Strain ST-14, characterized as a member of the genus Cupriavidus, was capable of utilizing 2- and 4-nitrobenzoates individually as sole sources of carbon and energy. Biochemical studies revealed the assimilation of 2- and 4-nitrobenzoates via 3-hydroxyanthranilate and protocatechuate, respectively. Screening of a genomic fosmid library of strain ST-14 constructed in Escherichia coli identified two gene clusters, onb and pob-pca, to be responsible for the complete degradation of 2-nitrobenzoate and protocatechuate, respectively. Additionally, a gene segment (pnb) harboring the genes for the conversion of 4-nitrobenzoate to protocatechuate was unveiled by transposome mutagenesis. Reverse transcription-PCR analysis showed the polycistronic nature of the gene clusters, and their importance in the degradation of 2- and 4-nitrobenzoates was ascertained by gene knockout analysis. Cloning and expression of the relevant pathway genes revealed the transformation of 2-nitrobenzoate to 3-hydroxyanthranilate and of 4-nitrobenzoate to protocatechuate. Finally, incorporation of functional 3-nitrobenzoate dioxygenase into strain ST-14 allowed the recombinant strain to utilize 3-nitrobenzoate via the existing protocatechuate metabolic pathway, thereby allowing the degradation of all three isomers of mononitrobenzoate by a single bacterial strain.Mononitrobenzoates are toxic chemicals largely used for the production of various value-added products and enter the ecosystem through industrial wastes. Bacteria capable of degrading mononitrobenzoates are relatively limited. Unlike other contaminants, these man-made chemicals have entered the environment since the last century, and it is believed that bacteria in nature evolved not quite efficiently to assimilate these compounds; as a consequence, to date, there are only a few reports on the bacterial degradation of one or more isomers of mononitrobenzoate. In the present study, fortunately, we have been able to isolate a Cupriavidus sp. strain capable of assimilating both 2- and 4-nitrobenzoates as the sole carbon source. Results of the biochemical and molecular characterization of catabolic genes responsible for the degradation of mononitrobenzoates led us to manipulate a single enzymatic step, allowing the recombinant host organism to expand its catabolic potential to assimilate 3-nitrobenzoate.
Based on the sole information of structural genes of the 2-nitrobenzoate (2NBA) utilizing catabolic gene cluster (onbX1X2FCAR1EHJIGDBX3), 2NBA-sensing bioreporters were constructed by incorporating egfp into the onb gene cluster of Cupriavidus sp. strain ST-14. Incorporation of reporter gene in proximal to the hypothesized promoter region in conjunction with the disruption of the gene encoding inducer-metabolizing enzyme was turned out to be advantageous in reporter gene expression at low inducer concentration. The bioreporter strain was capable of expressing EGFP from the very 1st hour of induction and could detect 2NBA at (sub) nanomolar level exhibiting a strict specificity toward 2NBA, displaying no response to EGFP expression from its meta- and para-isomers as well as from a number of structurally related compounds. The present study is a successful demonstration of the development of a 2NBA-sensing bioreporter with respect to ease of construction, inducer specificity, and sensitivity, without prior knowledge of the associated inducer-responsive promoter-regulator elements. The present approach can be used as a model for the development of bioreporters for other environmental pollutants.
Stabilization of PTEN by targeting CHIP can be a novel therapeutic approach in cancer regulation.The tumor suppressor, PTEN is key to the regulation ofdiverse cellular processes, making it a prime candidate to betightlyregulated.ThePTENleveliscontrolledinamajorwaybyE3 ligase-mediated degradation through the Ubiquitin-Protea-some System (UPS). Nedd 4-1, XIAP, and WWP2 have beenshown to maintain PTEN turnover. Here, we report that CHIP,the chaperone-associated E3 ligase, induces ubiquitination andregulates the proteasomal turnover of PTEN. It was apparentfrom our findings that PTEN transiently associates with themolecularchaperonesandtherebygetsdivertedtothedegrada-tion pathway through its interaction with CHIP. The TPRdomain of CHIP and parts of the N-terminal domain of PTENarerequiredfortheirinteraction.OverexpressionofCHIPleadsto elevated ubiquitination and a shortened half-life of endoge-nousPTEN.Ontheotherhand,depletionofendogenousCHIPstabilizes PTEN. CHIP is also shown to regulate PTEN-depen-dent transcription presumably through its down-regulation.PTEN shared an inverse correlation with CHIP in human pros-tatecancerpatientsamples,therebytriggeringtheprospectsofamore complex mode of PTEN regulation in cancer.
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