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

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.Lateral gene transfer between bacteria can potentially affect a variety of processes in soil, including the biodegradation of organic pollutants (7,8,10,12,17,27,32,55,56,61). Acquisition of catabolic genes can enhance contaminant biodegradation by increasing the diversity of organisms able to effect at least partial transformation of a compound or expanding on existing pathways so that degradation is more extensive or complete (mineralization). Pathway complementation is exemplified by strains engineered to possess the upper biphenyl degradation pathway as well as the lower chlorobenzoate and chlorocatechol pathways, resulting in an enhanced ability to mineralize polychlorinated biphenyls (18,25,35,46). Similar hybrid pathways could evolve naturally in the environment by lateral gene transfer and affect the activity of microbial communities mediating polychlorinated biphenyl (PCB) biodegradation, but relatively little is known about their occurrence.The recovery of PCB-mineralizing strains from bioreactors or soil inoc...

Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

“…An uncharacterized activity was also reported in a salicylate-grown strain of Moraxella (33). However, very recently a four-gene cluster corresponding to and closely homologous to nagAaGHAb has recently been identified in close physical association to genes for chlorobenzoate catabolism (17). The gene products have S5H activity, but the surrounding DNA is completely devoid of any residues of associated NDO-like genes (17).…”

“…However, very recently a four-gene cluster corresponding to and closely homologous to nagAaGHAb has recently been identified in close physical association to genes for chlorobenzoate catabolism (17). The gene products have S5H activity, but the surrounding DNA is completely devoid of any residues of associated NDO-like genes (17). Whereas we suggested that the nag oxygenase cluster arose by insertion of nagGH into a classical nah-like operon (12), this finding suggests that the four constituent S5H genes may also have an independent evolutionary history of their own.…”

Characterization of a C—C Bond Hydrolase fromSphingomonas wittichiiRW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites

“…The 5Ј end of a second open reading frame was deduced downstream of the dxnB2 gene. This partial open reading frame encodes a fragment of 311 amino acid residues that shares sequence identity with salicylate hydroxylases (e.g., 65% amino acid sequence identity to the ␣ subunit of salicylate hydroxylase from Pseudomonas aeruginosa JB2 [16]). This result is consistent with DxnB2 being involved in dibenzofuran catabolism, as salicylate would be one of the products of the hydrolase-catalyzed reaction in such a pathway.…”

Salicylic Acid Biosynthesis and Metabolism

“…Currently, the plant genes/enzymes involved in 2,3-DHBA and 2,5-DHBA synthesis are unknown. However, based on bacterial pathways, it is possible that isochorismatase catalyzes the formation of 2,3-DHBA directly from IC (Rusnak et al, 1990), and hydroxylation of SA could be performed by a monoxygenase similar to those identified in Pseudomonas and Ralstonia (Hickey et al, 2001 and references therein).…”