The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24-48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0.
- MeSH
- bakteriální proteiny metabolismus genetika chemie izolace a purifikace MeSH
- biodegradace * MeSH
- fenol * metabolismus MeSH
- fylogeneze MeSH
- katechol-1,2-dioxygenasa * metabolismus genetika MeSH
- koncentrace vodíkových iontů MeSH
- Pseudomonas putida * enzymologie genetika metabolismus MeSH
- půdní mikrobiologie * MeSH
- RNA ribozomální 16S genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Diethyl phthalate (DEP) is one of the extensively used plasticizers which has been considered a priority hazardous pollutant due to its carcinogenic, endocrine disrupter, and multi-toxic effects on humans. The identification of DEP in different parts of the ecosphere has increased the global community's attention to the elimination of this pollutant in a bio-eco-friendly way. In this research, a novel aerobic bacterial strain nominates as ShA (GenBank accession number: MN298858) capable of consuming DEP as carbon and energy sources, was isolated from the upper phase (0-10 cm) of Anzali international wetland sediments by enrichment culture method. Morphological characteristics and 16S rRNA gene sequence analysis demonstrated that strain ShA belonged to Pseudomonas putida. The substrate utilization test demonstrated that strain ShA was able to grow in mineral salt medium containing dimethyl phthalate (DMP) and phthalic acid (PA) isomers including terephthalic and isophthalic acid. Degradation assay showed strain ShA completely degraded 200 mg/L DEP within 22 h (pH 7.0, 30 °C). Surprisingly, PA as the main intermediate of DEP biodegradation was identified by GC-FID. Moreover, the rapid degradation of 2000 mg/L PA to CO2 and H2O was viewed in 22 h by strain ShA. The possible route of DEP degradation was DEP directly to PA and then PA consumption for growth. This study obtained results that provide a great contribution to applying strain ShA in the biodegradation of low molecular weight of PAEs and PA isomers in natural ecosystems. This is the first report of a P. putida strain able to degrade DEP and PA.
- MeSH
- biodegradace MeSH
- ekosystém MeSH
- látky znečišťující životní prostředí * MeSH
- lidé MeSH
- Pseudomonas putida * genetika MeSH
- RNA ribozomální 16S genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A revised model of the aromatic binding A domain of the σ54 -dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR and DmpR. The resulting frame was instrumental for mapping a number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. Yet, several loop regions of the A domain could provide the flexibility required for widening such a tunnel for passage of aromatic ligands. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Structural and proteomic analyses confirmed the predicted in/out distribution of residues but also supported two additional possible scenarios of interaction of the A domain with aromatic effectors: a dynamic interaction of the fully structured yet flexible protein with the aromatic partner and/or inducer-assisted folding of the A domain.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- modely strukturální MeSH
- plazmidy MeSH
- proteomika MeSH
- Pseudomonas putida * genetika metabolismus MeSH
- regulace genové exprese u bakterií MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as lignocellulosic residues or plastics. However, P. putida on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to address this limitation by adopting a recombinant cellulosome strategy for this host. In this work, we report an essential step in this endeavor-a display of designer enzyme-anchoring protein "scaffoldins", encompassing cohesin binding domains from divergent cellulolytic bacterial species on the P. putida surface. Two P. putida chassis strains, EM42 and EM371, with streamlined genomes and differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were optimally delivered to their surface with one of four tested autotransporter systems (Ag43 from Escherichia coli), and the efficient display was confirmed by extracellular attachment of chimeric β-glucosidase and fluorescent proteins. Our results not only highlight the value of cell surface engineering for presentation of recombinant proteins on the envelope of Gram-negative bacteria but also pave the way toward designer cellulosome strategies tailored for P. putida.
- MeSH
- beta-glukosidasa metabolismus MeSH
- celulosa metabolismus MeSH
- celulozómy metabolismus MeSH
- chromozomální proteiny, nehistonové chemie MeSH
- Escherichia coli metabolismus MeSH
- genom bakteriální * MeSH
- membránové proteiny metabolismus MeSH
- metabolické inženýrství metody MeSH
- proteinové domény MeSH
- proteiny buněčného cyklu chemie MeSH
- proteiny z Escherichia coli metabolismus MeSH
- Pseudomonas putida genetika metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- vnější bakteriální membrána metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
N-butanol, a valued solvent and potential fuel extender, could possibly be produced by fermentation using either native producers, i.e. solventogenic Clostridia, or engineered platform organisms such as Escherichia coli or Pseudomonas species, if the main process obstacle, a low final butanol concentration, could be overcome. A low final concentration of butanol is the result of its high toxicity to production cells. Nevertheless, bacteria have developed several mechanisms to cope with this toxicity and one of them is active butanol efflux. This review presents information about a few well characterized butanol efflux pumps from Gram-negative bacteria (P. putida and E. coli) and summarizes knowledge about putative butanol efflux systems in Gram-positive bacteria.
