microbiology, Vol 145, 1461-1472, Copyright © 1999 by Society for General Microbiology

ARTICLES

The genes controlling sucrose utilization in Clostridium beijerinckii NCIMB 8052 constitute an operon

SJ Reid, MS Rafudeen and NG Leat
Department of Microbiology, University of Cape Town, Rondebosch 7700, South Africa

The sucrose operon of Clostridium beijerinckii NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBC(Scr) protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK). The scrARBK operon was cloned in Escherichia coli in three stages. Initial isolation was achieved by screening a C. beijerinckii genomic library in E. coli 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 scrB mutant constructed by targeted gene disruption. Substrate specificity assays confirmed that the sucrose hydrolase was a beta-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the scrK gene encoded an ATP/Mg2+-dependent fructokinase. Both enzyme activities were induced by sucrose in C. beijerinckii. Disruption of the scr operon of C. beijerinckii by targeted plasmid integration into either the scrR or the scrB 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 scr 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 scrA ATG start codon, immediately adjacent to the imperfect palindrome sequence proposed to be a repressor binding site. Disruption of the scrR gene resulted in constitutive transcription of the upstream scrA gene, suggesting that scrR encodes a transcriptional repressor which acts at the scrA operator sequence. The scrR gene is therefore itself negatively autoregulated as part of the polycistronic scrARBK mRNA.

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