5.
  2. Moss, J. and Lane, M.D., 1971. The biotin-dependent enzymes. Adv Enzymol Relat Areas Mol Biol, 35, pp.321-442.
  3. Bowers-Komro, D.M. and McCormick, D.B. 1985. Biotin uptake by isolated rat liver hepatocytes. In Biotin (K. Dakshinamurti and H.N. Bhagavan, eds.), New York Academy of Sciences, 1, pp. 350–358.
  4. Tatusova T., DiCuccio M., Badretdin A., Chetvernin V., Nawrocki E.P., Zaslavsky L., Lomsadze A., Pruitt KD., Borodovsky M., Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res, 19;44(14), pp. 6614-6624.