Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs.

The pathogenic protozoan parasite Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates. We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+- and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities. EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for anaerobic growth of E. coli. Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy. However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent. histolytica, and difficulties in quantitating drug efficacy in vitro. To approach this problem, we expressed the EhADH2 gene in a mutant strain of E. coli carrying a deletion of the adhE gene. Expression of EhADH2 restored the ability of the mutant E. coli strain to grow under anaerobic conditions. By screening compounds for the ability to inhibit the anaerobic growth of the E. coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity. Using bacteria to bypass the need for parasite culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases.

Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli.

Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins. We have found that polymerization of curlin to fimbriae-like structures (curli) on the surface of Escherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein. The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleator-presenting cell or by adjacent cells. The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation.

Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli.

Formation of deletions by recombination between short direct repeats is thought to involve either a break-join or a copy-choice process. The key step of the latter is slippage of the replication machinery between the repeats. We report that the main replicase of Escherichia coli, DNA polymerase III holoenzyme, slips between two direct repeats of 27 bp that flank an inverted repeat of approximately equal 300bp. Slippage was detected in vitro, on a single-stranded DNA template, in a primer extension assay. It requires the presence of a short (8 bp) G+C-rich sequence at the base of a hairpin that can form by annealing of the inverted repeats. It is stimulated by (i) high salt concentration, which might stabilize the hairpin, and (ii) two proteins that ensure the processivity of the DNA polymerase III holoenzyme: the single-stranded DNA binding protein and the beta subunit of the polymerase. Slippage is rather efficient under optimal reaction conditions because it can take place on >50% of template molecules. This observation supports the copy-choice model for recombination between short direct repeats.