The alkaline shock protein Asp23 was identified as a sigmaB-dependent protein in Staphylococcus aureus. In Bacillus subtilis, the asp23 promoter from S. aureus is regulated like other sigmaB-dependent promoters, which are strongly induced by heat and ethanol stress. However, almost no induction of asp23 expression was found after heat or ethanol stress in S. aureus MA13 grown in a synthetic medium, where the basal expression level of asp23 is high. Under the same experimental conditions the sigmaB gene itself showed a similar expression pattern: it was highly expressed in synthetic medium but not induced by heat or ethanol stress. In contrast, sigmaB activity was increased by heat stress when the cells were grown in a complex medium. The constitutive expression of sigB and sigmaB-dependent stress genes in S. aureus MA13 grown in a synthetic medium is in a sharp contrast to the regulation of sigmaB activity in B. subtilis, and needs further investigation. A deletion of 11 bp in the rsbU gene, which encodes the phosphatase that acts on RsbV (the anti-anti-sigma factor), in S. aureus NCTC 8325-4 might be responsible for the failure of heat stress to activate sigmaB in complex medium, and thus reduce the initiation of transcription at sigmaB-dependent promoters in this strain.
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...
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...
Gene therapeutic approaches clearly would benefit from techniques allowing the non-invasive monitoring of gene expression after in vivo gene transfer. Recently, the herpes virus thymidine type 1 kinase and radioiodinated 2'-fluoro-2'-deoxy-5-iodol-P-D-arabino-furanosyluracil (FIAU) were introduced as a reporter geneheporter probe combination [ 11. Various groups have demonstrated that this system can be used for visualization of gene expression after in v i m and in vivo gene transfer [2-41. However, little information is available about the correlation of regional radioactivity accumulation and the level of HSV-tk derived mRNA in tumors. We assessed the correlation between signal intensity and the level of gene expression. In addition, we studied the effect of ganciclovir (GCV) treatment and 9fluoro-2'-deoxyuridine (FdUrd) on the tracer uptake in vivo. Retrovirally transduced CMS-5 fibrosarcoma cells (CMS-STK) containing the HSVtk gene and the parental cells as negative control were used. Cell uptake studies were performed using different percentages (0, 25, 50, 75 and 100%) of HSV-tk expressing cells and respective parental cells. Cells were incubated with [14C]FIAU and tracer uptake was normalized to [3H]thymidine uptake (to correct for differences in cell proliferation). After lysis of the cells the amount of HSV-tk derived mRNA was determined by quantitative Northern Blots. Balb/c-mice were subcutaneously injected with lo6 cells consisting of different percentages of HSV-tk expressing cells at the right flank and negative control cells at the left flank. FAU was radiolabeled using the Iodogen method and purified by . Biodistribution studies (BD) and quantitative whole body autoradiography (QAR) were carried out at 2 h (BD) and 4 h (QAR) after i.v. tracer injection. Dissected tumors from BD were immediately deep frozen and HSV-tk mRNA was determined after homogenization of the tumor mass and isolation of the RNA. The effect of GCV on ['251]FIAU uptake was determined in a time (1 mg GCV per mouse i.p. 0, 1, 2 , 4 and 24 hours before [''51]FIAU injection) and concentration (increasing amounts of GCV: 0.005, 0.01, 0.05, and 0.1 mg/mouse together with ['2SI]FIAU) dependant manner using the tumor mouse model described above. Intracellular depletion of thymidine was accomplished by inhibition of the de novo synthesis by pretreatment with FdUrd (10 mg/kg) 30 min prior to tracer injection. h i v i m there is a linear correlation between the percentage of HSV-tk expressing cells and the normalized ['4C]FIAU uptake as well as the amount of HSV-tk mRNA. Analogous results were found in vivo. Biodistribution data as well as QAR I 1. Labelled Cpd Radiopham 44, Suppl. 1 (2001)
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