Strain PC1 Research Articles

The efficiency of Pseudomonas mendocina in biodegradation of chlorpyrifos insecticides

At present, extensive varieties of pesticides are being used, but the demand for Organophosphorus pesticides is increasing globally to control insects. Chlorpyrifos is a broad-spectrum, highly toxic, and chlorinated organophosphate insecticide that is synthetic and usually an ester or thiol derivative of phosphoric. The mode of action involves inhibiting acetyl-cholinesterase, leading to the accumulation of acetylcholine, causing neurotoxicity. Bacteria capable of degrading the pesticide chlorpyrifos were isolated from soil contaminated with pesticides. This way, three distinct chlorpyrifos degrading strains of p.mendocina were isolated and characterized using morphological and biochemical analysis. Strains exhibited the greatest chlorpyrifos degradation rate, reaching 100%, and were consequently selected for further investigation. Degradation of chlorpyrifos by strains was rapid at 20 and 37C. Bacteria species were able to effectively degrade chlorpyrifos in a sterilized medium using high inoculum levels. The maximum degradation rate of chlorpyrifos was calculated as 100% during 6-12 days. Bacteria such as strain PC1 that use chlorpyrifos as a carbon source could be employed in biodegradation sites contaminated with pesticides. Keywords: Biodegradation; organophosphate; Chlorpyrifos; pseudomonas putida; Hplc.

The efficiency of Pseudomonas mendocina in biodegradation of chlorpyrifos insecticides

At present, wide varieties of pesticides are being used, but the demand for Organophosphorus pesticides is increasing globally to control insects. Chlorpyrifos is a broad-spectrum, highly toxic, and chlorinated organophosphate insecticide that is synthetic in origin and is normally ester or thiol derivatives of phosphoric. The mode of action involves inhibiting acetyl-cholinesterase, leading to the accumulation of acetylcholine, causing neurotoxicity. Bacteria capable of degrading the pesticide chlorpyrifos were isolated from soil contaminated with pesticides. This way, three distinct chlorpyrifos degrading strains of p.mendocina were isolated and characterized using morphological and biochemical analysis. Strains exhibited the greatest chlorpyrifos degradation rate, reaching 100%, and were consequently selected for further investigation. Degradation of chlorpyrifos by strains was rapid at 20 and 37C. Bacteria species were able to effectively degrade chlorpyrifos in the sterilized medium using high inoculum levels. The maximum degradation rate of chlorpyrifos was calculated as 100% during 6-12 days. Bacteria such as strain PC1 that use chlorpyrifos as a carbon source could be employed for the biodegradation of sites contaminated with pesticides Keywords: Biodegradation, organophosphate, chlorpyrifos, Pseudomonas putida, Hplc.

Efficiency of Pseudomonas putida in bioremediation of chlorpyrifos toxicity

At present, extensive varieties of pesticides are being used but the demand for Organophosphorus pesticide is increasing globally to control insects. Chlorpyrifos is a broad-spectrum, highly toxic, and chlorinated organophosphate insecticide that is synthetic in origin and is normally ester or thiol derivatives of phosphoric. The mode of action involves inhibiting acetyl-cholinesterase leading to the accumulation of acetylcholine causing neurotoxicity. Bacteria capable of degrading the pesticide, chlorpyrifos, were isolated from soil contaminatied with pesticiedes. In this way, three distinct chlorpyrifosdegrading strains of Pseudomonas putidia were isolated, and were characterized using morphological and biochemical analysis. Strain PB1 exhibited the greatest chlorpyrifos degradation rate reach to 100% and was consequently selected for further investigation. Degradation of chlorpyrifos by strain PC1 was rapid at 20 and 37C. Strain PB1 was able to effectively degrade chlorpyrifos in sterilized medium using high inoculum levels. The maximum degradation rate of chlorpyrifos was calculated as 0.08µg/ml to 0.002µg/ml during 6-12 days. Bacteria such as strain PB1, that use chlorpyrifos as a carbon source, could be employed for the bioremediation of sites contaminated with pesticides.

