No abstract

Application of microbial metabolic potential (bioremediation) is accepted as an environmentally benign and economical measure for decontamination of polluted environments. Bioremediation methods are generally categorized into ex situ and in situ bioremediation. Although in situ bioremediation methods have been in use for two to three decades, they have not yet yielded the expected results. Their limited success has been attributed to reduced ecological sustainability under environmental conditions. An important determinant of sustainability of in situ bioremediation is pollutant bioavailability. Microbial chemotaxis is postulated to improve pollutant bioavailability significantly; consequently, application of chemotactic microorganisms can considerably enhance the performance of in situ degradation. The environmental fate of degradative microorganisms and the ecological consequence of intervention constitute other important descriptors for the efficiency and sustainability of bioremediation processes. Integrative use of culture-dependent, culture-independent methods (e.g. amplified rDNA restriction analysis, terminal restriction fragment length polymorphism, denaturing/thermal gradient gel electrophoresis, phospholipid fatty acid, etc.), computational and statistical analyses has enabled successful monitoring of the above aspects. The present review provides a detailed insight into some of the key factors that affect the efficiency of in situ bioremediation along with a comprehensive account of the integrative approaches used for assessing the ecological sustainability of processes. The review also discusses the possibility of developing suicidal genetically engineered microorganisms for optimized and controlled in situ bioremediation.

Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pathways in Gram positive and Gram negative bacteria. The former involves 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT), and maleylacetate (MA) as major degradation intermediates, whereas the later proceeds via formation of 1,4-benzoquinone (BQ) and hydroquinone (HQ). In the present study we identified a Gram negative organism viz. Burkholderia sp. strain SJ98 that degrades PNP via 4NC, BT, and MA. A 6.89 Kb genomic DNA fragment of strain SJ98 that encompasses seven putatively identified ORFs (orfA, pnpD, pnpC, orfB, orfC, orfD, and orfE) was cloned. PnpC is benzenetriol dioxygenase belonging to the intradiol dioxygenase superfamily, whereas PnpD is identified as maleylacetate reductase, a member of the Fe-ADH superfamily showing NADH dependent reductase activity. The in vitro activity assays carried out with purified pnpC and pnpD (btd and mar) gene products transformed BT to MA and MA to beta-ketoadipate, respectively. The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies ascertained the involvement of 4-NC, BT, and MA as degradation intermediates of PNP pathway in this strain. This is one of the first conclusive reports for 4-NC and BT mediated degradation of PNP in a Gram negative organism.

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.

BackgroundBurkholderia sp. strain SJ98 is known for its chemotaxis towards nitroaromatic compounds (NACs) that are either utilized as sole sources of carbon and energy or co-metabolized in the presence of alternative carbon sources. Here we test for the chemotaxis of this strain towards six chloro-nitroaromatic compounds (CNACs), namely 2-chloro-4-nitrophenol (2C4NP), 2-chloro-3-nitrophenol (2C3NP), 4-chloro-2-nitrophenol (4C2NP), 2-chloro-4-nitrobenzoate (2C4NB), 4-chloro-2-nitrobenzoate (4C2NB) and 5-chloro-2-nitrobenzoate (5C2NB), and examine its relationship to the degradation of such compounds.ResultsStrain SJ98 could mineralize 2C4NP, 4C2NB and 5C2NB, and co-metabolically transform 2C3NP and 2C4NB in the presence of an alternative carbon source, but was unable to transform 4C2NP under these conditions. Positive chemotaxis was only observed towards the five metabolically transformed CNACs. Moreover, the chemotaxis was induced by growth in the presence of the metabolisable CNAC. It was also competitively inhibited by the presence of nitroaromatic compounds (NACs) that it could metabolise but not by succinate or aspartate.ConclusionsBurkholderia sp. strain SJ98 exhibits metabolic transformation of, and inducible chemotaxis towards CNACs. Its chemotactic responses towards these compounds are related to its previously demonstrated chemotaxis towards NACs that it can metabolise, but it is independently inducible from its chemotaxis towards succinate or aspartate.

Gefitinib, an EGFR tyrosine kinase inhibitor, is used as FDA approved drug in breast cancer and non-small cell lung cancer treatment. However, this drug has certain side effects and complications for which the underlying molecular mechanisms are not well understood. By systems biology based in silico analysis, we identified off-targets of gefitinib that might explain side effects of this drugs. The crystal structure of EGFR-gefitinib complex was used for binding pocket similarity searches on a druggable proteome database (Sc-PDB) by using IsoMIF Finder. The top 128 hits of putative off-targets were validated by reverse docking approach. The results showed that identified off-targets have efficient binding with gefitinib. The identified human specific off-targets were confirmed and further analyzed for their links with biological process and clinical disease pathways using retrospective studies and literature mining, respectively. Noticeably, many of the identified off-targets in this study were reported in previous high-throughput screenings. Interestingly, the present study reveals that gefitinib may have positive effects in reducing brain and bone metastasis, and may be useful in defining novel gefitinib based treatment regime. We propose that a system wide approach could be useful during new drug development and to minimize side effect of the prospective drug.

An efficient 3,4-dichloroaniline (3,4-DCA)-mineralizing bacterium has been isolated from enrichment cultures originating from a soil sample with a history of repeated exposure to diuron, a major metabolite of which is 3,4-DCA. This bacterium, Bacillus megaterium IMT21, also mineralized 2,3-, 2,4-, 2,5-and 3,5-DCA as sole sources of carbon and energy. These five DCA isomers were degraded via two different routes. 2,3-, 2,4-and 2,5-DCA were degraded via previously unknown dichloroaminophenol metabolites, whereas 3,4-and 3,5-DCA were degraded via dichloroacetanilide.

Aerobic microbial degradation of p-nitrophenol (PNP) has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative bacteria, whereas in Gram-positive PNP degraders it proceed via ‘Benzenetriol (BT) pathway’. These pathways are characterized by the ring cleavage of HQ and BT as terminal aromatic intermediates respectively. Earlier reports on PNP degradation have indicated these pathways to be mutually exclusive. We report involvement of both ‘HQ’ and ‘BT’ ring cleavage pathways in PNP degradation by Burkholderia sp. strain SJ98. Genetic characterization of an ~41 Kb DNA fragment harboring PNP degradation gene cluster cloned and sequenced from strain SJ98 showed presence of multiple orfs including pnpC and pnpD which corresponded to previously characterized ‘benzenetriol-dioxygenase (BtD)’ and ‘maleylacetate reductase (MaR)’ respectively. This gene cluster also showed presence of pnpE1 and pnpE2, which shared strong sequence identity to cognate sub-units of ‘hydroquinone dioxygenase’ (HqD). Heterologous expression and biochemical characterization ascertained the identity of PnpE1 and PnpE2. In in vitro assay reconstituted heterotetrameric complex of PnpE1 and PnpE2 catalyzed transformation of hydroquinone (HQ) into corresponding hydroxymuconic semialdehyde (HMS) in a substrate specific manner. Together, these results clearly establish branching of PNP degradation in strain SJ98. We propose that strain SJ98 presents a useful model system for future studies on evolution of microbial degradation of PNP.