IDENTIFICATION OF A NOVEL GENE, AUT, INVOLVED IN AUTOTROPHIC GROWTH OF ALCALIGENES-EUTROPHUS

The aerobic facultative chemoautotroph Alcaligenes eutrophus was found to possess a novel gene, designated auf, required for both lithoautot rophic (hydrogen plus carbon dioxide) and organoautotrophic (formate) growth (Aut(+) phenotype). Insertional mutagenesis by transposon Tn5-M ob localized the gene on a chromosomal 13-kbp EcoRI fragment. Physiolo gical characterization of various Aut(-) mutants revealed pleiotropic effects caused by the transposon insertion. Heterotrophic growth of th e mutants on substrates catabolized via the glycolytic pathway was slo wer than that of the parent strains, and the colony morphology of the mutants was altered when grown on nutrient agar. The heterotrophic der epression of the cbb operons encoding Calvin cycle enzymes was abolish ed, although their expression was still inducible in the presence of f ormate. Apparently, the mutation did not affect the cbb genes directly but impaired the autotrophic growth in a more general manner. The con jugally transferred wild-type EcoRI fragment allowed phenotypic in tra ns complementation of the mutants. Further subcloning and sequencing i dentified a single open reading frame (aut) of 495 bp that was suffici ent for complementation. The monocistronic aut gene was constitutively transcribed into a 0.65-kb mRNA. However, its expression appeared to be low. Heterologous expression of aut was achieved in Escherichia col i, resulting in overproduction of an 18-kDa protein. Database searches yielded weak partial sequence similarities bf the deduced Aut protein sequence to some cytidylyltransferases, but no indication for the exa ct function of the aut gene was obtained. Hybridizing DNA sequences th at might be similar to the aut gene were detected by Southern hybridiz ation in the genome of two other autotrophic bacteria.