Exploring the survival tactics and plant growth promising traits of root-associated bacterial strains under Cd and Pb stress: A modelling based approach

The study represents a microbial method for reducing heavy metal stress in terrestrial environment. Two rhizobacterial strains Pantoea agglomerance (PC1) and Pseudomonas aeruginosa (SA) having the ability to tolerate Cd2+ and Pb2+ ions stress, were employed in this study. The growth promotion and survival tactics of the strains under metal stress were explored through kinetic growth model using logistic equation, Luedeking-Piret model and Box Behnken design. Study also involves the interaction of strains with Zea mays L. under Cd2+ and Pb2+ ions stress. Results revealed that both strains have the potential to tolerate 500 mg L−1 of Cd2+ and Pb2+, ions and maintained the plant growth promoting traits. The Luedeking-Piret model estimated the maximum value of IAA on biomass growth (YP/X) 5.377 μg g−1 and 10.3 μg g−1 under Cd2+ ions, while 7.742 μg g−1 and 18.071 μg g−1 under Pb2+ ions stress for strains SA and PC1, respectively. Further, phosphate solubilization activity was optimized with the help of response surface methodology using Box Behnken Design. The optimum solubilization by strain PC1 and SA was achieved at 100 and 150 mg L−1 of Cd2+, and 150 and 200 mg L−1 of Pb2+ ion concentration at the pH range 6.75 and 7.5 respectively. The interactive study with Zea mays L. showed significant increase in seed germination in the presence of Cd2+ and Pb2+ ions thereby proving them as potent plant growth promoters and metal stress reducing biological agents. Hence, the findings of the study suggest that rhizobacterial strains could be a sustainable tool for restoration of metal contaminated sites.

Astaxanthin overproduction in yeast by strain engineering and new gene target uncovering

BackgroundAstaxanthin is a natural carotenoid pigment with tremendous antioxidant activity and great commercial value. Microbial production of astaxanthin via metabolic engineering has become a promising alternative. Although great efforts have been conducted by tuning the heterologous modules and precursor pools, the astaxanthin yields in these non-carotenogenic microorganisms were still unsatisfactory for commercialization, indicating that in addition to targeted tailoring limited targets guided by rationally metabolic design, combining more globe disturbances in astaxanthin biosynthesis system and uncovering new molecular mechanisms seem to be much more crucial for further development. Since combined metabolic engineering with mutagenesis by screening is a powerful tool to achieve more global variations and even uncover more molecular targets, this study would apply a comprehensive approach integrating heterologous module engineering and mutagenesis by atmospheric and room temperature plasma (ARTP) to promote astaxanthin production in Saccharomyces cerevisiae.ResultsHere, compared to the strain with β-carotene hydroxylase (CrtZ) from Alcaligenes sp. strain PC-1, involving new CrtZ from Agrobacterium aurantiacum enhanced astaxanthin yield to 1.78-fold and increased astaxanthin ratio to 88.7% (from 66.6%). Astaxanthin yield was further increased by 0.83-fold (to 10.1 mg/g DCW) via ARTP mutagenesis, which is the highest reported yield at shake-flask level in yeast so far. Three underlying molecular targets (CSS1, YBR012W-B and DAN4) associated with astaxanthin biosynthesis were first uncovered by comparative genomics analysis. To be noted, individual deletion of CSS1 can recover 75.6% improvement on astaxanthin yield achieved by ARTP mutagenesis, indicating CSS1 was a very promising molecular target for further development. Eventually, 217.9 mg/L astaxanthin (astaxanthin ratio was 89.4% and astaxanthin yield was up to 13.8 mg/g DCW) was obtained in 5-L fermenter without any addition of inducers.ConclusionsThrough integrating rational engineering of pathway modules and random mutagenesis of hosts efficiently, our report stepwise promoted astaxanthin yield to achieve the highest reported one in yeast so far. This work not only breaks the upper ceiling of astaxanthin production in yeast, but also fulfills the underlying molecular targets pools with regard to isoprenoid microbial overproductions.

Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae

The conversion of β-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps of ketolation are catalyzed by β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) respectively. Here, astaxanthin biosynthesis pathway was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high β-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE_Sc118060 with CrtW from Brevundimonas vesicularis DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Sc118060 was replaced from FBA1p to TEF1p, the astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW). In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtW were observed at early exponential phase in medium with 4% (w/v) glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% (w/v) glucose, the transcription level of both crtZ and crtW were actually decreased during the whole time course, consequently leading to no significant improvement on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance desired compounds accumulation by employing the key enzyme(s) in microbes. Open image in new window

Characterization of a strain of Pseudomonas putida isolated from agricultural soil that degrades cadusafos (an organophosphorus pesticide)

Bacteria capable of degrading the pesticide, cadusafos, were isolated from agricultural soil using an enrichment method. In this way, five distinct cadusafos-degrading strains of Pseudomonas putidia were isolated, and were characterized using morphological and biochemical analysis, as well as 16S rRNA sequencing. Strain PC1 exhibited the greatest cadusafos degradation rate and was consequently selected for further investigation. Degradation of cadusafos by strain PC1 was rapid at 20 and 37°C, but was greatly reduced (~1.5-fold) by the presence of carbon sources. Strain PC1 was able to effectively degrade cadusafos in sterilized soil using low inoculum levels. The maximum degradation rate of cadusafos (V ( max )) was calculated as 1.1 mg l(-1 )day(-1), and its saturation constant (K ( s )) was determined as 2.5 mg l(-1). Bacteria such as strain PC1, that use cadusafos as a carbon source, could be employed for the bioremediation of sites contaminated with pesticides.

