An acid, 2,2-dichloropropionic acid (2,2-DCP) is an active ingredient in herbicide (Dalapon®). Using 2,2-DCP as a model substrate, an alkalotolerant bacterium was successfully isolated from the Blue Lake, Turkey. This bacterium is a potential bioremediation agent of recalcitrant xenobiotic halogenated compounds. This study aimed to prove the efficacy of the alkalotolerance Bacillus megaterium BHS1 in degrading 2,2-DCP as the sole source of carbon. Biolog GEN III system and 16S rRNA analysis were used for the identification of the bacterium. It was discovered that the strain BHS1 is Bacillus megaterium, and the bacterium that was observed to thrive in alkaline conditions (pH 7.0−14.0), supplemented with varying concentrations of 2,2-DCP (from 20 to 60 mM). Growth of strain BHS1 was exceptional in 40 mM of 2,2-DCP at pH 9, corresponding to a cell doubling time of 17.7 hour, whereas was fully inhibited at 50 mM 2,2-DCP. Since halogenated pollutants can make their way into highly alkaline environments, therefore, identifying threshold levels of strain BHS1 with respect to alkaline-tolerance and maximum level of 2,2-DCP may prove pertinent. This is to ensure that an optimal environment is created for the bacteria to degrade 2,2-DCP-contaminated water. In addition, this is the first study exploring a Bacillus species isolated from an alkaline environment adept in utilizing 2,2-DCP as a sole source of carbon. Hence, the ability of this strain to degrade other types of haloalkanoic acids constitutes a worthy future study.

Purpose Bacillus megaterium strain BHS1, isolated from an alkaline water sample taken from Mavi Gölü (Blue Lake, Turkey), can grow on minimal medium containing 2,2-dichloropropionic acid. We characterized this bacterium at the genomic level. Methods The HiSeq platform was used to carry out genome sequencing, de novo assembly, and scaffolding with strain BHS1. Next, genome data were analyzed to demarcate DNA regions containing protein-coding genes and determine the function of certain BHS1 genes. Finally, results from a colorimetric chloride ion–release assay demonstrated that strain BHS1 produces dehalogenase. Results De novo assembly of the BHS1 genomic sequence revealed a genome size of ~ 5.37 Mb with an average G+C content of 38%. The predicted nuclear genome harbors 5509 protein-coding genes, 1353 tRNA genes, 67 rRNA genes, and 6 non-coding (mRNA) genes. Genomic mapping of strain BHS1 revealed its amenability to synthesize two families of dehalogenases (Cof-type haloacid dehalogenase IIB family hydrolase and haloacid dehalogenase type II), suggesting that these enzymes can participate in the catabolism of halogenated organic acids. The mapping identified seven Na+/H+ antiporter subunits that are vital for adaptation of the bacterium to an alkaline environment. Apart from a pairwise analysis to the well-established L-2-haloacid dehalogenases, whole-cell analysis strongly suggested that the haloacid dehalogenase type II might act stereospecifically on L-2-chloropropionic acid, D,L-2-chloropropionic acid, and 2,2-dichloropropionic acid. Whole-cell studies confirmed the utilization of these three substrates and the gene’s role in dehalogenation. Conclusions To our knowledge, this is the first report of the full genome sequence for strain BHS1, which enabled the characterization of selected genes having specific metabolic activities and their roles in the biodegradation of halogenated compounds.

This is the first structural model of L-haloacid dehalogenase (DehLBHS1) isolated from alkalotolerant Bacillus megaterium BHS1, which has been known to degrading halogenated environmental contaminants. The study suggested five important key amino acid residues of DehLBHS1, namely Arg40, Phe59, Asn118, Asn176 and Trp178 important for catalysis and molecular recognition of haloalkanoic acid. Alkatolerant DehLBHS1was modeled by I-TASSER with the best C-score 1.23. Model validation was carried out utilising PROCHECK to produce the Ramachandran map with 89.2 percent of its residues were found in the most preferred region, indicating that the model was appropriate. The Molecular docking (MD) simulation found that the DehLBHS1 preferred 2,2DCP more than other substrates and formed one hydrogen bond with Arg40 and minimum energy -2.5 kJ/ mol. Molecular dynamics has verified the substrate preference towards 2,2DCP based on RMSD, RMSF, Gyration, Hydrogen bond and Molecular distance. This structural knowledge from DehLBHS1 structural perspective gives insights into substrate specificity and catalytic function to exploit DehLBHS1 of BHS1 strain in degrading 2,2-DCP in the polluted alkaline environments.