Bheong-Uk Lee

A role in the regulation of transcription by light for RCO-1 and RCM-1, the Neurospora homologs of the yeast Tup1-Ssn6 repressor.

The activation of gene transcription by light is transient since light-dependent mRNA accumulation ceases after long exposures to light. This phenomenon, photoadaptation, has been observed in plants and fungi, and allows the perception of changes in light intensities. In the fungus Neurosporacrassa photoadaptation involves the transient binding of the photoresponsive White Collar Complex (WCC) to the promoters of light-regulated genes. We show that RCO-1 and RCM-1, the Neurospora homologs of the components of the yeast Tup1-Ssn6 repressor complex, participate in photoadaptation. Mutation in either rco-1 or rcm-1 result in high and sustained accumulation of mRNAs for con-10 and other light-regulated genes after long exposures to light. The mutation of rco-1 increased the sensitivity to light for con-10 activation and delayed synthesis and/or degradation of con-10 and con-6 mRNAs without altering the amount or the light-dependent phosphorylation of the photoreceptor WC-1. RCO-1 and RCM-1 are located in the Neurospora nuclei were they regulate gene transcription. We show that RCO-1 and RCM-1 participate in the light-transduction pathway of Neurospora and has a role in photoadaptation by repressing gene transcription after long exposures to light.

Comparative analysis of 2,4,6-trinitrotoluene (TNT)-induced cellular responses and proteomes in Pseudomonas sp. HK-6 in two types of media

TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.

Purification and characterization of the NAD(P)H-nitroreductase for the catabolism of 2,4,6-trinitrotoluene (TNT) inPseudomonas sp. HK-6

The NAD(P)H-nitroreductase of thePseudomonas sp. HK-6 which is capable of catabolizing 2,4,6-trinitrotoluene (TNT), was purified and biochemically characterized. The specific activity of the purified TNT nitroreductase was approximately 1.47 units/mg, and was concentrated to 10.1-fold compared to the crude extract. The optimal temperature and pH of the highest nitroreductase activity was 30°C and 7.5, respectively. The substrate specificity test revealed that the nitroreductase exhibited the highest enzyme activity for the TNT substrate of the nitroaromatic compounds tested in this study. Moreover, the molecular weight of the TNT nitroreductase was approximately 27 kDa on the SDS-PAGE. The N-terminal amino acid sequence of the purified protein was 5′-MDTVSLAKRRYTTKAYDASR, which is identical topnrB ofPseudomonas putida JLR11, and is capable of TNT reduction. The molecular analysis of the approximately 650-bp PCR product, orginating from the HK-6, revealed that the oxygen-insensitive NAD(P)H-nitroreductase gene, which transforms TNT in strain HK-6 with five unique amino acid sequences and diverges from the nitroreductases identified so far inPseudomonas, Burkholderia, andRalstonia, is frequently found amidst the powerful degraders of aromatic compounds.

Comparative analysis of explosive RDX-induced proteomes in the Pseudomonas sp. HK-6 wild-type strain and its rpoH mutant strain

Pseudomonas sp. HK-6 can utilize the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as the sole nitrogen source under aerobic conditions. It is known that HK-6 is capable of completely degrading 50 μM RDX within 50 days, while the rpoH mutant degrades less than 10% of that amount in the same period of time. The proteomes of the HK-6 and the rpoH mutant strains grown under RDX stress conditions were compared using 2-dimensional electrophoresis (2-DE). A total of 14 upregulated and down-regulated unambiguous protein spots were analyzed using MALDI-TOF MS. Several down-regulated proteins connected with energy metabolism, including NirB, RimO, and NahH, and a transport and binding protein (AapJ) were less expressed in the rpoH genetic background than in the wild-type, and certain proteins connected with the cell envelope, including OprQ and Alg8, were more highly expressed in the rpoH mutant than in the wild-type. It was shown that certain proteins such as GroEL were not expressed in rpoH cells. These results provide insight into survival and the role of the rpoH gene for RDX degradation under RDX stress conditions. In addition to the proteome analysis, the 16S rRNA of HK-6 was cloned and sequenced to draw a phylogenetic tree for precise species identification. The 16S rRNA sequence of HK-6 is closely related to that of Pseudomonas putida.