RAPID ISOLATION, IDENTIFICATION AND PHYLOGENETIC ANALYSIS OF PECTOBACTERIUM CAROTOVORUM ssp.

Thirty-two strains of Pectobacterium carotovorum ssp. (Pcc) were isolated from Chinese cabbage and other vegetables. These strains were subject to morphological observation, biochemical and physiological identification as well as 16S rDNA sequence analysis. All strains except D4, Z1-2, Z2-1, Z2-2 and Z3-2 manifested the same PCR patterns as indicated by amplifying 16S-23S rDNA ITS and ITS-RFLP. The phylogenetic analysis of the 16S rDNA sequences indicated that these five strains that clustered with Pcc strain PC1 and close to P. betavasculorum and P. wasabiae were distinct from other strains in the Pcc groups. However, the whole-cell fatty acid analysis and the biochemical and physiological characteristics suggested that these five strains could still be identified as Pcc. In conclusion, our results suggest that these five strains are probably from a new evolutionary taxonomy of Pectobacterium carotovorum ssp.

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.

Biodegradation efficiency of functionally important populations selected for bioaugmentation in phenol- and oil-polluted area

Denaturing gradient gel electrophoresis of amplified fragments of genes coding for 16S rRNA and for the largest subunit of multicomponent phenol hydroxylase (LmPH) was used to monitor the behaviour and relative abundance of mixed phenol-degrading bacterial populations ( Pseudomonas mendocina PC1, P. fluorescens strains PC18, PC20 and PC24) during degradation of phenolic compounds in phenolic leachate- and oil-amended microcosms. The analysis indicated that specific bacterial populations were selected in each microcosm. The naphthalene-degrading strain PC20 was the dominant degrader in oil-amended microcosms and strain PC1 in phenolic leachate microcosms. Strain PC20 was not detectable after cultivation in phenolic leachate microcosms. Mixed bacterial populations in oil-amended microcosms aggregated and formed clumps, whereas the same bacteria had a planktonic mode of growth in phenolic leachate microcosms. Colony hybridisation data with catabolic gene specific probes indicated that, in leachate microcosms, the relative proportions of bacteria having meta (PC1) and ortho (PC24) pathways for degradation of phenol and p-cresol changed alternately. The shifts in the composition of mixed population indicated that different pathways of metabolism of aromatic compounds dominated and that this process is an optimised response to the contaminants present in microcosms.

Reversible accumulation of p-hydroxybenzoate and catechol determines the sequential decomposition of phenolic compounds in mixed substrate cultivations in pseudomonads

Accumulation of key catabolic intermediates during degradation of phenol, p-cresol and benzoate was studied in two-substrate batch cultivations by the strains Pseudomonas mendocina PC1, Pseudomonas fluorescens PC18 and P. fluorescens PC24. According to sequence analysis of 16S rRNA genes the strains belonged to different monophyletic clusters of Pseudomonas. The catechol 2,3-dioxygenase (C23O) gene, xylE, of strain PC1 and the phenol monooxygenase gene, pheA, of PC24 were localised on the chromosome, while the C23O gene, xylE, of strain PC18 and the p-cresol methylhydroxylase gene, pchF, of strains PC18 and PC24 were on plasmids. It was shown that, if the substrates were degraded from mixtures using either catechol meta, catechol ortho or catechol ortho and protocatechuate ortho pathways, then both substrates were catabolised simultaneously (nondiauxic growth) without the accumulation of intermediates. Exceptionally, degradation of phenol and benzoate via the catechol ortho pathway caused irreversible accumulation of cis,cis-muconate without detectable effect on simultaneous consumption of substrates. When the substrates were degraded from mixtures through meta and ortho catabolic pathways, the sequential consumption of substrates (diauxic growth) was observed due to the reversible accumulation of the catabolic intermediates p-hydroxybenzoate or catechol. Regulation of parallel catabolic pathways by the accumulation of catabolic intermediates depended on the concentration of growth substrates. At low concentrations simultaneous degradation occurred and the antagonistic effect of p-hydroxybenzoate on the degradation of phenol was diminished. In strain PC18 only the accumulation of p-hydroxybenzoate during growth on a phenol–p-cresol mixture seems to be directly metabolically regulated because phenol also induces the catabolic pathway for p-cresol degradation. Partial sequencing of the pchF genes of strains PC18 and PC24 showed considerable differences.

