Semi‐Rational Engineering of Toluene Dioxygenase from Pseudomonas putida F1 towards Oxyfunctionalization of Bicyclic Aromatics

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.

Identification and functional analysis of the activator gene involved in the biosynthesis of Co-type nitrile hydratase from Aurantimonas manganoxydans

Pseudomonas putida F1 is unable to grow on styrene due to the accumulation of 3-vinylcatechol, a toxic metabolite that is produced through the toluene degradation (tod) pathway and causes catechol-2,3-dioxygenase (C23O) inactivation. In this study, we characterized a spontaneous F1 mutant, designated SF1, which acquired the ability to grow on styrene and did not accumulate 3-vinylcatechol. Whereas adaptation to new aromatic substrates has typically been shown to involve increased C23O activity or the acquisition of resistance to C23O inactivation, SF1 retained wild-type C23O activity. Surprisingly, SF1 grew more slowly on toluene, its native substrate, and exhibited reduced toluene dioxygenase (TDO) activity (approximately 50 % of that of F1), the enzyme responsible for ring hydroxylation and subsequent production of 3-vinylcatechol. DNA sequence analysis of the tod operon of SF1 revealed a single base pair mutation in todA (C479T), a gene encoding the reductase component of TDO. Replacement of the wild-type todA allele in F1 with todA(C479T) reduced TDO activity to SF1 levels, obviated vinylcatechol accumulation, and conferred the ability to grow on styrene. This novel 'less is more' strategy - reduced catechol production as a means to expand growth substrate range - sheds light on an alternative approach for managing catechol toxicity during the metabolism of aromatic compounds.

Hydrocarbons and Methylotrophy

Toluene dioxygenase (tod) is a multicomponent enzyme system in Pseudomonas putida F1. Tod can mediate the degradation of Trichloroethylene (TCE), a widespread pollutant. In this study, we try to explore the TCE-regulated tod expression by using real-time qRT-PCR. The minimal culture media were supplemented with glucose, toluene, or a mixture of glucose/toluene respectively as carbon and energy sources. The TCE was injected into each medium after a 12-hour incubation period. The TCE injection severely affected bacterial growth when cultured with toluene or toluene/glucose mixtures. The cell density dropped 61 % for bacteria growing in toluene and 36 % for bacteria in the glucose/toluene mixture after TCE injection, but the TCE treatment had little effect on bacteria supplied with glucose alone. The decrease in cell number was caused by the cytotoxicity of the TCE metabolized by tod. The results from the real-time qRT-PCR revealed that TCE was capable of inducing tod expression in a toluene-dependent manner and that the tod expression level increased 50 times in toluene and 3 times in the toluene/glucose mixture after 6 hours of TCE treatment. Furthermore, validation of the rpoD gene as a reference gene for P. putida F1 was performed in this study, providing a valuable foundation for future studies to use real-time qRT-PCR in the analysis of the P. putida F1 strain.

For this study we chose a series of commonly encountered bacterial and fungal microorganisms: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida F1, Klebsiella pneumoniae, Staphylococcus aureus, Micrococcus luteus and Candida albicans. For their analysis using infrared spectrophotometry. Interpretation of data from the Spectrophotometer reading was done with the program Origin version 7. The results showed detectable differences between the spectra taken on bacteria and fungi. Could differentiate the chromatogram peaks characteristics for bacteria and fungi. The graphics made it was found that the combination of polysaccharide region (1200-900 cm-1) with “fingerprint region” (900-700 cm-1) and mixed region (1500-1200 cm-1) spectra and their derivatives primarily were most useful to characterize the FT-IR spectra, and to differentiate microorganisms. From the research done on the microorganisms: Staphylococcus aureus, Micrococcus luteus, Pseudomonas putida F1, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Candida albicans can concluded that they can differentiate using FT-IR technique. FT - IR Identification techniques is a fast and accurate of microorganisms identification techniques (organisms composition differs, molecular composition in generals is different and FT-IR spectra of them will be different) with shortening of identification time. Method FT - IR can be used for rapid identification of microorganisms. Being only need a small amount of culture for 24 hours and 5 minutes to read Spectrophotometer IR light.

Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. However, deletion of the gene encoding the P. putida F1 orthologue (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 mcpS-deletion mutant. Expression of mcfS in the same mcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.

