BRADYRHIZOBIUM-JAPONICUM TLPA, A NOVEL MEMBRANE-ANCHORED THIOREDOXIN-LIKE PROTEIN INVOLVED IN THE BIOGENESIS OF CYTOCHROME-AA3 AND DEVELOPMENT OF SYMBIOSIS

We report the discovery of a bacterial gene, tlpA, that codes for a hi therto unknown type of thioredoxin-like protein. The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nod ule symbiont Bradyrhizobium japonicum. The TlpA protein shared up to 3 1% amino acid sequence identity with various eukaryotic and prokaryoti c thioredoxins and protein disulfide isomerases, and possessed a chara cteristic active-site sequence, Trp-Cys-Val-Pro-Cys. In contrast to al l members of the thioredoxin family known to date, TlpA was shown to b e anchored to the cytoplasmic membrane by means of an N-terminal trans membrane domain, while the active site-containing part of the protein faced the periplasm. The tlpA mutant had a pleiotropic phenotype in th at it was defective in the development of a nitrogen fixing endosymbio sis and exhibited a strongly decreased oxidase activity, as compared w ith the wild-type. Holocytochrome aa3 was spectroscopically undetectab le in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic m embrane. Since cytochrome aa3 is not a prerequisite for the developmen t of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential perip lasmic step in the maturation of cytochrome aa3.