Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.

Macrolides are complex polyketides of microbial origin that possess an extraordinary variety of pharmacological properties, paired with an impressive structural diversity. Bioassays for specific detection of such compounds will be of advantage for a class-specific drug screening. The current paper describes a cell-based microbial biosensor, assigning a luminescence response to natural or chemically modified macrolides, independent from their biological activity. This biosensor is based on the coupling of the structural luciferase genes of Vibrio fischeri to the regulatory control mechanism of a bacterial erythromycin resistance operon. The bioassays is easy to handle and can be applied to various screening formats. The feasibility of the test system for natural products screening is exemplified by the isolation and characterization of picromycin from a Streptomyces species. Biosensor-guided screening for macrolides is based on macrolide-promoted expression of lux genes and induction of luminescence (independent of macrolide antibiotic activity).

No abstract

In Alcaligenes eutrophus CH34, determinants encoding inducible resistance to chromate (chr) and to cobalt and nickel (cnr) are located adjacent to each other on plasmid pMOL28. To develop metal-sensing bacterial strains, a cloned part of plasmid pMOL28, which contains both determinants, was mutated with Tn5-lacZ. The chr::lacZfusions were specifically induced by chromium; cnr was induced best by Ni2+ but was also induced by Co2+, Mn2+, chromate, Cu2+, Cd2+, and Zn2+. The broad-host-range IncP1 plasmid pEBZ141, which contains achr::lux fusion, was constructed.A. eutrophus AE104(pEBZ141), carrying achr::lux transcriptional fusion, could be used as a biosensor for chromate when cultivated in glycerol as an optimal carbon source. Chromate and bichromate were the best inducers; induction by Cr3+ was 10 times lower, and other ions induced only a little or not at all. Interactions among induction of the chr resistance determinant, chromate reduction, chromate accumulation, and the sulfate concentration of the growth medium were demonstrated.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic-mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid, Nickel deficiency correlated with a low level of the nickelcontaining hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic-mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic-mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normafly mediates the uptake of magnesium.Bartha and Ordal (1) were the first to report a specific nickel requirement for chemolithoautotrophic growth of Hydrogenomonas strains. Later it was shown that nickel is essential for catalytically active hydrogenase of Alcaligenes eutrophus (formerly Hydrogenomonas eutropha) (6) and is incorporated into the enzyme during protein synthesis (8). Subsequently, nickel was shown to be an essential trace element for many microorganisms, in which it is involved in at least four biological processes: hydrolysis of urea, hydrogen uptake and hydrogen evolution, methanogenesis, and acetogenesis (9).In order to provide the cells with sufficient nickel, the metal ion has to be actively transported into the bacterial cell. Two energy-dependent processes have been reviewed recently (9). Nickel ion transport may be catalyzed by a magnesium transporter and by a high-affinity, nickel-specific system. The latter was first shown to exist in strains of A. eutrophus (26). Lohmeyer and Friedrich (17) The characterization of nickel-deficient mutants and their derivatives was conducted as follows. NiCl2-free FN-medium (10 ml) was inoculated with cells from an agar plate. The medium for growing transconjugants was routinely supplied with 7.5 p.g of tetracycline per ml. The cells were grown overnight and diluted in 30 ml of FN-medium as specified above to an optical density (OD) of approximately 35 Klett units. After growth to mid-exponential phase, the cells were washed once with 36 mM sodium potassium phosphate buffer, pH 7.0. Mineral salts medium (10 ml) with additions of Ni2+ and Mg2+ at concentrations specified in the