Isolation and Characterization a Novel Catabolic Gene Cluster Involved in Chlorobenzene Degradation in Haloalkaliphilic Alcanivorax sp. HA03

SIMPLE SUMMARY: This study has demonstrated for the first time the metabolic pathway of chlorobenzene (CB) in haloalkaliphilic bacteria Alcanivorax sp. HA03 isolated from soda lakes in Wadi E1Natrun-Egypt. The complete chlorobenzene dioxygenase gene cluster (α and β subunits, ferredoxin and ferredoxin reductase) was detected by PCR amplification and confirmed by DNA sequencing and expression. This gene cluster has the capability to ring-hydroxylating CB transformed into 3-chlorocatechol during the first steps of biodegradation; with further chloride release and subsequent paths, HA03 showed capability for CB mineralization. ABSTRACT: Chlorobenzene (CB) poses a serious risk to human health and the environment, and because of its low degradation rate by microorganisms, it persists in the environment. Some bacterial strains can use CB as growth substrates and their degradative pathways have evolved; very little is known about these pathways and the enzymes for CB degradation in high pH and salinity environments. Alcanivorax sp. HA03 was isolated from the extremely saline and alkaline site. HA03 has the capability to degrade benzene, toluene and chlorobenzene (CB). CB catabolic genes were isolated from HA03, which have a complete gene cluster comprising α and β subunits, ferredoxin and ferredoxin reductase (CBA1A2A3A4), as well as one gene-encoding enzyme for chlorocatechol 1,2-dioxygenase (CC12DOs). Based on the deduced amino acid sequence homology, the gene cluster was thought to be responsible for the upper and lower catabolic pathways of CB degradation. The CBA1A2A3A4 genes probably encoding a chlorobenzene dioxygenase was confirmed by expression during the growth on CB by RT-PCR. Heterologous expression revealed that CBA1A2A3A4 exhibited activity for CB transformation into 3-chlorocatechol, while CC12DOs catalyze 3-chlorocatechol, transforming it into 2-chloromucounate. SDS-PAGE analysis indicated that the sizes of CbA1 and (CC12DOs) gene products were 51.8, 27.5 kDa, respectively. Thus, Alcanivorax sp. HA03 constitutes the first bacterial strain described in the metabolic pathway of CB degradation under high pH and salinity conditions. This finding may have obvious potential for the bioremediation of CB in both highly saline and alkaline contaminated sites.