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Pseudomonas putida
About: Pseudomonas putida is a research topic. Over the lifetime, 6854 publications have been published within this topic receiving 230572 citations.
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TL;DR: Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toLUene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to the tod genes.
Abstract: Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to the tod genes. The ttgDEF genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in P. putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn5 insertion in the ttgABC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the ttgD gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1, and a double mutant with knock outs in the ttgD and ttgA genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the single ttgD mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type and ttgD mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in P. putida DOT-T1E.
127 citations
TL;DR: Adapted Pseudomonas putida strains grew in the presence of up to 6% (vol/vol) butanol, the highest reported butanol concentration tolerated by a microbe.
Abstract: Adapted Pseudomonas putida strains grew in the presence of up to 6% (vol/vol) butanol, the highest reported butanol concentration tolerated by a microbe. P. putida might be an alternative host for biobutanol production, overcoming the primary limitation of currently used strains—insufficient product titers due to low butanol tolerance.
127 citations
TL;DR: Strain PKO1 compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation.
Abstract: We studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. Three Pseudomonas isolates, P. pickettii PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K(m) and Vmax) for catechol 2,3-dioxygenase (C230), a key shared enzyme of the toluene-degradative pathway for these strains, and compared these parameters with those for the analogous enzymes from archetypal toluene-degrading pseudomonads which did not show enhanced, nitrate-dependent toluene degradation. C230 purified from strains W31, PKO1, and CFS215 had a significantly greater affinity for oxygen as well as a significantly greater rate of substrate turnover than found for the analogous enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleotide and deduced amino acid sequences of C23O from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the subfamily of C23Os that preferentially cleave monocyclic substrates. Moreover, deletion analysis of the nucleotide sequence upstream of the translational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of sequences critical for regulated expression of tbuE, including a sequence homologous to the ANR-binding site of Pseudomonas aeruginosa PAO. When present in cis, this site enhanced expression of tbuE under oxygen-limited conditions. Taken together, these results suggest the occurrence of a novel group of microorganisms capable of oxygen-requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and xylenes in hypoxic environments. Strain PKO1, which exemplifies this novel group of microorganisms, compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation.
127 citations
TL;DR: It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA.
Abstract: Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h−1 but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (μ = 0.15 h−1) produced 70 g l−1 biomass containing 75% PHA. At a higher exponential feed rate (μ = 0.25 h−1), the overall productivity was increased but less biomass (56 g l−1) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.
126 citations
TL;DR: Wild-type PpG7 degraded naphthalene more rapidly than two mutant strains, one deficient in chemotaxis to naphthaene and the other deficient in motility, as all three strains degraded nphthalene at similar rates in a well-mixed system.
Abstract: Chemotaxis has the potential to enhance the bacterial degradation of organic pollutants in systems in which the pollutants are distributed heterogeneously. However, experimental evidence to confirm this potential has not been documented. In the present study, we evaluated the role of chemotaxis in naphthalene degradation by Pseudomonas putida G7 (PpG7) in aqueous systems that supplied naphthalene from a glass capillary tube. Wild-type PpG7 degraded naphthalene more rapidly than two mutant strains, one deficient in chemotaxis to naphthalene and the other deficient in motility. This result was not due to differences in inherent naphthalene degradation kinetics, as all three strains degraded naphthalene at similar rates in a well-mixed system. In the heterogeneous system, a 90% reduction in the amount of naphthalene initially present took 6 h with the wild-type PpG7 at an initial concentration of 4 × 106 cfu/mL, while a similar reduction with either mutant strain at the same concentration took approximately ...
126 citations