In Bacillus subtilis, aryl-␤-glucosides such as salicin and arbutin are catabolized by the gene products of bglP and bglH, encoding an enzyme II of the phosphoenolpyruvate sugar-phosphotransferase system and a phospho-␤-glucosidase, respectively. These two genes are transcribed from a single promoter. The presence of a transcript of about 4,000 nucleotides detected by Northern (RNA) blot analysis indicates that bglP and bglH are part of an operon. However, this transcript is only present when cells are grown in the presence of the inducing substrate, salicin. In the absence of the inducer, a transcript of about 110 nucleotides can be detected, suggesting that transcription terminates downstream of the promoter at a stable termination structure. Initiation of transcription is abolished in the presence of rapidly metabolized carbon sources. Catabolite repression of bglPH expression involves the trans-acting factors CcpA and HPr. In a ccpA mutant, transcription initiation is relieved from glucose repression. Furthermore, we report a catabolite responsive element-CcpA-independent form of catabolite repression requiring the ribonucleic antiterminator-terminator region, which is the target of antitermination, and the wild-type HPr protein of the phosphotransferase system. Evidence that the antitermination protein LicT is a crucial element for this type of regulation is provided.The presence of glucose in culture medium was observed to repress the synthesis of enzymes for the utilization of less rapidly metabolized sugar substrates in a number of bacterial species. Moreover, synthesis of secondary metabolites, e.g., antibiotics, as well as developmental pathways, e.g., spore formation and synthesis of extracellular enzymes, is subject to carbon catabolite repression (4,12,26).The mechanism by which Escherichia coli accomplishes catabolite repression is well understood. The positive regulator CAP (catabolite activator protein) in complex with cyclic AMP activates transcription of catabolite repression-sensitive operons by binding to a specific site upstream of the Ϫ35 region of the respective promoters (25). Enzyme IIA Glc of the phosphotransferase system (PTS) turned out to be the central regulatory protein, since it controls both the intracellular level of cyclic AMP and several non-PTS permeases (31).Carbon catabolite repression in Bacillus subtilis obviously applies different mechanisms, as very low concentrations of cyclic AMP are present and a negative regulatory mechanism triggers this type of regulation (3). Efforts to identify cis-acting elements mediating catabolite repression of several genes led to the establishment of a consensus sequence for a catabolite responsive element (CRE) (18, 42). Mutations in CRE-homologous sequences found in front of the amyE gene (30) and in the xyl (19), gnt (29), licS (23), hut (44), and bgl (22) operons, as well as in the acsA-acuABC promoter region (13), result in loss of glucose repression.B. subtilis mutants relieved from glucose repression have been isolated. The crsA mut...

The expression of the putative operon bglPH of Bacillus subtilis was studied by using bglP'-lacZ transcriptional fusions. The bglP gene encodes an aryl-beta-glucoside-specific enzyme II of the phosphoenolpyruvate sugar:phosphotransferase system, whereas the bglH gene product functions as a phospho-beta-glucosidase. Expression of bglPH is regulated by at least two different mechanisms: (i) carbon catabolite repression and (ii) induction via an antitermination mechanism. Distinct deletions of the promoter region were created to determine cis-acting sites for regulation. An operatorlike structure partially overlapping the -35 box of the promoter of bglP appears to be the catabolite-responsive element of this operon. The motif is similar to that of amyO and shows no mismatches with respect to the consensus sequence established as the target of carbon catabolite repression in B. subtilis. Catabolite repression is abolished in both ccpA and ptsH1 mutants. The target of the induction by the substrate, salicin or arbutin, is a transcriptional terminator located downstream from the promoter of bglP. This structure is very similar to that of transcriptional terminators which regulate the induction of the B. subtilis sacB gene, the sacPA operon, and the Escherichia coli bgl operon. The licT gene product, a member of the BglG-SacY family of antitermination proteins, is essential for the induction process. Expression of bglP is under the negative control of its own gene product. The general proteins of the phosphoenolpyruvate-dependent phosphotransferase system are required for bglP expression. Furthermore, the region upstream from bglP, which reveals a high AT content, exerts a negative regulatory effect on bglP expression.

