THIOREDOXIN IS ESSENTIAL FOR RHODOBACTER-SPHAEROIDES GROWTH BY AEROBIC AND ANAEROBIC RESPIRATION

To investigate the biological role of thioredoxin in the facultative p hotosynthetic bacterium Rhodobacter sphaeroides, attempts were made to construct a thioredoxin-deficient mutant by site-specific mutagenesis , using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TrxA(-) mu tation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is e ssential for aerobic growth of R. sphaeroides. An ORF of unknown funct ion was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene r eplacement to introduce a Cys33 to Ser mutation into the trxA chromoso mal copy. The strain carrying this mutation produced a thioredoxin imp aired in its protein-disulfide reductase activity and was also not via ble. These data suggest that the physiological function of R. sphaeroi des thioredoxin is redox-dependent Thioredoxin purified from R. sphaer oides was shown to have a glutathione-disulfide oxidoreductase activit y typical of glutaredoxins. This unexpected finding suggests that R. s phaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, h as the potential to act in GSH-dependent processes. Thus, the fundamen tal role of R. sphaeroides thioredoxin in cell growth probably origina tes from the multiple functions it can serve in vivo.