Biomedical subjects
C Diorio
Publications and source records attributed to C Diorio.
An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria.
Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional gene fusion library, we have identified an Escherichia coli operon whose expression is induced by cellular exposure to sodium arsenite at concentrations as low as 5 micrograms/liter. This chromosomal operon was cloned, sequenced, and found to consist of three cistrons which we named arsR, arsB, and arsC because of their strong homology to plasmid-borne ars operons. Mutants in the chromosomal ars operon were found to be approximately 10- to 100-fold more sensitive to sodium arsenate and arsenite exposure than wild-type E. coli, while wild-type E. coli that contained the operon cloned on a ColE1-based plasmid was found to be at least 2- to 10-fold more resistant to sodium arsenate and arsenite. Moreover, Southern blotting and high-stringency hybridization of this operon with chromosomal DNAs from a number of bacterial species showed homologous sequences among members of the family Enterobacteriaceae, and hybridization was detectable even in Pseudomonas aeruginosa. These results suggest that the chromosomal ars operon may be the evolutionary precursor of the plasmid-borne operon, as a multicopy plasmid location would allow the operon to be amplified and its products to confer increased resistance to this toxic metalloid.
Metallothionein-II and ferritin H mRNA levels are increased in arsenite-exposed HeLa cells.
Arsenite is extremely toxic and, though non-mutagenic, is a carcinogen. To determine the effects of arsenite on changes in cell physiology, we searched for genes in HeLa cells whose mRNAs are more abundant after cellular exposure to arsenite. A cDNA subtraction was performed between cDNA synthesized from HeLa cells grown in the absence and presence of 5 microM sodium arsenite. Isolation and sequencing of three clones that showed a higher hybridization signal to RNA from arsenite-exposed cells, versus unexposed cells, revealed that two of the cDNAs coded for human ferritin H chain and the other coded for metallothionein-II. These results suggest the possibility that arsenite exposure may lead to increased levels of oxygen radicals, which augmented metallothionein and ferritin can act to detoxify.
Transcription of the Escherichia coli fliC gene is regulated by metal ions.
luxAB gene fusions in the Escherichia coli genome were used to screen for clones displaying transcriptional changes in the presence of aluminum. One clone was found that contained a luciferase gene fusion in which transcription was increased in the presence of aluminum and which was subsequently shown to be induced by copper, iron, and nickel. Cloning of the metal-regulated gene, hybridization to the ordered phage lambda bank of the E. coli chromosome, and sequencing of DNA adjacent to the luxAB fusion revealed that the insertion occurred within the fliC (hag) gene of E. coli. This gene encodes flagellin, the filament subunit of the bacterial motility organ, and is under the control of several regulatory cascades. These results suggest that environmental metals may play a role in the regulation of the motility potential of E. coli and that this bioluminescent gene fusion clone (or derivatives thereof) may be used to prepare a biosensor for the rapid detection of metal contamination in water samples.