Heterogeneous distribution of lysine 6-aminotransferase during cephamycin C biosynthesis in Streptomyces clavuligerus demonstrated using green fluorescent protein as a reporter

The cellular distribution of the cephamycin biosynthetic enzyme lysine 6-am inotransferase (LAT) has been studied in Streptomyces clavuligerus hyphae b y confocal microscopy using the S65T mutant of green fluorescent protein (G FP) as a reporter, LAT mediates the first committed step in the biosynthesi s Of the secondary metabolite cephamycin C by S. clavuligerus. The enzymic activity of LAT varies with time during the growth of S, clavuligerus in li quid medium. To investigate if this temporal variation occurs uniformly amo ngst all hyphae, S, clavuligerus was transformed with a plasmid containing the LAT-encoding gene translationally fused to the GFP-encoding gene. The L AT-GFP fusion product displayed fluorescence spectral characteristics of GF P, and showed similar temporal characteristics of LAT activity compared to the wild-type strain of S, clavuligerus, The transformed strain exhibited a heterogeneous distribution of fluorescence in mycelia grown in liquid cult ures. This distribution varied significantly as the batch progressed: only a fraction of the mycelia fluoresced in the early growth phase, whereas nea rly all hyphae fluoresced by the late growth phase. Thereafter, a non-unifo rm distribution of fluorescence was again observed in the declining growth phase. A large fraction of the non-fluorescent cells in the declining growt h phase were found to be non-viable. Observations of S, clavuligerus coloni es grown on solid agar also showed variation of LAT-GFP expression at diffe rent stages of growth. These observations in the solid phase can be explain ed in terms of nutrient deprivation and signalling molecules. The results s uggest that physiological differentiation of S, clavuligerus mycelia leadin g to cephamycin C biosynthesis is both temporally and spatially distributed . The findings also revealed that the observed heterogeneity was independen t of the position of individual cell compartments within the hypha, The pot ential of GFP as a reporter for the quantitative study of cephamycin biosyn thesis at the cellular level has also been demonstrated.