Time-lapsed confocal microscopy reveals temporal and spatial expression of the lysine epsilon-aminotransferase gene in Streptomyces clavuligerus.
To investigate the temporal and spatial expression patterns of the gene (lat ) encoding lysine epsilon-aminotransferase (LAT) for cephamycin C biosynthesis, a mutant form of green fluorescent protein (mut1GFP) was integrated into the Streptomyces clavuligerus chromosome (strain LH369), resulting in a translational fusion with lat. LAT activity and fluorescence profiles of the recombinant protein paralleled the native LAT enzyme activity profile in wild-type S. clavuligerus, which peaked during exponential growth phase and decreased slowly towards stationary phase. These results indicate that the LAT-Mut1GFP fusion protein retains both LAT and GFP functionality in S. clavuligerus LH369. LH369 produced wild-type levels of cephamycin C in minimal medium culture conditions supplemented with lysine. Time-lapsed confocal microscopy of the S. clavuligerus LH369 strain revealed the temporal and spatial characteristics of lat gene expression and demonstrated that physiological development of S. clavuligerus colonies leading to cephamycin C biosynthesis is limited to the substrate mycelia.
Han
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Department of Microbiology and Biological Process Technology Institute, University of Minnesota, Minneapolis 55455, USA.
Khetan
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A
Hu
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Sherman
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DH
D015139:Blotting, Southern; D015153:Blotting, Western; D002513:Cephamycins / Q000096:biosynthesis; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins; D051306:L-Lysine 6-Transaminase; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism*
D002513:Cephamycins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000637:Transaminases; D051306:L-Lysine 6-Transaminase; C024925:cephamycin C