Search

Abstract The phenylurea herbicides are persistent in soil and water, necessitating the creation of methods for removing them from the environment. This study aimed to examine the soil microbial diversity, searching for local bacterial isolates able to efficiently degrade the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea (IPU). The best isolates able to effectively degrade IPU were selected, characterized, and identified as Pseudomonas putida and Acinetobacter johnsonii. The catechol 1, 2-dioxygenase enzyme's catA gene was amplified, cloned, and expressed in E. coli M15. The Expressed E. coli showed high degradation efficiency (44.80%) as analyzed by HPLC after 15 days of inoculation in comparison to P. putida (21.60%). The expression of the catA gene in P. putida and expressed E. coli was measured using quantitative polymerase chain reaction (qPCR). The results displayed a significant increase in the mRNA levels of the catA gene by increasing the incubation time with IPU. Hydrophilic interaction chromatography (HILIC) mass spectrometry analysis revealed that three intermediate metabolites, 1-(4-isopropylphenyl)-3-methylurea (MDIPU), 4-Isopropylaniline (4-IA) and 1-(4-isopropylphenyl) urea (DDIPU) were generated by both P. putida and expressed E. coli. In addition, IPU-induced catA activity was detected in both P. putida and expressed E. coli. The supernatant of both P. putida and expressed E. coli had a significant influence on weed growth. The study clearly exhibited that P. putida and expressed E. coli were capable of metabolizing IPU influentially and thus could be utilized for bioremediation and biodegradation technology development.

The phenylurea herbicides are persistent in soil and water, necessitating the creation of methods for removing them from the environment. This study aimed to examine the soil microbial diversity, searching for local bacterial isolates able to efficiently degrade the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea (IPU). The best isolates able to effectively degrade IPU were selected, characterized, and identified as Pseudomonas putida and Acinetobacter johnsonii. The catechol 1, 2-dioxygenase enzyme's catA gene was amplified, cloned, and expressed in E. coli M15. The Expressed E. coli showed high degradation efficiency (44.80%) as analyzed by HPLC after 15 days of inoculation in comparison to P. putida (21.60%). The expression of the catA gene in P. putida and expressed E. coli was measured using quantitative polymerase chain reaction (qPCR). The results displayed a significant increase in the mRNA levels of the catA gene by increasing the incubation time with IPU. Hydrophilic interaction chromatography (HILIC) mass spectrometry analysis revealed that three intermediate metabolites, 1-(4-isopropylphenyl)-3-methylurea (MDIPU), 4-Isopropylaniline (4-IA) and 1-(4-isopropylphenyl) urea (DDIPU) were generated by both P. putida and expressed E. coli. In addition, IPU-induced catA activity was detected in both P. putida and expressed E. coli. The supernatant of both P. putida and expressed E. coli had a significant influence on weed growth. The study clearly exhibited that P. putida and expressed E. coli were capable of metabolizing IPU influentially and thus could be utilized for bioremediation and biodegradation technology development. [ABSTRACT FROM AUTHOR]

Background and Objective: Duplex PCR is a widespread molecular biology technique that has the ability in specific and high sensitivity detection of microorganisms. This study was performed to evaluate the molecular identification of Pseudomonas stutzeri using duplex PCR. Methods: In this descriptive-laboratory study, Pseudomonas stutzeri ATCC 17588 bacteria was purchased from genetic resources center and after culturing the bacteria, DNA was extracted in the exponential growth phase using boiling method. Duplex PCR was carried out for specific identification of the bacteria subsequently. The primers were designed using catA and nirP gene sequences. Sensitivity and specificity of duplex PCR technique were investigated using 5 bacteria. Results: The amplification of two bands of 512 bp and 249 bp for catA and nirP genes were observed, respectively. The specificity was 100% .The sensitivity of 0.048 ng/µL of genomic DNA was determined for catA and nirP genes, respectively. Conclusion: Duplex-PCR molecular method with its sensitivity and proper feature and high potential for identification of Pseudomonas bacteria can be applied as a routine method in well-equipped laboratories by expert technician to identify suspicious cases.

Background and Objective: Duplex PCR is a widespread molecular biology technique that has the ability in specific and high sensitivity detection of microorganisms. This study was performed to evaluate the molecular identification of Pseudomonas stutzeri using duplex PCR. Methods: In this descriptive-laboratory study, Pseudomonas stutzeri ATCC 17588 bacteria was purchased from genetic resources center and after culturing the bacteria, DNA was extracted in the exponential growth phase using boiling method. Duplex PCR was carried out for specific identification of the bacteria subsequently. The primers were designed using catA and nirP gene sequences. Sensitivity and specificity of duplex PCR technique were investigated using 5 bacteria. Results: The amplification of two bands of 512 bp and 249 bp for catA and nirP genes were observed, respectively. The specificity was 100% .The sensitivity of 0.048 ng/μL of genomic DNA was determined for catA and nirP genes, respectively. Conclusion: Duplex-PCR molecular method with its sensitivity and proper feature and high potential for identification of Pseudomonas bacteria can be applied as a routine method in well-equipped laboratories by expert technician to identify suspicious cases. [ABSTRACT FROM AUTHOR]

Fifteen actinomycete strains were evaluated for their potential use in removal of polycyclic aromatic hydrocarbons (PAH). Their capability to degrade of naphthalene, phenanthrene, and pyrene was tested in minimal medium (MM) and MM with glucose as another substrate. Degradation of naphthalene in MM was observed in all isolates at different rates, reaching maximum values near to 76% in some strains of Streptomyces, Rhodococcus sp. 016 and Amycolatopsis tucumanensis DSM 45259. Maximum values of degradation of phenanthrene in MM occurred in cultures of A. tucumanensis DSM 45259 (36.2%) and Streptomyces sp. A12 (20%), while the degradation of pyrene in MM was poor and only significant with Streptomyces sp. A12 (4.3%). Because of the poor performance when growing on phenanthrene and pyrene alone, Rhodococcus sp. 20, Rhodococcus sp. 016, A. tucumanensis DSM 45259, Streptomyces sp. A2, and Streptomyces sp. A12 were challenged to an adaptation schedule of successive cultures on a fresh solid medium supplemented with PAHs, decreasing concentration of glucose in each step. As a result, an enhanced degradation of PAHs by adapted strains was observed in the presence of glucose as co-substrate, without degradation of phenanthrene and pyrene in MM while an increase to up to 50% of degradation was seen with these strains in glucose amended media. An internal fragment of the catA gene, which codes for catechol 1,2-dioxygenase, was amplified from both Rhodococcus strains, showing the potential for degradation of aromatic compounds via salycilate. These results allow us to propose the usefulness of these actinomycete strains for PAH bioremediation in the environment. [ABSTRACT FROM AUTHOR]

We isolated and sequenced a genomic clone ( CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice ( Oryza sativa L.). The 5′-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5′-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects. The 1.9 kb 5′-upstream fragment (−1559 to +342) of the CatA gene was fused with the Escherichia coli β-glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between −1564 to −699. Abscisic acid (ABA) at a final concentration of 10 M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5′-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at −266 to −254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light. The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5α, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM 110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay. [ABSTRACT FROM AUTHOR]