Redundancy of Enzymes for Formaldehyde Detoxification in Pseudomonas putida

Abstract

Pseudomonas putida KT2440 exhibits redundant formaldehyde dehydrogenases and formate dehydrogenases that contribute to the detoxification of formaldehyde, a highly toxic compound. Physical and transcriptional analyses showed that the open reading frame (ORF) PP0328, encoding one of the formaldehyde dehydrogenases, is self-sufficient, whereas the other functional formaldehyde dehydrogenase gene (ORF PP3970) forms an operon with another gene of unknown function. Two formate dehydrogenase gene clusters (PP0489 to PP0492 and PP2183 to PP2186) were identified, and genes in these clusters were found to form operons. All four transcriptional promoters were mapped by primer extension and revealed the presence of noncanonical promoters expressed at basal level in the exponential growth phase and at a higher level in the stationary phase regardless of the presence of extracellular formaldehyde or formate. These promoters were characterized by a 5'-AG-CCA-C/A-CT-3' conserved region between -7 and -16. To determine the contribution of the different gene products to formaldehyde and formate mineralization, mutants with single and double mutations of formaldehyde dehydrogenases were generated, and the effect of the mutations on formaldehyde catabolism was tested by measuring (14)CO(2) evolution from (14)C-labeled formaldehyde. The results showed that both enzymes contributed to formaldehyde catabolism. A double mutant lacking these two enzymes still evolved CO(2) from formaldehyde, suggesting the presence of one or more still-unidentified formaldehyde dehydrogenases. Mutants with single and double mutations in the clusters for formate dehydrogenases were also generated, and all of them were able to metabolize [(14)C]formate to (14)CO(2), suggesting a redundancy of functions that was not limited to only the annotated genes. Single and double mutants deficient in formaldehyde dehydrogenases and formate dehydrogenases exhibited longer lag phases than did the parental strain when confronted with concentrations of formaldehyde close to the MICs. This suggests a role for the detoxification system in tolerance to sublethal concentrations of formaldehyde.