Reversible accumulation of p-hydroxybenzoate and catechol determines the sequential decomposition of phenolic compounds in mixed substrate cultivations in pseudomonads

Accumulation of key catabolic intermediates during degradation of phenol, p-cresol and benzoate was studied in two-substrate batch cultivations by the strains Pseudomonas mendocina PC1, Pseudomonas fluorescens PC18 and P. fluorescens PC24. According to sequence analysis of 16S rRNA genes the strains belonged to different monophyletic clusters of Pseudomonas. The catechol 2,3-dioxygenase (C23O) gene, xylE, of strain PC1 and the phenol monooxygenase gene, pheA, of PC24 were localised on the chromosome, while the C23O gene, xylE, of strain PC18 and the p-cresol methylhydroxylase gene, pchF, of strains PC18 and PC24 were on plasmids. It was shown that, if the substrates were degraded from mixtures using either catechol meta, catechol ortho or catechol ortho and protocatechuate ortho pathways, then both substrates were catabolised simultaneously (nondiauxic growth) without the accumulation of intermediates. Exceptionally, degradation of phenol and benzoate via the catechol ortho pathway caused irreversible accumulation of cis, cis-muconate without detectable effect on simultaneous consumption of substrates. When the substrates were degraded from mixtures through meta and ortho catabolic pathways, the sequential consumption of substrates (diauxic growth) was observed due to the reversible accumulation of the catabolic intermediates p-hydroxybenzoate or catechol. Regulation of parallel catabolic pathways by the accumulation of catabolic intermediates depended on the concentration of growth substrates. At low concentrations simultaneous degradation occurred and the antagonistic effect of p-hydroxybenzoate on the degradation of phenol was diminished. In strain PC18 only the accumulation of p-hydroxybenzoate during growth on a phenol– p-cresol mixture seems to be directly metabolically regulated because phenol also induces the catabolic pathway for p-cresol degradation. Partial sequencing of the pchF genes of strains PC18 and PC24 showed considerable differences.

Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay.

An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained: (a) 3,3'-hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3-hydroxyechinenone to 4-ketozeaxanthin (adonixanthin), 3'-hydroxyechinenone to 4-ketozeaxanthin, as well as echinenone to 4-ketozeaxanthin via 3- and 3'-hydroxyechinenone; (b) 4,4'-Oxygenase (from A. aurantiacum, Alcaligenes sp. strain PC-1 and Haematococcus pluvialis) converted 4-ketozeaxanthin to astaxanthin, 3-hydroxyechinenone to phoenicoxanthin, 3'-hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin. Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono-hydroxylated and ketolated products are enzymically feasible.

In Vitro Characterization of Astaxanthin Biosynthetic Enzymes

Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes. Specific enzyme assays indicated that all of the enzymes are bifunctional, in that the CrtZ enzymes formed zeaxanthin from beta-carotene via beta-cryptoxanthin, as well as astaxanthin from canthaxanthin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from beta-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin. Comparison of maximum catalytic activities as well as selectivity experiments carried out in the presence of both utilizable substrates indicated that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwinia CrtZ possessed a favorability to the formation of zeaxanthin from beta-carotene. The CrtW/BKT enzymes were not so defined in their substrate preference, responding readily to fluctuations in substrate levels. Other properties obtained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of 2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to activity. Based on enzymological data, a predicted pathway for astaxanthin biosynthesis is described, and it is proposed that CrtZ-like enzymes be termed carotenoid 3, (3')-beta-ionone ring hydroxylase and CrtW/BKT carotenoid 4, (4')-beta-ionone ring oxygenase.

Taxonomic Confusion of Pseudomonas cissicola Originated from Mislabeling by International Type Culture Collections

The type strain (strain ICMP 4289) and two reference strains (strains ICMP 4290 and ICMP 4291) of Pseudomonas cissicola (Takimoto 1939) Burkholder 1948 were obviously mislabeled at the Plant Disease Division Culture Collection (presently the International Collection of Microorganisms from Plants) in New Zealand when they were deposited in 1975 or subsequently when they were coded for the catalog of the collection. The strains were also mislabeled at the National Collection of Plant Pathogenic Bacteria in England when strain NCPPB 2982T (T = type strain) was transferred from the Plant Disease Division Culture Collection. Recently, these errors have caused considerable confusion in the taxonomic placement of this bacterium, not only in workers who received cultures from the culture collections but also in workers who are directly responsible for the culture collections. The type strain of the bacterium should be strain PC1 in the culture collection of plant-pathogenic bacteria at the Laboratory of Plant Pathology, Shizuoka University, strain ATCC 33616 in the American Type Culture Collection, or strain CCM 2888 in the Czechoslovak Collection of Microorganisms.

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties

Spectinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc 1-11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1-11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.