MULTIPLE POSTTRANSCRIPTIONAL REGULATORY FEATURES CONTROL EXPRESSION OF ETHANOLAMINE UTILIZATION GENES IN ENTEROCOCCUS FAECALIS

Enterococcus faecalis is a Gram-positive bacterium that lives as a commensal organism in the mammalian gastrointestinal tract, but can behave as an opportunistic pathogen. Our lab discovered that mutation of the eutK gene attenuates virulence of E. faecalis in the C. elegans model host. eutK is part of the ethanolamine metabolic pathway which was previously unknown in E. faecalis. I discovered the presence of two unique posttranscriptional regulatory features that control expression of eut locus genes. The first feature I found is an AdoCBL riboswitch, a cis-acting RNA regulatory element that acts as a positive regulator of gene expression. The second feature I discovered is a unique two-component system, EutVW. The EutV response regulator contains an ANTAR family domain, which binds RNA to trigger transcriptional antitermination. I determined that induction of expression of several genes in the eut locus is dependent on ethanolamine, AdoCBL and the two-component system. AdoCBL and ethanolamine are both required for induction of eut locus gene expression. Additionally, I discovered eutG is regulated by a unique mechanism of antitermination. Both the AdoCBL riboswitch and EutV response regulator control the expression of the downstream gene eutG. EutV potentially acts through a novel antitermination mechanism in which a dimer of EutV binds to a pair of mRNA stem loops forming an antitermination complex. My data show a unique mechanism by which two environmental signals are integrated by two different posttranscriptional regulators to regulate a single locus.

UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^