No abstract

A bacterial strain Rhodococcus imtechensis RKJ300 (= MTCC 7085(T) = JCM 13270(T)) was isolated from pesticide-contaminated soil of Punjab by the enrichment technique on minimal medium containing 4-nitrophenol. Strain RKJ300 is capable of utilizing 4-nitrophenol, 2-chloro-4-nitrophenol, and 2,4-dinitrophenol as sole sources of carbon and energy. The strain involved both oxidative and reductive catabolic mechanisms for initial transformation of these compounds. In the case of 2-chloro-4-nitrophenol, colorimetric analysis indicated that nitrite release was followed by stoichiometric elimination of chloride ions. Experiments using whole cells and cell-free extracts showed chlorohydroquinone and hydroquinone as the intermediates of 2-chloro-4-nitrophenol degradation. This is the first report of degradation on 2-chloro-4-nitrophenol by a bacterium under aerobic condition to the best of our knowledge. However, pathways for degradation of 4-nitrophenol and 2,4-dinitrophenol were similar to those reported in other strains of Rhodococcus. Laboratory-scale soil microcosm studies demonstrated that the organism was capable of degrading a mixture of nitrophenols simultaneously, indicating its applicability toward in situ bioremediation of contaminated sites. The fate of the augmented strain as monitored by the plate-counting method and hybridization technique was found to be fairly stable throughout the period of microcosm experiments.

Acinetobacter sp. strain YAA is able to use aniline and o-toluidine as the sole carbon and energy source. This strain has several different plasmids and acridine orange curing suggested that aniline utilization in strain YAA was Sall fragment from the insert in E. coli resulted in the accumulation of catechol. Southern hybridization studies indicated that the aniline oxygenase gene (atdA) was present on one of the plasmids, pYA1. These results suggest that in strain YAA aniline is degraded via catechol through a pathway involving meta-cleavage of the benzene-ring by plasmid-encoded genes including atdA.

Background: Biodegradation of polyamides is important from the industrial and environmental point of view. Results: We identified the catalytic residue of nylon hydrolase as Thr-267 and enhanced the protein thermostability by 36°C (T m ϭ 88°C) by introducing mutations at the subunit interfaces of tetramer structure. Conclusion:We revealed the mechanism of nylon-6 hydrolysis. Significance: We established an approach to biodegrade polymeric nylon-6.

Delftia tsuruhatensis AD9 was isolated as an aniline-degrading bacterium from the soil surrounding a textile dyeing plant. The gene cluster involved in aniline degradation was cloned from the total DNA of strain AD9 into Escherichia coli JM109. After shotgun cloning, two recombinant E. coli strains showing aniline oxidation activity or catechol meta-cleavage activity were obtained by simple plate assays. These strains contained 9?3 kb and 15?4 kb DNA fragments, respectively. Sequence analysis of the total 24?7 kb region revealed that this region contains a gene cluster (consisting of at least 17 genes, named tadQTA1A2BRD1C1D2C2EFGIJKL) responsible for the complete metabolism of aniline to TCA-cycle intermediates. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR) were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode meta-cleavage pathway enzymes for catechol degradation. In addition, it was found that the gene cluster is surrounded by two IS1071 sequences, indicating that it has a class I transposon-like structure. PFGE and Southern hybridization analyses confirmed that the tad gene cluster is encoded on the chromosome of strain AD9 in a single copy. These results suggest that, in strain AD9, aniline is degraded via catechol through a meta-cleavage pathway by the chromosome-encoded tad gene cluster. The tad gene cluster showed significant similarity in nucleotide sequence and genetic organization to the plasmid-encoded aniline degradation gene cluster of Pseudomonas putida UCC22.

A carboxylesterase with a β‐lactamase fold from Arthrobacter possesses a low level of hydrolytic activity (0.023 μmol·min−1·mg−1) when acting on a 6‐aminohexanoate linear dimer byproduct of the nylon‐6 industry (Ald). G181D/H266N/D370Y triple mutations in the parental esterase increased the Ald‐hydrolytic activity 160‐fold. Kinetic studies showed that the triple mutant possesses higher affinity for the substrate Ald (Km = 2.0 mm) than the wild‐type Ald hydrolase from Arthrobacter (Km = 21 mm). In addition, the kcat/Km of the mutant (1.58 s−1·mm−1) was superior to that of the wild‐type enzyme (0.43 s−1·mm−1), demonstrating that the mutant efficiently converts the unnatural amide compounds even at low substrate concentrations, and potentially possesses an advantage for biotechnological applications. X‐ray crystallographic analyses of the G181D/H266N/D370Y enzyme and the inactive S112A‐mutant–Ald complex revealed that Ald binding induces rotation of Tyr370/His375, movement of the loop region (N167–V177), and flip‐flop of Tyr170, resulting in the transition from open to closed forms. From the comparison of the three‐dimensional structures of various mutant enzymes and site‐directed mutagenesis at positions 266 and 370, we now conclude that Asn266 makes suitable contacts with Ald and improves the electrostatic environment at the N‐terminal region of Ald cooperatively with Asp181, and that Tyr370 stabilizes Ald binding by hydrogen‐bonding/hydrophobic interactions at the C‐terminal region of Ald.

4-Nitrophenol (4-NP

Burkholderia sp. strain SJ98 (DSM 23195) was previously isolated and characterized for degradation and co-metabolic transformation of a number nitroaromatic compounds. In the present study, we evaluated its metabolic activity on chlorinated nitroaromatic compounds (CNACs). Results obtained during this study revealed that strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon, nitrogen, and energy under aerobic conditions. The cells of strain SJ98 removed 2C4NP from the growth medium with sequential release of nearly stoichiometric amounts of chloride and nitrite in culture supernatant. Under aerobic degradation conditions, 2C4NP was transformed into the first intermediate that was identified as p-nitrophenol by high-performance liquid chromatography, LCMS-TOF, and GC-MS analyses. This transformation clearly establishes that the degradation of 2C4NP by strain SJ98 is initiated by "reductive dehalogenation"; an initiation mechanism that has not been previously reported for microbial degradation of CNAC under aerobic conditions.