A new gene cluster, designated sepABC and a divergently transcribed sepR, was found downstream of the two-component todST phosphorelay system that regulates toluene degradation (the tod pathway) in Pseudomonas putida F1 (PpF1). The deduced amino acid sequences encoded by sepABC show a high homology to bacterial proteins known to be involved in solvent efflux or multidrug pumps. SepA, SepB and SepC are referred to be periplasmic, inner membrane and outer membrane efflux proteins respectively. Effects on growth of various PpF1 mutants compared to that of the wild type in the presence of toluene indicated a possible protective role of the solvent efflux system in a solvent-stressed environment. Growth tests with the complemented mutants confirmed the involvement of the Sep proteins in conferring solvent tolerance. The sepR gene encodes a 260-residue polypeptide that is a member of the E. coli IclR repressor protein family. The repressor role of SepR was established by conducting tests with a sep-lacZ transcriptional fusion in Escherichia coli and PpF1, expression of SepR as a maltose-binding fusion protein in a DNA binding assay, and mRNA analysis. Southern hybridization experiments and analysis of the P. putida KT2440 genome sequence indicated that sepR is a relatively rare commodity compared to homologues of the sepABC genes. We developed a whole-cell bioluminescent biosensor, PpF1G4, which contains a chromosomally based sep-lux transcriptional fusion. The biosensor showed significant induction of the sepABC genes by a wide variety of aromatic molecules, including benzene, toluene, ethylbenzene, and all three isomers of xylene (BTEX), naphthalene, and complex mixtures of aliphatic and aromatic hydrocarbons. PpF1G4 represents a second-generation biosensor that is not based on a catabolic promoter but is nonetheless inducible by aromatic pollutants and moreover functional under nutrient-rich conditions.

Pseudomonas putida F1 can assimilate benzene, toluene and ethylbenzene using the toluene degradation pathway, and can also utilize p-cymene via p-cumate using the p-cymene and p-cumate catabolic pathways. In the present study, P. putida F1 strains were isolated that were adapted to assimilate new substrates such as n-propylbenzene, n-butylbenzene, cumene and biphenyl, and the molecular mechanisms of genetic adaptation to an expanded range of aromatic hydrocarbons were determined. Nucleotide sequence analyses showed that the selected strains have mutations in the cymR gene but not in todF gene. The impairment of the repressor CymR by mutation led to the constitutive expression of CmtE, a meta-cleavage product hydrolase from the cmt operon. This study also showed that CmtE has a broad range of substrates and can hydrolyse meta-cleavage products formed from biphenyl and other new growth substrates via the toluene degradation pathway. However, the artificially constructed strain P. putida F1(cymR : : Tc(r)) and a recombinant P. putida F1, which expressed CmtE constitutively, could not grow on the new substrates. The adapted strains possess the tod operon, which is induced by new growth substrates that are poor inducers of wild-type P. putida F1. When the todS gene from the adapted strains was introduced in a trans manner to P. putida F1(cymR : : Tc(r)), the resulting recombinant strains were able to grow on biphenyl and other new substrates. This finding indicates that the TodS sensor was altered to recognize these substrates and this conclusion was confirmed by nucleotide sequence analyses. Amino acid substitutions were found in the regions corresponding to the receiver domain and the second PAS domain and their boundaries in the TodS protein. These results showed that P. putida F1 adapted strains capable of growth on n-propylbenzene, n-butylbenzene, cumene and biphenyl possess mutations to employ CmtE and to induce the tod catabolic operon by the new growth substrates.

Recently, the development and optimization of a flow injection analysis (FIA) operated bacterial biosensor based on the aerobic catabolism of Pseudomonas putida ML2 was reported in the literature (Lanyon et al. 2004, 2005). By adapting information from these reports, we investigated whether operating parameters and procedures of the benzene biosensor could be directly applied to a new system based on a different bacterial strain for the detection of the whole benzene, toluene, ethylbenzene, and xylenes range. Cells of the investigated bacterial strain, Pseudomonas putida F1, were immobilized between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen electrode. The P. putida F1 aerobically degrades benzene, toluene, and ethylbenzene (BTE) (Cho et al. 2000). The BTE biosensor in kinetic mode FIA displayed a linear range of 0.02–0.14 mM benzene (response time: 5 min, base-line recovery time: 15 min), 0.05–0.2 mM toluene (response time: 8 min, baseline recovery time: 20 min), and 0.1–0.2 mM ethylbenzene (response time: 12 min, baseline recovery time: 30 min), respectively. Due to the differences in sensitivity, response, and baseline recovery times for BTE, it was possible to differentiate each compound in mixtures of these volatile organic compounds (VOCs). No response for xylenes could be obtained since they cannot be completely metabolized by this bacterial strain. However, it was reported that the range of compounds degradable by P. putida F1 can possibly be expanded by cultivating the cells on different carbon sources (Choi et al. 2003). The sensor showed good intra‐ and interassay reproducibility, and all obtained results were comparable with those reported in the literature. The demonstrated reproducibility and the simplicity and ease of use as well as the portability for in situ measurements indicates that the biosensor could be suitable as a reliable initial warning device for elevated BTE levels in indoor and outdoor environments.

