The sucrose operon of NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBC protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK). The operon was cloned in in three stages. Initial isolation was achieved by screening a genomic library in for clones able to utilize sucrose, while the remainder of the operon was isolated by inverse PCR and by plasmid rescue of flanking regions from a mutant constructed by targeted gene disruption. Substrate specificity assays confirmed that the sucrose hydrolase was a β-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the gene encoded an ATP/Mg-dependent fructokinase. Both enzyme activities were induced by sucrose in Disruption of the operon of by targeted plasmid integration into either the or the gene resulted in strains unable to utilize sucrose, indicating that this was the only inducible sucrose catabolic pathway in this organism. RNA analysis confirmed that the genes of the operon were co-transcribed on a 5 kb mRNA transcript and that transcription was induced by sucrose, but not by glucose, fructose, maltose or xylose. Primer extension experiments identified the transcriptional start site as lying 44 bp upstream of the ATG start codon, immediately adjacent to the imperfect palindrome sequence proposed to be a repressor binding site. Disruption of the gene resulted in constitutive transcription of the upstream gene, suggesting that encodes a transcriptional repressor which acts at the operator sequence. The gene is therefore itself negatively autoregulated as part of the polycistronic mRNA.


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