Characterization and Expression Analysis of Extradiol and Intradiol Dioxygenase of Phenol-Degrading Haloalkaliphilic Bacterial Isolates

Abstract

Haloalkophilic bacteria have a potential advantage as a bioremediation organism of high oil-polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotype script, biochemical characters, and sequence analysis of bacterial-16S rRNA were used to identify the bacterial isolates; Halomonas HA1 and Marinobacter HA2. These strains required high concentrations of NaCl to ensure bacterial growth, especially Halomonas HA1 strain. Notably, both isolates can degrade phenol at optimal pH values, between 8 and 9, with the ability to grow in pH levels up to 11, like what was seen in the Halomonas HA1 strain. Moreover, both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway, while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Interestingly, Marinobacter HA2 isolate eliminated the added phenol within an incubation period of 72 h, while the Halomonas HA1 isolate invested 96 h in degrading 84% of the same amount of phenol. Phylogenetic analysis of these 1,2 CTD (catechol dioxygenase) sequences clearly showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae. In comparison, 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Furthermore, semi-quantitative RT-PCR analysis proved the constitutive expression pattern of both dioxygenase genes. These findings provide new isolates of Halomonas sp. and Marinobacter sp. that can degrade phenol at high salt and pH conditions via two independent mechanisms.