Deletion or overexpression of tpx gene alters doubling time and response to stress agents and affects asphaltene biotransformation in Micrococcus sp. IITD107

Thiol peroxidases have been identified as antioxidant enzymes in different organisms. However, there have been only a few reports on deleting or overexpressing its gene to characterize its function. Recently, the ability of the enzyme thiol peroxidase belonging to the Micrococcus sp. IITD107 on asphaltene biotransformation has been reported. Here, we report the construction of deletion and overexpression strains of tpx and compared them with the wild-type strain of Micrococcus sp. IITD107. For this purpose, whole-genome sequencing of the strain was carried out. The genome sequence revealed the presence of only one copy of the tpx gene and several other genes which might play a role in stress response, polyaromatic hydrocarbon (PAH) degradation, etc. The tpx deletion mutant was constructed by homologous recombination. For overexpression, a pRC4 replicon from Rhodococcus was used, and the gene for tpx was expressed under the control of an IPTG-inducible tac promoter. The replicon was found to be stable in Micrococcus. The deletion mutant showed coccoid morphology, and the doubling time was found to be 7.2 h as compared to the doubling times of 3.5 h for the wild-type strain and 4.5 h for the overexpression strain in LB medium. The deletion strain was unable to grow in the presence of high salt or hydrogen peroxide concentrations, whereas the overexpression strain showed less growth as compared to the wild type. Further, about a 50% decrease and only a 5% increase in the rate of asphaltene biotransformation in the deletion and overexpression strains, respectively, were observed. Overall, our results suggest that thiol peroxidase helps not only in asphaltene biotransformation but also aids the bacterium to survive in the presence of stress agents such as salt and hydrogen peroxide. KEY POINTS: • Gene for thiol peroxidase confirmed to enhance asphaltene biotransformation • Change in growth and morphology • Alteration in halotolerance and stress response.