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.

In vivo role of FdhD and FdmE in formate metabolism in Pseudomonas putida: Redundancy and expression in the stationary phase

“…3). The time‐course expression of PP0257 was similar to that described before for formate dehydrogenase clusters, the expression of which has been previously shown to be partially dependent on RpoS (Roca et al. , 2009).…”

“…, 2008) when cells were grown in the presence of sub‐lethal concentrations of formaldehyde. We also found that P. putida formaldehyde dehydrogenase and formate dehydrogenases are preferentially expressed at the onset of the stationary phase and that their expression seems to be mediated by RNA polymerase with σ 38 (Roca et al. , 2009).…”

“…Our previous transcriptomic studies showed that expression of the ORF encoding PP0257 was among those that are induced the most in P. putida KT2440 (Roca et al, 2008) when cells were grown in the presence of sub-lethal concentrations of formaldehyde. We also found that P. putida formaldehyde dehydrogenase and formate dehydrogenases are preferentially expressed at the onset of the stationary phase and that their expression seems to be mediated by RNA polymerase with s 38 (Roca et al, 2009).…”

“…, 2008). In addition, this strain detoxifies formaldehyde via the coupled action of formaldehyde dehydrogenase/formate dehydrogenase systems (Roca et al. , 2009).…”

“…Pseudomonas putida KT2440 reacts to formaldehyde and formate via the activation of DNA-repair systems, chaperones and the extrusion of chemicals via the MexEF/OprN efflux pump (Roca et al, 2008). In addition, this strain detoxifies formaldehyde via the coupled action of formaldehyde dehydrogenase/ formate dehydrogenase systems (Roca et al, 2009). The original sequencing and annotation of the KT2440 strain predicted two NAD + -linked formaldehyde dehydrogenases that oxidize formaldehyde to formate (Nelson et al, 2002), although other, yet unidentified, dehydrogenase is also present as deduced from the fact that a double mutant in the two annotated formaldehyde dehydrogenases is still able to oxidize formaldehyde (Roca et al, 2009).…”