The target of the induction by sucrose of the levansucrase gene is a transcription terminator (sacRt) located upstream from the coding sequence, sacB. The two-gene locus sacX-sacY (formerly sacS) and the ptsI gene were previously shown to be involved in this induction. ptsI encodes enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. SacX is strongly homologous to sucrose-specific phosphotransferase system-dependent permeases. SacY is a positive regulator of sacB. Here we show that SacY is probably an antiterminator interacting directly with sacRt, since in Escherichia coli the presence of the sacY gene stimulates the expression of a reporter gene fused downstream from sacRt. Missense mutations affecting sacY were sequenced, and the sacB regulation was studied in isogenic strains carrying these mutations or in vitro-generated mutations affecting sacX, sacY, or ptsI. The phenotype of double mutants suggests a model in which SacX might be a sucrose sensor that would be phosphorylated by the phosphotransferase system and, in this state, could inhibit the SacY antiterminator. Exogenous sucrose, or a mutation inactivating the phosphotransferase system, would dephosphorylate SacX and allow antitermination at sacRt.
The adjacent sacX and sacY genes are involved in sucrose induction of the BaciUus subtilis sacB gene by an antitermination mechanism. sacB, encoding the exoenzyme levansucrase, is also subject to regulation by the DegS-DegU signalling system. Using sacXY'-acZ and sacX'-acZ fusions, we show that the transcription of the sacX and sacY genes is both inducible by sucrose and regulated by DegU. sacX and sacY appear to constitute an operon, since the deletion of the sacX leader region abolished the expression of a sacXY'-acZ fusion. The degU-dependent promoter was located by deletion analysis and reverse transcriptase mapping 300 nucleotides upstream from the sacX initiator codon. Sucrose induction of the sacX-acZ fusion requires either SacY or the homologous SacT antiterminator, which is involved in sucrose induction of the intracellular sucrase gene (sacPA operon). Sequence analysis of the sacX leader region revealed (20 nucleotides downstream from the transcription start site) a putative binding site for these regulators; however, no structure resembling a rho-independent terminator could be found overlapping this site, unlike the situation for sacPA and sacB. Deletion of a segment of the leader region located 100 nucleotides downstream from this site led to constitutive expression of the sacXY'-acZ and sacX'-acZ fusions. These results suggest that the mechanism of sucrose induction of sacXY is different from that of sacPA and sacB.
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