CLONING AND CHARACTERIZATION OF THE GENES ENCODING NITRILOTRIACETATE MONOOXYGENASE OF CHELATOBACTER-HEINTZII ATCC-29600

A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate ( NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotid e sequence contained three major open reading frames (ORFs), Two of th e ORFs, which were oriented divergently with an intergenic region of 3 07 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were ident ical with those determined for the purified components. We therefore n amed these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B), The ntaA and ntaB genes could he expressed in Esche richia coil DH5 alpha, and the gene products were visualized after Wes tern blotting (immunoblotting) and incubation with polyclonal antibodi es against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution o f a functional NTA monooxygenase complex was possible. The deduced gen e product of ntaA showed only significant homology to SoxA (involved i n dibenzothiophene degradation) and to SnaA (involved in pristamycin s ynthesis); that of ntaB shared weak homologies in one domain with othe r NADH:flavine mononucleotide oxidoreductases. These homologies provid e no conclusive answer as to the possible evolutionary origin of the N TA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial re gulator proteins and therefore may encode a regulator protein, possibl y involved in regulation of ntaA and ntaB expression.