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SummaryBy deleting the radA open reading frame of an extreme halophile, Haloferax volcanii, we created and characterized a recombination-deficient archaeon. This strain, Hf. volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type. These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA/RAD51 family by function as well as sequence homology. In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids. Despite its slower growth rate, Hf. volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination-deficient background is desired.

The gene encoding dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been subcloned and overexpressed in the parent organism by using the halophilic archaeal rRNA promoter. The recombinant protein has been purified to homogeneity and characterized with respect to its kinetic, molecular, and salt-dependent properties. A dihydrolipoamide dehydrogenase-minus mutant of H. volcanii has been created by homologous recombination with the subcloned gene after insertion of the mevinolin resistance determinant into the protein-coding region. To explore the physiological function of the dihydrolipoamide dehydrogenase, the growth properties of the mutant halophile have been examined. Dihydrolipoamide dehydrogenase catalyzes the oxidation ofIn members of the domains Bacteria and Eucarya, the enzyme normally performs this function as an integral component of the pyruvate, 2-oxoglutarate, and branchedchain 2-oxoacid dehydrogenase multienzyme complexes (19,20) or of the glycine cleavage system (16). In all of these systems, the lipoyl moiety is covalently bound to the enzyme complexes, where its reduction and reoxidation are part of the catalytic cycle.Whereas the role in these complexes is the only established function of dihydrolipoamide dehydrogenase, the enzyme has now been found in a variety of Bacteria, Eucarya, and Archaea in the apparent absence of the multienzyme systems (10,11,23,24,26). Such observations are indicative of a new function for the dehydrogenase (8), although its precise nature is still to be established. Clearly the identification of a new role requires evidence from both molecular genetics and protein structure, and to this end we have chosen to study in detail the dihydrolipoamide dehydrogenase from the moderately halophilic archaeon, Haloferax volcanii.The discovery of dihydrolipoamide dehydrogenase and its substrate lipoic acid in the halophilic Archaea, and of the apparent absence of the 2-oxoacid dehydrogenase complexes in these organisms, is reviewed in references 9 and 12. The gene encoding the enzyme has been cloned and sequenced from H. volcanii (29), and from sequence alignments it is clearly related to the complexed dihydrolipoamide dehydrogenases from Bacteria and Eucarya. In addition, shuttle vectors with antibiotic resistance markers for H. volcanii are now available (13-15) to enable the necessary molecular biological analysis of this halophilic gene and its enzyme product.In this paper, we report the subcloning and expression of the H. volcanii gene, the purification and characterization of the recombinant dihydrolipoamide dehydrogenase, and the creation and growth analysis of a H. volcanii strain that lacks the functional enzyme. MATERIALS AND METHODSReagents and enzymes. Bovine serum albumin, DL-lipoamide, RNase A, lowmelting-point agarose, novobiocin, and polyethylene glycol (PEG) 600 were purchased from Sigma. PEG 600 was purified by the method of Klebe et al. (17) DL-Dihydrolipoamide was prepared by the reduction of DL-lipoamide with NaBH 4 (22); t...

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In the present study, we utilised a polymerase-chain-reaction-coupled capillary electrophoresis (CE) approach to investigate the epidemiology of Eimeria species on a broiler-breeder farm in Victoria, Australia. The Eimeria populations of two flocks vaccinated against coccidiosis were followed over an 11-week period. All seven recognised Eimeria species of chickens were detected in both flocks. One flock suffered increased morbidity and mortality in its eighth week and had consistently higher Eimeria oocyst counts, species prevalences and rates of co-infections. Four Eimeria species included in the vaccine administered occurred at higher prevalences before the disease outbreak in the flock. Using the CE approach, two new, previously undescribed Eimeria genotypes were discovered in both chicken flocks, one of which dominated toward the end of the study period. The molecular approach proved versatile and capable of providing useful epidemiological data which could be used to investigate and interpret coccidiosis outbreaks.