Thioredoxin is essential for Rhodobacter sphaeroides growth by aerobic and anaerobic respiration.
To investigate the biological role of thioredoxin in the facultative photosynthetic 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- mutation 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 essential for aerobic growth of R. sphaeroides. An ORF of unknown function 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 replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulfide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in vivo.
Pasternak
Cécile
C
Laboratoire de Technologie Enzymatique, URA 1442 du CNRS, Université de Technologie de Compiègne, BP 649, 60206 Compiègne Cedex, France.
Assemat
Karine
K
Laboratoire de Technologie Enzymatique, URA 1442 du CNRS, Université de Technologie de Compiègne, BP 649, 60206 Compiègne Cedex, France.
Clément-Métral
Jenny D
JD
Laboratoire de Technologie Enzymatique, URA 1442 du CNRS, Université de Technologie de Compiègne, BP 649, 60206 Compiègne Cedex, France.
Klug
Gabriele
G
Institut für Mikrobiologie und Molekularbiologie, Frankfurter Strasse 107, 35392 Giessen, Germany.
D000332:Aerobiosis; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001483:Base Sequence; D004220:Disulfides / Q000378:metabolism; D005798:Genes, Bacterial; D005804:Genes, Lethal; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010788:Photosynthesis / Q000502:physiology*; D011490:Protein Disulfide Reductase (Glutathione); D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000254:growth & development*; D017422:Sequence Analysis, DNA; D013879:Thioredoxins / Q000235:genetics*
D004220:Disulfides; D054477:Glutaredoxins; D013879:Thioredoxins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione