Construction and partial characterization of an L-amino acid oxidase-free Synechococcus PCC 7942 mutant and localization of the L-amino acid oxidase in the corresponding wild type.
The gene (aoxA) coding for an L-amino acid oxidase (L-AOX) with high specificity for basic L-amino acids (L-arginine being the best substrate) in the cyanobacterium Synechococcus PCC 6301 has previously been identified, sequenced and analysed (Bockholt, R., Masepohl, M., Kruft, V., Wittmann-Liebold, B. and Pistorius, E.K. (1995) Biochim. Biophys. Acta 1264, 289-293). Here we report on the inactivation of the aoxA gene in the closely related Synechococcus PCC 7942 by interrupting the gene with a kanamycin resistance cassette from Tn5. The mutant called D6 has no detectable L-AOX activity and no detectable L-AOX protein. Characterization of the mutant showed that in contrast to Synechococcus PCC 7942 wild-type (WT) cells the mutant cells can not grow on L-arginine as sole N-source, suggesting that the L-AOX is essential for growth on L-arginine. Mutant cells can grow on nitrate or ammonium as N-source under photoautotropic conditions with a growth rate of about 75% of the WT rate. Under these conditions the photosynthetic O2 evolving activity is reduced by about the same amount, and the pigment content, especially the phycobiliprotein content, is much lower than in WT cells, indicating that the mutant suffers from some type of deficiency. Immunocytochemical investigations and extraction of the soluble proteins from periplasma after plasmolysing the cell wall gave evidence that the L-AOX is predominantly located in the periplasma with only a small amount being intracellularly located. A model of the possible function of the L-AOX in Synechococcus PCC 6301/7942 will be given.
Bockholt
R
R
Biologie VIII:Zellphysiologie, Universität Bielefeld, Germany.
Scholten-Beck
G
G
Pistorius
E K
EK
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D003470:Culture Media; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D007150:Immunohistochemistry; D050830:L-Amino Acid Oxidase; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009566:Nitrates / Q000378:metabolism; D010788:Photosynthesis
D001426:Bacterial Proteins; D003470:Culture Media; D009566:Nitrates; D001120:Arginine; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase