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Physiological responses of Pseudomonas putida to formaldehyde during detoxification.

ABSTRACT: Pseudomonas putida KT2440 exhibits two formaldehyde dehydrogenases and two formate dehydrogenase complexes that allow the strain to stoichiometrically convert formaldehyde into CO(2). The strain tolerated up to 1.5 mM formaldehyde and died in the presence of 10 mM. In the presence of 0.5 mM formaldehyde, a sublethal concentration of this chemical, the growth rate decreased by about 40% with respect to growth in the absence of the toxicant. Transcriptomic analysis revealed that in response to low formaldehyde concentrations, a limited number of genes (52) were upregulated. Based on the function of these genes it seems that sublethal concentrations of HCOH trigger responses to overcome DNA and protein damage, extrude this toxic compound, and detoxify it by converting the chemical to CO(2). In strains bearing mutations of the upregulated genes we analysed growth inhibition by 1.5 mM HCOH and killing rates by 10 mM HCOH. Mutants in the MexEF/OprN efflux pump and in the DNA repair genes recA and uvrB were hypersensitive to 10 mM HCOH, the killing rate being three to four orders of magnitude higher than those in the wild-type strain. Mutants in other upregulated genes died at slightly higher or at similar rates to the parental strain. Regarding growth inhibition, we found that mutants in glutathione biosynthesis, stress response mediated by 2-hydroxy acid dehydrogenases and two efflux pumps of the MSF family were unable to grow in the presence of 1.5 mM HCOH. In an independent screening test we searched for mutants which were hypersensitive to formaldehyde, but whose expression did not change in response to this chemical. Two mutants with insertions in recD and fhdA were found which were unable to grow in the presence of 1.5 mM HCOH. The recD mutant was hypersensitive to 10 mM HCOH and died at a higher rate than the parental strain.

SUBMITTER: Roca A

PROVIDER: S-EPMC3864449 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Physiological responses of Pseudomonas putida to formaldehyde during detoxification.

Roca Amalia A Rodríguez-Herva José-Juan JJ Duque Estrella E Ramos Juan L JL

Microbial biotechnology 20080301 2

Pseudomonas putida KT2440 exhibits two formaldehyde dehydrogenases and two formate dehydrogenase complexes that allow the strain to stoichiometrically convert formaldehyde into CO(2). The strain tolerated up to 1.5 mM formaldehyde and died in the presence of 10 mM. In the presence of 0.5 mM formaldehyde, a sublethal concentration of this chemical, the growth rate decreased by about 40% with respect to growth in the absence of the toxicant. Transcriptomic analysis revealed that in response to low ...[more]

PMID: 21261833

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Project description: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.

Project description:BackgroundPolyhydroxyalkanoates (PHAs) have attracted much attention in recent years as natural alternatives to petroleum-based synthetic polymers that can be broadly used in many applications. Pseudomonas putida KT2440 is a metabolically versatile microorganism that is able to synthesize medium-chain-length PHAs (mcl-PHAs). The phenomena that drive mcl-PHAs synthesis and accumulation seems to be complex and are still poorly understood. Therefore, here we determine new insights into cellular responses of Pseudomonas putida KT2440 during biopolymers production using two-dimensional difference gel-electrophoresis (2D-DIGE) followed by MALDI TOF/TOF mass spectrometry.ResultsThe maximum mcl-PHAs content in Pseudomonas putida KT2440 cells was 24% of cell dry weight (CDW) and was triggered by nitrogen depletion. Proteomic analysis allowed the detection of 150 and 131 protein spots differentially regulated at 24 h and 48 h relative to the cell growth stage (8 h), respectively. From those, we successfully identified 84 proteins that had altered expression at 24 h and 74 proteins at 48 h of the mcl-PHAs synthesis process. The protein-protein interactions network indicated that the majority of identified proteins were functionally linkage. The abundance of proteins involved in carbon metabolism were significantly decreased at 24 h and 48 h of the cultivations. Moreover, proteins associated with ATP synthesis were up-regulated suggesting that the enhanced energy metabolism was necessary for the mcl-PHAs accumulation. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis and transport was observed. Our results indicate that mcl-PHAs accumulated in the bacterial cells changed the protein abundance involved in stress response and cellular homeostasis.ConclusionsThe presented data allow us to investigate time-course proteome rearrangement in response to nitrogen limitation and biopolyesters accumulation. Our results have pointed out novel proteins that might take part in cellular responses of mcl-PHA-accumulated bacteria. The study provides an additional knowledge that could be helpful to improve the efficiency of the bioprocess and make it more economically feasible.

Project description:Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings.IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.

Dataset Information

Physiological responses of Pseudomonas putida to formaldehyde during detoxification.

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Physiological responses of Pseudomonas putida to formaldehyde during detoxification.

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