Time-lapsed confocal microscopy reveals temporal and spatial expression ofthe lysine epsilon-aminotransferase gene in Streptomyces clavuligerus

To investigate the temporal and spatial expression patterns of the gene (la t) encoding lysine epsilon-aminotransferase (LAT) for cephamycin C biosynth esis, a mutant form of green fluorescent protein (mut1GFP) was integrated i nto the Streptomyces clavuligerus chromosome (strain LH369), resulting in a translational fusion with lat. LAT activity and fluorescence profiles of t he recombinant protein paralleled the native LAT enzyme activity profile in wild-type S, clavuligerus, which peaked during exponential growth phase an d decreased slowly towards stationary phase. These results indicate that th e LAT-Mut1GFP fusion protein retains both LAT and GFP functionality in S, c lavuligerus LH369. LH369 produced wild-type levels of cephamycin C in minim al medium culture conditions supplemented with lysine. Time-lapsed confocal microscopy of the S. clavuligerus LH369 strain revealed the temporal and s patial characteristics of laf gene expression and demonstrated that physiol ogical development of S. clavuligerus colonies leading to cephamycin C bios ynthesis is limited to the substrate mycelia.