Characterization of TsaR, an Oxygen-Sensitive LysR-Type Regulator for the Degradation of p-Toluenesulfonate in Comamonas testosteroni T-2

TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaR^His. TsaR^His was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used. Gel filtration showed TsaR^His to be a monomer in solution. Overexpressed TsaR^His bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD. The dissociation constant (K_D) for the whole promoter region was about 0.9 μM, and the interaction was a function of the concentration of the ligand TSA. A regulatory model for this LysR-type regulator is proposed on the basis of these data.     

An additional regulator,TsaQ, is involved with TsaR in regulation of transport during the degradation of<Emphasis Type="Italic"> p</Emphasis>-toluenesulfonate in<Emphasis Type="Italic"> Comamonas testosteroni</Emphasis> T-2

The degradation of p-toluenesulfonate (TSA) by Comamonas testosteroni T-2 is initiated by a transport system (TsaST) and enzymes (TsaMBCD) encoded on the tsa transposon, Tntsa, on the TSA plasmid (pTSA). Tntsa comprises an insert of 15 kb between two IS1071 elements. The left-hand 6 kb and the right-hand 6 kb are nearly mirror images. The regulator of the tsaMBCD₁ genes (right-hand side) is the centrally located LysR-type TsaR, which is encoded upstream of tsaMBCD₁ on the reverse strand. The other centrally located genes are tsaS and tsaT, encoded downstream of tsaR and on the same strand as both tsaR and tsaMBCD₂. The latter four genes are not expressed. Downstream of tsaD₁ (tsaD₂) is tsaQ₁ (tsaQ₂) and another open reading frame of unknown function. The tsaQ genes have identical sequences. Sequence analysis indicated that TsaQ could be an IclR-type regulator, whose expression during degradation of TSA was proven by data from RT-PCR. Both copies of tsaQ could be knocked-out by homologous recombination. Double mutants failed to grow with TSA but grew with p-toluenecarboxylate (TCA), which is also degraded via TsaMBCD. This showed TsaQ to be essential for the degradation of TSA but not TCA. We attributed this to regulation of the transport of TSA, especially to regulation of the expression of tsaT, which was expressed solely during growth with TSA. Seven independently isolated bacteria containing the tsa operon were available. Those six which contained tsaT on Tntsa also contained tsaQ. The promoter region of tsaT was found to be a target of the regulator TsaR. Band-shift data indicate that TsaR is required for the expression of tsaT, which suggests that tsaR and tsaQ_1,2, together with tsaMBCD₁, belong to a common regulatory unit.     

Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.     

Perinuclear positioning of the inactive human cystic fibrosis gene depends on CTCF, A-type lamins and an active histone deacetylase

The nuclear positioning of mammalian genes often correlates with their functional state. For instance, the human cystic fibrosis transmembrane conductance regulator (CFTR) gene associates with the nuclear periphery in its inactive state, but occupies interior positions when active. It is not understood how nuclear gene positioning is determined. Here, we investigated trichostatin A (TSA)-induced repositioning of CFTR in order to address molecular mechanisms controlling gene positioning. Treatment with the histone deacetylase (HDAC) inhibitor TSA induced increased histone acetylation and CFTR repositioning towards the interior within 20 min. When CFTR localized in the nuclear interior (either after TSA treatment or when the gene was active) consistent histone H3 hyperacetylation was observed at a CTCF site close to the CFTR promoter. Knockdown experiments revealed that CTCF was essential for perinuclear CFTR positioning and both, CTCF knockdown as well as TSA treatment had similar and CFTR-specific effects on radial positioning. Furthermore, knockdown experiments revealed that also A-type lamins were required for the perinuclear positioning of CFTR. Together, the results showed that CTCF, A-type lamins and an active HDAC were essential for perinuclear positioning of CFTR and these components acted on a CTCF site adjacent to the CFTR promoter. The results are consistent with the idea that CTCF bound close to the CFTR promoter, A-type lamins and an active HDAC form a complex at the nuclear periphery, which becomes disrupted upon inhibition of the HDAC, leading to the observed release of CFTR.     

Novel Biochemical Pathways for 5-Fluorouracil in Managing Experimental Hepatocellular Carcinoma in Rats

Five fluorouracil (5-FU) is extensively used in the treatment of hepatocellular carcinoma (HCC). It is well documented that 5-FU and its metabolites inhibit DNA synthesis through inhibition of thymidylate synthetase. Little is known about additional pathways for 5-FU in managing HCC. The present experiment was mainly designed to study possible biochemical pathways that can be added to 5-FU’s mechanisms of action. Four groups of rats constituted a control group (given saline only), a trichloroacetic acid group (TCA, 0.5 g/kg/day for 5 days, orally), a 5-FU-positive group (75 mg/kg body weight, intraperitoneally, once weekly for 3 weeks) and a TCA-treated with 5-FU group (24 h from last TCA dose). We executed both biochemical—serum alpha-fetoprotein (AFP), liver tissue contents of total glycosaminoglycan (TGAGs), collagen (represented as hydroxyproline), total sialic acid (TSA), free glucosamine (FGA) and proteolytic enzyme activity (as pepsin and free cathepsin-D—and histological examinations of the liver tissue. The results revealed histological changes such as central vein congestion and irregularly shaped, substantially enlarged, vesiculated and binucleated hepatocytes. The nuclei were mostly polymorphic and hyperchromatic, and several vacuolation was noticed in the cytoplasm encircling the nucleus with masses of acidophilic material. 5-FU greatly corrected these changes, except that some necrotic and cytotoxic effects of 5-FU were still shown. AFP was significantly elevated in TCA-intoxicated, but reversed in 5-FU-treated, groups. Increased proteolytic activity by TCA was reversed by 5-FU, which also restored TGAG contents to normal; but both TCA and 5-FU depleted collagen content. TCA significantly elevated FGA but depressed TSA; this action was reversed by 5-FU treatment. In conclusion, it is possible that proteolytic activity, expressed as upregulated pepsin and free cathepsin-D activities, is increased in HCC. This is accompanied by extracellular matrix macromolecular disturbance, manifested as decreased TGAGs, collagen and TSA, with marked increase in FGA liver tissue content. The elevated FGA with depressed TSA content of liver tissue may be attributed to a cancer-hampered N-acetylation of FGA into SA. 5-FU administration markedly depressed hepatic tissue proteolysis, possibly reactivated N-acetylation of FGA into SA and elevated TGAGs without stopping tissue fibrosis as collagen was not affected. This study explores additional pathways for the mechanism of action of 5-FU, through conservation of extracellular matrix composition in situ, inhibiting invasion and metastasis in addition to its DNA-disturbing pathway.     

Cell cycle specific changes in the human cyclin B1 gene regulatory region as revealed by response to trichostatin A

The human cyclin Bl gene is cell cycle regulated with maximal activity during G(2)/M. We examined the role of histone deacetylation in cyclin Bl regulation using the histone deacetylase inhibitor trichostatin A (TSA). TSA treatment (100 ng/ml) of NIH3T3 cells containing the luciferase reporter construct pCycB(-287)-LUC caused an increase in promoter activity in G(0) and G(1) but no significant change in G(2). Removal of upstream sequences including an E-box and Sp1 site eliminated the TSA induced increase in G(0) and G(1), and caused a decrease in promoter activity during S and G(2). Promoter activity increased only 2-fold following TSA treatment of G(0) cells containing the construct pCycB(MUT-E-Box)-LUC with an E-box mutation, and a decrease in activity was detected during G(2). We conclude that histone deacetylation contributes to the repression of cyclin B1 expression in G(0) and G(1), and that this mechanism requires, in part, the E-box. TSA reduction of cyclin B1 promoter activity in G(2), however, involves sequences within the first 119 bp. A working model for cyclin B1 regulation is provided.