Cloning, Nucleotide Sequencing, and Functional Analysis of a Novel, Mobile Cluster of Biodegradation Genes from Pseudomonas aeruginosa Strain JB2

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Abstract
We have identified in Pseudomonas aeruginosa strain JB2 a novel cluster of mobile genes encoding degradation of hydroxy- and halo-aromatic compounds. Nineteen open reading frames were located and, based on sequence similarities, were putatively identified as encoding a ring hydroxylating oxygenase ( hybABCD ), an ATP-binding cassette-type transporter, an extradiol ring-cleavage dioxygenase, transcriptional regulatory proteins, enzymes mediating chlorocatechol degradation, and transposition functions. Expression of hybABCD in Escherichia coli cells effected stoichiometric transformation of 2-hydroxybenzoate (salicylate) to 2,5-dihydroxybenzoate (gentisate). This activity was predicted from sequence similarity to functionally characterized genes, nagAaGHAb from Ralstonia sp. strain U2 (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522–2530, 1998), and is the second confirmed example of salicylate 5-hydroxylase activity effected by an oxygenase outside the flavoprotein group. Growth of strain JB2 or Pseudomonas huttiensis strain D1 (an organism that had acquired the 2-chlorobenzoate degradation phenotype from strain JB2) on benzoate yielded mutants that were unable to grow on salicylate or 2-chlorobenzoate and that had a deletion encompassing hybABCD and the region cloned downstream. The mutants' inability to grow on 2-chlorobenzoate suggested the loss of additional genes outside of, but contiguous with, the characterized region. Pulsed-field gel electrophoresis revealed a plasmid of >300 kb in strain D1, but no plasmids were detected in strain JB2. Hybridization analyses confirmed that the entire 26-kb region characterized here was acquired by strain D1 from strain JB2 and was located in the chromosome of both organisms. Further studies to delineate the element's boundaries and functional characteristics could provide new insights into the mechanisms underlying evolution of bacterial genomes in general and of catabolic pathways for anthropogenic pollutants in particular.