- MeSH
- bakteriální proteiny MeSH
- biologický transport MeSH
- Escherichia coli * MeSH
- membránové transportní proteiny MeSH
- metabolické inženýrství MeSH
- mikrobiální viabilita MeSH
- n-butanol * analýza metabolismus toxicita MeSH
- proteiny z Escherichia coli MeSH
- Pseudomonas putida * MeSH
- rozpouštědla MeSH
- transportní proteiny MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
In this study, we show the repetitive detection of toluene on a tapered optical fiber element (OFE) with an attached layer of Pseudomonas putida TVA8 bioluminescent bioreporters. The bioluminescent cell layer was attached on polished quartz modified with (3-aminopropyl)triethoxysilane (APTES). The repeatability of the preparation of the optical probe and its use was demonstrated with five differently shaped OFEs. The intensity of measured bioluminescence was minimally influenced by the OFE shape, possessing transmittances between 1.41% and 5.00%. OFE probes layered with P. putida TVA8 were used to monitor liquid toluene over a two-week period. It was demonstrated that OFE probes layered with positively induced P. putida TVA8 bioreporters were reliable detectors of toluene. A toluene concentration of 26.5 mg/L was detected after <30 min after immersion of the probe in the toluene solution. Additional experiments also immobilized constitutively bioluminescent cells of E. coli 652T7, on OFEs with polyethyleneimine (PEI). These OFEs were repetitively induced with Lauria-Bertani (LB) nutrient medium. Bioluminescence appeared 15 minutes after immersion of the OFE in LB. A change in pH from 7 to 6 resulted in a decrease in bioluminescence that was not restored following additional nutrient inductions at pH 7. The E. coli 652T7 OFE probe was therefore sensitive to negative influences but could not be repetitively used.
Co-production of two or more desirable compounds from low-cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co-production strategy are still scarce. In this study, the ability of genome-edited strain Pseudomonas putida EM42 to simultaneously valorize d-xylose and d-cellobiose - two important lignocellulosic carbohydrates - by converting them into the platform chemical d-xylonate and medium-chain-length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with P. putida resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with a high yield. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic β-glucosidase BglC from Thermobifida fusca on d-cellobiose. This disaccharide turned out to be a better co-substrate for xylose-to-xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in P. putida. Co-production of extracellular xylose-born xylonate and intracellular cellobiose-born medium-chain-length polyhydroxyalkanoates was established in proof-of-concept experiments with P. putida grown on the disaccharide. This study highlights the potential of P. putida EM42 as a microbial platform for the production of xylonate, identifies cellobiose as a new substrate for mcl-PHA production, and proposes a fresh strategy for the simultaneous valorization of xylose and cellobiose.
1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.
- MeSH
- biodegradace MeSH
- biotransformace MeSH
- exprese genu MeSH
- hydrolasy genetika metabolismus MeSH
- látky znečišťující životní prostředí metabolismus MeSH
- metabolické inženýrství * MeSH
- metabolické sítě a dráhy genetika MeSH
- plazmidy MeSH
- propan analogy a deriváty metabolismus MeSH
- Pseudomonas putida genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- selekce (genetika) MeSH
- transpozibilní elementy DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT) activity. The assay is based on the transamination of L-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining in polyacrylamide gels with the Salkowski reagent is also presented.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- enzymatické testy metody MeSH
- indikátory a reagencie MeSH
- kinetika MeSH
- kolorimetrie metody MeSH
- Pseudomonas putida chemie enzymologie MeSH
- transaminasy chemie metabolismus MeSH
- tryptofan metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Ecotoxicity and genotoxicity of widely used veterinary antimicrobials oxytetracycline and flumequine was studied with six model organisms (Vibrio fischeri, Pseudomonas putida, Pseudokirchneriella subcapitata, Lemna minor, Daphnia magna, Escherichia coli). Overall median effective concentration (EC50) values ranged from 0.22 mg/L to 86 mg/L. Pseudomonas putida was the most sensitive organism (EC50 values for 16-h growth inhibition were 0.22 and 0.82 mg/L for oxytetracycline and flumequine, respectively), followed by duckweed Lemna minor (7-d growth inhibition, EC50 2.1 and 3.0 mg/L) and green alga Pseudokirchneriella subcapitata (4-d growth inhibition, EC50 3.1 and 2.6 mg/L). The least sensitive organism was Daphnia magna (48-h immobilization, lowest-observed-effect concentration [LOEC] of oxytetracycline of 400 mg/L). Oxytetracycline showed limited genotoxicity (SOS-chromotest with Escherichia coli, minimal genotoxic concentration of 500 mg/L), and flumequine was genotoxic at 0.25 mg/L. Based on the reported measured concentrations (MECs) and predicted no-effect concentrations (PNECs), oxytetracycline may be concluded to be of ecotoxicological concern (calculated risk quotient = 8), whereas flumequine seems to represent lower risk.
- MeSH
- Aliivibrio fischeri účinky léků MeSH
- antiinfekční látky toxicita MeSH
- chemické látky znečišťující vodu toxicita MeSH
- Chlorophyta MeSH
- Daphnia účinky léků MeSH
- Escherichia coli účinky léků MeSH
- fluorochinolony toxicita MeSH
- mutageny toxicita MeSH
- oxytetracyklin toxicita MeSH
- Pseudomonas putida účinky léků MeSH
- vodní hospodářství MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