Gene licS of Bacillus subtilis encodes an excreted ␤-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two-to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in ␤-glucan utilization.Evolutionarily conserved mechanisms of gene regulation by means of transcriptional antitermination have recently been described for sucrose metabolism in Bacillus subtilis and for ␤-glucoside utilization in Escherichia coli. The ␤-glucoside (bgl) operon of E. coli consists of three genes (33) which are preceded by a catabolite gene activator protein-cyclic AMPdependent promoter (24). Interestingly, in wild-type strains, the bgl promoter is kept silent in vivo (28) and utilizes insertion sequences as transcriptional enhancers to gain full activity (24,25,31). The first gene of the operon, bglG, encodes the antiterminator protein. It is flanked by two transcriptional terminators (t1 and t2) at which the BglG protein acts to alleviate termination (20,29). Terminators t1 and t2 share a highly conserved sequence motif proximal to and extending into their stem-loop structures (33). It has been shown for terminator t1 that BglG specifically binds to this sequence motif (now termed RAT [3]) at the mRNA level (16), suggesting that binding prevents formation of the terminator structure, thereby allowing transcription to proceed.The second gene of the operon, bglF, encodes the ␤-glucoside-specific transport protein, enzyme II Bgl , which is part of the phosphoenolpyruvate sugar-phosphotransferase system (PTS) and phosphorylates its substrates concomitantly with their transport (6,32,33). Enzyme II Bgl additionally functions as negative regulator of the operon: in the absence of ␤-glucosidic substrates, it phosphorylates the BglG protein, thereby inhibiting its antiterminator activity. This phosphorylation is reversible, allowing induction of the operon upon addition of sugar substrate (1, 2, 29, 30). The third gene, bglB, codes for the hydrolyzing enzyme, a phospho-␤-glucosidase.In B. subtilis, two systems involved in sucrose metabolism are likewise controlled by transcriptional antitermi...

A new catabolic system in Bacillus subtilis involved in utilization of ␤-glucosidic compounds has been investigated. It consists of five genes encoding phosphotransferase system (PTS) enzyme II (licB and licC) and enzyme IIA (licA), a presumed 6-phospho-␤-glucosidase (licH), as well as a putative regulator protein (licR). The genes map around 334؇ of the B. subtilis chromosome, and their products are involved in the uptake and utilization of lichenan degradation products. These five genes are organized in two transcriptional units. A weak promoter precedes gene licR, and transcription is obviously terminated at a secondary structure immediately downstream of the reading frame, as shown by Northern RNA blot analysis. Genes licB, licC, licA, and licH constitute an operon. Initiation of transcription at the promoter in front of this operon presumably requires activation by the gene product of licR. The LicR protein shows an unusual domain structure, i.e., similarities to (i) the conserved transcriptional antiterminator BglG family signature and (ii) PTS enzyme II. Using RNA techniques and transcriptional lacZ fusions, we have shown that the expression of the licBCAH operon is inducible by products of lichenan hydrolysis, lichenan and cellobiose. The presence of excess glucose prevents the induction of this operon, indicating the control by carbon catabolite repression. Moreover, the expression of the operon requires the general PTS components and seems to be negatively controlled by the specific lic PTS enzymes.

The Bacillus subtilis sacY and sacT genes encode antiterminator proteins, similar to the Escherichia coli bglG gene product and required for transcription of sucrose metabolism genes. A Tn10 insertion into bglP (formerly sytA) has been previously identified as restoring sucrose utilization to a strain with deletions of both sacY and sacT. The nucleotide sequence of bglP showed a high degree of homology with the E. coli bglF gene (BglF is a ␤-glucoside permease of the phosphotransferase system and also acts as a negative regulator of the BglG antiterminator). Complementation studies of an E. coli strain with a deletion of the bgl operon showed that BglP was a functional ␤-glucoside permease. In B. subtilis, bglP complemented in trans both the bglP::Tn10 original insertion and a phenotypically similar bglP deletion. Disruption of licT abolished the suppressor phenotype in a bglP mutant. LicT is a recently identified third B. subtilis antiterminator of the BglG/SacY family. These observations indicated that BglP was also a negative regulator of LicT. Both LicT and BglP seem to be involved in the induction by ␤-glucosides of an operon containing at least two genes, bglP itself and bglH, encoding a phospho-␤-glucosidase. Other ␤-glucoside genes homologous to bglP and bglH have been recently described in B. subtilis. Thus, B. subtilis possesses several sets of ␤-glucoside genes, like E. coli, but these genes do not appear to be cryptic.The products of the Bacillus subtilis sacPA operon mediate transport and hydrolysis of sucrose. Mutants affected in sacPA, or in the sacT gene which encodes an antiterminator protein (AT) involved in sacPA induction by sucrose, are impaired for utilization of this sugar (8,12). This defect is not absolute because B. subtilis can also assimilate sucrose through an extracellular pathway involving the exoenzyme levansucrase encoded by sacB (35). This gene is also induced by sucrose through an antitermination mechanism involving SacY, a second AT structurally similar to SacT, but fully activated only in the presence of a high sucrose concentration (Ͼ1%) (7). When activated, SacY can partially replace SacT for induction of sacPA (36); this appears to be due to the structural similarity of the two ATs and of their targets within the sacPA and sacB leader regions (4). The model for induction by sucrose of sacB (7) is similar to that proposed for induction by ␤-glucosides of the Escherichia coli bgl operon (19, 25): an enzyme II bgl (BglF), a permease of the phosphotransferase system (PTS) specific for the inducer, inactivates the AT (BglG) by phosphorylating it, and in the presence of the inducer, its permeation and phosphorylation lead to the concomitant dephosphorylation of BglF and then to that of BglG. The activated (dephosphorylated) AT binds to a specific imperfect palindromic RNA target sequence (RAT sequence), which overlaps the 5Ј part of a regulatory terminator located in the leader region of the regulated gene. This binding stabilizes the RAT secondary structure and therefore preven...