Bärbel Friedrich

A topological model for the high-affinity nickel transporter of Alcaligenes eutrophus

The gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy‐dependent uptake of nickel ions. Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis. To distinguish between these possibilities varying segments of the amino‐terminal end of the transporter were fused to the Escherichia coli enzymes aikaline phosphatase (PhoA) or β‐galactosidase (LacZ). The enzymatic activity of 16 HoxN‐PhoA and 15 HoxN‐LacZ fusions was determined. On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two‐dimensional model for the nickel transporter was developed. This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices. The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.

Mutants of pseudomonas facilis defective in lithoautotrophy

SUMMARY: Mutants of Pseudomonas facilis impaired in lithoautotrophic metabolism (Aut-) were obtained by Tn5 mutagenesis, mitomycin C treatment or incubation at sublethal temperature. A colony assay provided a fast screening method for hydrogenase-negative mutants. Three classes of mutants were identified: the first had lost the ability to grow autotrophically with H2 and heterotrophically with NO3n- as nitrogen source; the second lacked both hydrogenase and ribulose-1,5-bisphosphate carboxylase activity but nitrate metabolism was unaffected; the third retained the activities of the key enzymes of lithoautotrophic metabolism but failed to grow with H2 and CO2. All of the mutants showed the plasmid pattern of the wild-type.