A tod-lux transcriptional fusion bioluminescent reporter strain, Pseudomonas putida B2, was developed to permit on-line analysis of trichloroethylene (TCE) transformation by toluene dioxygenase (todC1C2BA) in Pseudomonas putida F1. Strain B2 was exposed to toluene in growing and resting cell bioluminescence assays. The growing cells showed a direct correlation between bioluminescence and toluene concentration, while resting cells showed reproducible bioluminescence with repeated toluene exposures. In addition, P. putida B2 was encapsulated in alginate beads and used in a packed bed flow-through differential volume reactor. The TCE feed into the differential volume reactor was constant at 20 mg L-1 and toluene was pulsed in square-wave perturbations at 10 mg L-1. The system showed a direct correlation between the expression of the tod operon (as monitored by light output) and the co-metabolism of TCE. Approximately 20% of the TCE and 50% of the toluene was removed at a flow rate of 0.4 ml min-1. This approach allowed the on-line monitoring of tod gene expression and its relation to TCE biotransformation.

Expression and longevity of toluene dioxygenase in Pseudomonas putida F1 induced at different dissolved oxygen concentrations

[This corrects the article DOI: 10.1186/s13068-016-0452-y.].

BackgroundPseudomonas putida is a promising host for the bioproduction of chemicals, but its industrial applications are significantly limited by its obligate aerobic character. The aim of this paper is to empower the anoxic metabolism of wild-type Pseudomonas putida to enable bioproduction anaerobically, with the redox power from a bioelectrochemical system (BES).ResultsThe obligate aerobe Pseudomonas putida F1 was able to survive and produce almost exclusively 2–Keto-gluconate from glucose under anoxic conditions due to redox balancing with electron mediators in a BES. 2-Keto-gluconate, a precursor for industrial anti-oxidant production, was produced at an overall carbon yield of over 90 % based on glucose. Seven different mediator compounds were tested, and only those with redox potential above 0.207 V (vs standard hydrogen electrode) showed interaction with the cells. The productivity increased with the increasing redox potential of the mediator, indicating this was a key factor affecting the anoxic production process. P. putida cells survived under anaerobic conditions, and limited biofilm formation could be observed on the anode’s surface. Analysis of the intracellular pools of ATP, ADP and AMP showed that cells had an increased adenylate energy charge suggesting that cells were able to generate energy using the anode as terminal electron acceptor. The analysis of NAD(H) and NADP(H) showed that in the presence of specific extracellular electron acceptors, the NADP(H) pool was more oxidised, while the NAD(H) pool was unchanged. This implies a growth limitation under anaerobic conditions due to a shortage of NADPH and provides a way to limit biomass formation, while allowing cell maintenance and catalysis at high purity and yield.ConclusionsFor the first time, this study proved the principle that a BES-driven bioconversion of glucose can be achieved for a wild-type obligate aerobe. This non-growth bioconversion was in high yields, high purity and also could deliver the necessary metabolic energy for cell maintenance. By combining this approach with metabolic engineering strategies, this could prove to be a powerful new way to produce bio-chemicals and fuels from renewables in both high yield and high purity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0452-y) contains supplementary material, which is available to authorized users.

Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was used to determine intact phospholipid profiles for five reference pseudomonad strains harboring different (aerobic) toluene catabolic pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. These five strains contained a predominant pool of phosphatidylethanolamines. Other phospholipids identified include phosphatidylglycerol, phosphatidylserine, phosphatidylmethylethanolamine, and phosphatidyldimethylethanolamine. There was a clear separation in phospholipid profiles that allows for the differentiation between the Pseudomonas and Burkholderia genera. Factor analysis of the phospholipid profiles showed that B. cepacia G4, P. putida mt-2, and B. pickettii PKO1 were clearly separated, while P. putida F1 and P. mendocina KR1 were clustered as a group. These results suggest that intact phospholipid profiling could be used to evaluate the relative abundance of specific degraders in bioreactors or in aquifer material. Nevertheless, the usefulness of this technique for taxonomic characterization of such complex samples remains to be demonstrated because of potential confounding effects of overlapping profiles and potential changes in phospholipid composition due to different growth conditions.