Biodegradation of isoproturon by Escherichia coli expressing a Pseudomonas putida catechol 1,2-dioxygenase gene.

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.

Draft genome of Raoultella planticola, a high lead resistance bacterium from industrial wastewater.

Isolation of heavy metals-resistant bacteria from their original habitat is a crucial step in bioremediation. Six lead (Pb) resistant bacterial strains were isolated and identified utilizing 16S rRNA to be Enterobacter ludwigii FACU 4, Shigella flexneri FACU, Microbacterium paraoxydans FACU, Klebsiella pneumoniae subsp. pneumonia FACU, Raoultella planticola FACU 3 and Staphylococcus xylosus FACU. It was determined that all these strains had their Minimum inhibitory concentration (MIC) to be 2500 ppm except R. planticola FACU 3 has a higher maximum tolerance concentration (MTC) up to 2700 ppm. We evaluated the survival of all six strains on lead stress, the efficiency of biosorption and lead uptake. It was found that R. planticola FACU 3 is the highest MTC and S. xylosus FACU was the lowest MTC in this evaluation. Therefore, transmission electron microscopy (TEM) confirmed the difference between the morphological responses of these two strains to lead stress. These findings led to explore more about the genome of R. planticola FACU 3 using illumine Miseq technology. Draft genome sequence analysis revealed the genome size of 5,648,460 bp and G + C content 55.8% and identified 5526 CDS, 75 tRNA and 4 rRNA. Sequencing technology facilitated the identification of about 47 genes related to resistance to many heavy metals including lead, arsenic, zinc, mercury, nickel, silver and chromium of R. planticola FACU 3 strain. Moreover, genome sequencing identified plant growth-promoting genes (PGPGs) including indole acetic acid (IAA) production, phosphate solubilization, phenazine production, trehalose metabolism and 4-hydroxybenzoate production genes and a lot of antibiotic-resistant genes.

Draft genome analysis for Enterobacter kobei, a promising lead bioremediation bacterium.

Lead pollution of the environment poses a major global threat to the ecosystem. Bacterial bioremediation offers a promising alternative to traditional methods for removing these pollutants, that are often hindered by various limitations. Our research focused on isolating lead-resistant bacteria from industrial wastewater generated by heavily lead-containing industries. Eight lead-resistant strains were successfully isolated, and subsequently identified through molecular analysis. Among these, Enterobacter kobei FACU6 emerged as a particularly promising candidate, demonstrating an efficient lead removal rate of 83.4% and a remarkable lead absorption capacity of 571.9 mg/g dry weight. Furthermore, E. kobei FACU6 displayed a remarkable a maximum tolerance concentration (MTC) for lead reaching 3,000 mg/L. To further investigate the morphological changes in E. kobei FACU6 in response to lead exposure, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed. These analyses revealed significant lead adsorption and intracellular accumulation in treated bacteria in contrast to the control bacterium. Whole-genome sequencing was performed to gain deeper insights into E. kobei's lead resistance mechanisms. Structural annotation revealed a genome size of 4,856,454 bp, with a G + C content of 55.06%. The genome encodes 4,655 coding sequences (CDS), 75 tRNA genes, and 4 rRNA genes. Notably, genes associated with heavy metal resistance and their corresponding regulatory elements were identified within the genome. Furthermore, the expression levels of four specific heavy metal resistance genes were evaluated. Our findings revealed a statistically significant upregulation in gene expression under specific environmental conditions, including pH 7, temperature of 30°C, and high concentrations of heavy metals. The outstanding potential of E. kobei FACU6 as a source of diverse genes related to heavy metal resistance and plant growth promotion makes it a valuable candidate for developing safe and effective strategies for heavy metal disposal.

Identification and characterization of novel bacterial polyaromatic hydrocarbon-degrading enzymes as potential tools for cleaning up hydrocarbon pollutants from different environmental sources.

Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant hazardous environmental contaminants. Various strategies, including chemical and physical like oxidation, fixation, leaching, and electrokinetic or biological-based techniques are used for remediation of polluted sites. Bioremediation of PAHs, via PAH-degrading endophytic and rhizospheric microbes, represent a time-/cost-effective way for ecorestoration. Four bacterial strains were isolated from contaminated soil on MSM supplemented with anthracene, alpha-naphthalene or catechol as sole carbon sources. These isolates were identified with 16S rRNA as Bacillus anthracis, B. cereus, B. mojavensis and B. subtilis. The degradation efficiency on the selected aromatic compounds was tested by HPLC analysis. B. subtilis showed the highest degradation efficiency of anthracene (99%) after five days of incubation. B. subtilis showed the highest catechol 1, 2 dioxygenase activity in MSM supplemented with anthracene. The enzyme was purified by gel filtration chromatography and characterized (70 kD, Km 2.7 µg and Vmax 178U/mg protein). The catechol 1,2 dioxygenase gene from the identified four bacterial strains were isolated and submitted to GenBank (accession numbers MG255165-MG255168). The gene expression level of catechol 1,2 dioxygenase was upregulated 23.2-fold during the 72 h of incubation period. Furthermore, B. subtilis is a promising strain to be used in bioremediation of aromatic compounds-contaminated environments.