Browsing by Author "Dang, Phuong Nga"

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Determining the functions of transcriptional regulatory genes of the npd gene cluster encoding 2,4,6-trinitrophenol degradation in Rhodococcus opacus HL PM-1
(2004) Dang, Phuong Nga; Knackmuss, Hans-Joachim (Prof. Dr.)
Rhodococcus opacus HL PM-1 utilises 2,4,6-trinitrophenol (TNP) and 2,4-dinitrophenol (DNP) as a sole carbon, nitrogen and energy source. The TNP metabolic enzymes are encoded by the npd gene cluster. The initial attack on TNP is two hydrogenation reactions, which are catalysed by hydride transferase I (encoded by npdC) and hydride transferase II (encoded by npdI) and the NADPH-dependent F420 reductase (encoded by npdG). In this work, three open reading frames, orfA1, orfA2 and npdR were of interest. Database searches with the deduced amino acid sequences from npdR, orfA1 and orfA2 suggested that npdR may encode for a transcriptional regulator of the IclR family, and orfA1 and orfA2 may encode for two components of a signal transduction protein. The npdR gene was expressed in E. coli, the protein was purified and shown to bind to intergenic regions between orfA1 and orfB, and between npdH and npdI. DNP, TNP, 2-chloro-4,6-dinitrophenol, and 2-methyl-4,6-dinitrophenol induced the expression of npdI and npdC by reducing the binding affinity of NpdR to the DNA-binding regions. Both intergenic regions were cloned upstream of a reporter gene (xylE), causing the expression of xylE. Hence, both intergenic regions contain promoters. Two direct repeats were identified immediately downstream of the two promoter regions. Gel retardation assays with one of the direct repeats between orfA1 and orfB demonstrated that it was the binding site for NpdR. The sequences of the two direct repeats were 82 % identical, suggesting that the second direct repeat between npdH and npdI may be a second binding site for NpdR. This coincided with gel retardation assays, which showed that NpdR might bind to two sites in each intergenic region. A deletion mutant for npdR in R. opacus HL PM-1 was constructed, in which an internal part of npdR was deleted. The expression of npdC and npdI were induced by DNP in the wild-type strain, but were constitutive in the mutant. Hence, NpdR is a repressor involved in TNP degradation. In vivo deletion mutants for orfA1 or orfA2 were also constructed, in which the entire orfA1 or a part of orfA2 was deleted. If orfA1 and orfA2 encode for transcriptional regulators involved in TNP degradation, an effect on expression of the npd genes or on the growth with TNP would be expected in the mutants. However, there was no change in the expression of npdC or npdI in both mutant strains. Resting cells of the deletion mutants for orfA1 or orfA2 with TNP as a substrate did not show any difference in TNP conversion compared to the wild-type cells. Further growth rate of the mutants on TNP as nitrogen source was also similar to that of the wild-type strain. In order to eliminate the possibility of a second copy of orfA1 or orfA2 with full activity in strain HL PM-1, the location of npd genes was determined. npdC, npdI, npdR, orfA1 and orfA2 were localised on the chromosomal DNA in strain HL PM-1. Hybridisation analysis of total DNA from strain HL PM-1 with npdR, orfA1 or orfA2 probes revealed that these genes are present as a single copy in the strain. Hence, orfA1 and orfA2 seem not to be involved in TNP degradation. To conclude, it was shown that NpdR is a repressor of TNP degradation, that two promoter regions are present in the npd gene cluster, and that orfA1 and orfA2 are not involved in metabolism of TNP as nitrogen source.