Transcriptional analysis of bglPH expression in Bacillus subtilis: evidence for two distinct pathways mediating carbon catabolite repression

Abstract

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...

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…CCR of the gnt operon carrying a cre in the gntR-coding region is partially promoterindependent, and the amounts of the transcripts containing regions downstream of the cre decrease considerably on the addition of glucose, 7) implying that transcriptional roadblock might be involved in this catabolite repression. This is supported by the finding 88) that a mutation of mfd encoding a transcription-repair coupling factor, Mfd, 89) relieves CCR of hut and gnt expression at the cis-acting cre sequences located downstream of their transcriptional start sites, but does not affect CCR at the promoter-proximal cre sites, such as in amyE 3) and bglP, 27) suggesting that the Mfd protein displaces RNA polymerase stalled at downstream cre sites to which the CcpA/P-Ser-HPr (or -Crh) is bound. Nonetheless, CCR of acsA expression is not affected by an mfd mutation in spite of the location of the acsA cre 44 bp downstream of the acsA transcriptional initiation sites, but CCR is relieved by it if the cre is placed 161 bp downstream of the initiation site.…”

“…These proteins (SacY, SacT, LicT, and GlcT) positively control the genes involved in the metabolism of carbohydrates that are taken up by the PTS; SacY and T are involved in sucrose metabolism, LicT in the metabolism of oligo--glucoside and aryl--glucoside, and GlcT in glucose assimilation. SacY and T [140][141][142] are antiterminators of the sacB (encoding levansucrase)-levB (endolevanase)-yveA, sacX (IIBC SacX )-sacY, and sacP (IIBC SacP )-sacA (sucrose-6-P hydrolase)-ywdA operons, whereas LicT 27,143) is an antiterminator of the licS gene (endo-1,3-1,4--glucanase) and the bglP (IIBCA Bgl )-bglH (6-P--glucosidase)-yxiE operon, and GlcT 144,145) is that of the ptsG (IICBA Glc )-ptsH (HPr)-ptsI (EI) operon. In the presence of the cognate PTS-sugars that activate these antiterminators, they bind to a conserved motif called the ribonucleic antiterminator (RAT), which is present in the untranslated leader-mRNAs of their target genes.…”

“…The operons involved in the catabolism of secondary carbon sources are as follows (the carbon sources in parentheses): gntRKPZ (gluconate), 16,35,36) xylAB (xylose), 9) iolABCDEFGHIJ (myo-inositol), 40,41,102) trePAR (trehalose), 25,47,103) galKT (galactose), 25) glpFK (glycerol), 33) glvARC (6-P--glucoside), 34) bglPH ( -glucoside), 26,27) yjlBCD-uxaC-yjmBCD-uxuA-yjmFexuTR-uxaBA (hexuronate), 25,48) xynPB ( -xyloside), 65) yxjC-scoAE-bdh ( -hydroxybutyrate), 25,49) ara-ABDLMNPQ-abfA (arabinose) 24,52,53) and abnA xsa (arabinose), 50) kdgRKAT (hexuronate), 25,42) and kduID (galacturonate). 43) The yxkF-msmX operon probably involved in the transport of unknown sugars has been found to be under CcpA-mediated CCR.…”

Transcription–repair coupling factor is involved in carbon catabolite repression of the Bacillus subtilis hut and gnt operons

“…Expression of the bglPH operon, which encodes two enzymes involved in ␤-glucoside assimilation, is induced by salicin and repressed by glucose (Le Coq et al, 1995;Krü ger and Hecker, 1995). CCR of bglPH expression is mediated both at the level of induction and at a cre that overlaps the ¹35 region of the promoter (Krü ger et al, 1996). To examine the affect of the mfd22 mutation on CCR mediated at the bgl cre, a bgl-lacZ transcriptional fusion, BGL2, was constructed.…”

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT

“…This latter type of control has so far only been identified in B. subtilis and is implicated in catabolite repression (Arnaud et al, 1996;Krü ger et al, 1996;Martin-Verstraete et al, 1995;Stü lke et al, 1995).…”