The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1alpha (hepatocyte nuclear factor 1alpha) transcription factor. HNF1alpha, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1alpha augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1 alpha transcription decreased the CFTR mRNA levels. Hnf1 alpha knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene.
D-Mannitol is taken up by Bacillus stearothermophilusand phosphorylated via a phosphoenolpyruvatedependent phosphotransferase system (PTS). Transcription of the genes involved in mannitol uptake in this bacterium is regulated by the transcriptional regulator MtlR, a DNA-binding protein whose affinity for DNA is controlled by phosphorylation by the PTS proteins HPr and IICB mtl . The mutational and biochemical studies presented in this report reveal that two domains of MtlR, PTS regulation domain (PRD)-I and PRD-II, are phosphorylated by HPr, whereas a third IIA-like domain is phosphorylated by IICB mtl . An involvement of PRD-I and the IIA-like domain in a decrease in affinity of MtlR for DNA and of PRD-II in an increase in affinity is demonstrated by DNA footprint experiments using MtlR mutants. Since both PRD-I and PRD-II are phosphorylated by HPr, PRD-I needs to be dephosphorylated by IICB mtl and mannitol to obtain maximal affinity for DNA. This implies that a phosphoryl group can be transferred from HPr to IICB mtl via MtlR. Indeed, this transfer could be demonstrated by the phosphoenolpyruvate-dependent formation of [ 3 H]mannitol phosphate in the absence of IIA mtl . Phosphoryl transfer experiments using MtlR mutants revealed that PRD-I and PRD-II are dephosphorylated via the IIA-like domain. Complementation experiments using two mutants with no or low phosphoryl transfer activity showed that phosphoryl transfer between MtlR molecules is possible, indicating that MtlRMtlR interactions take place. Phosphorylation of the same site by HPr and dephosphorylation by IICB mtl have not been described before; they could also play a role in other PRD-containing proteins.Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) 1 (1-3). Recently, the mannitol operon of Bacillus stearothermophilus was cloned (4) and shown to consist of four genes, mtlA, mtlR, mtlF, and mtlD, coding for the mannitol transporter IICB mtl , the transcriptional regulator MtlR, the phosphotransferase IIA mtl , and mannitol-1-phosphate dehydrogenase, respectively. Analysis of the mannitol promoter revealed a catabolite response element overlapping the mannitol promoter, indicating that this operon is sensitive to catabolite repression. When favorable catabolites like glucose are utilized, HPr is phosphorylated by a kinase on a specific serine (5) that forms a complex with the CcpA repressor. Binding of this complex to catabolite response element sites located in or near the promoter regions of catabolic operons will prevent expression of these operons (6). In addition to catabolite repression, the expression of the mannitol operon is probably also regulated by the mannitol regulator MtlR (7). Domains in this protein show similarity to domains of two types of transcriptional regulators: DNA-binding proteins and anti-terminators. A helix-turn-helix motif is situated at the N terminus that is similar to those of DNA-binding transcriptional regulators of the DeoR family. The ...
A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA Mtl . Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII Mtl were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB Mtl of S. carnosus and the IICB part of the IICBA Mtl s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60؇C of B. stearothermophilus IICB Mtl expressed in E. coli was two times lower than that of E. coli IICB Mtl . IICB Mtl in B. stearothermophilus is maximally active at 85؇C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Two general cytoplasmic proteins, EI and HPr, are responsible for the transfer of the phosphoryl group from PEP to different sugar-specific PTS proteins (15,18,22 In general, the genes of proteins involved in the uptake and phosphorylation of a specific sugar are located in a single operon. To date, the mtlA genes of E. coli (13), S. carnosus (7), and Bacillus subtilis (1) and the cryptic mtlA gene of E. coli (29), all encoding homologous mannitol transporters, have been cloned. Our attempts to crystallize the E. coli protein have been unsuccessful, possibly because of limited stability. Since membrane proteins in thermophilic bacteria were found to be more stable than their mesophilic counterparts, a search for a mannitol PTS was done in these organisms. A mannitol PTS had been found in the obligate anaerobic thermophile Clostridium thermocellum (21). This paper reports the identification of a mannitol PTS in the aerobic thermophile Bacillus stearothermophilus and presents the cloning of the mannitol operon, sequencing of the mtlA gene, functional expression of B. stearothermophilus IICB Mtl in E. coli, and purification and partial characterization of the mannitol transporter. MATERIALS AND METHODSStrains and plasmids used in this study are listed in Table 1. Restriction enzymes, Taq DNA polymerase, Klenow enzyme, T4 DNA ligase, digoxigenin (DIG) labeling mix, and DIG nucleic acid detection kit were purchased from Boehringer Mannheim. The DNA sequencing kit was obtained from Pharmacia. Sodium deoxycholate (DOC), n-decyl--D-maltopyranoside (d-maltopyranoside), PEP, and mannitol...
D-Mannitol is taken up by Bacillus stearothermophilusand phosphorylated via a phosphoenolpyruvatedependent phosphotransferase system (PTS). The genes involved in the mannitol uptake were recently cloned and sequenced. One of the genes codes for a putative transcriptional regulator, MtlR. The presence of a DNA binding helix-turn-helix motif and two antiterminatorlike PTS regulation domains, suggest that MtlR is a DNA-binding protein, the activity of which can be regulated by phosphorylation by components of the PTS. Many bacteria transport D-mannitol and other carbohydrates via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) 1 (1-3). Two general cytoplasmic proteins, EI and HPr, are responsible for the transfer of the phosphoryl group from PEP to different sugar-specific PTS proteins. Each sugar-specific system consists of three parts: IIA, IIB, and IIC. IIC is the transmembrane transporter, responsible for the transfer of the sugar across the cytoplasmic membrane. The transported sugar is phosphorylated by IIB while it is still bound to IIC. IIA is responsible for the transfer of the phosphoryl group from HPr to IIB. Different fusions between IIA, IIB, and IIC occur naturally in the PTS. For example, in the mannitol uptake system of Escherichia coli, these proteins are covalently linked as one polypeptide chain, IICBA mtl , whereas in B. stearothermophilus, the same system consists of a soluble IIA mtl and a membrane-bound IICB mtl . Recently, the entire mannitol operon of Bacillus stearothermophilus was cloned (4). Four open reading frames, mtlA, mtlR, mtlF, and mtlD, were identified within the operon, coding for the mannitol transporter (IICB mtl ), a putative transcriptional regulator (MtlR), enzyme IIA mtl , and the mannitol-1-phosphate dehydrogenase (MPDH), respectively (Fig. 1). The mannitol transporter IICB mtl was expressed, and its involvement in the uptake of mannitol by B. stearothermophilus was confirmed (4). The sequence of the mtlR gene resembles that of transcriptional regulators, such as antiterminators and repressors, and is, therefore, expected to be involved in the regulation of the mannitol operon. In this paper, we report the functional expression and isolation of the B. stearothermophilus EI, HPr, IIA mtl , and MtlR and the analysis of the mtlR gene product, MtlR, that reveals its function as a transcriptional regulator, involved in the regulation of the mannitol operon. MATERIALS AND METHODSRestriction enzymes, Taq-DNApolymerase, nucleotides, oligonucleotide-kinase, pyruvate-kinase, IPTG, 5-bromo-4-chloro-3-indolyl--Dgalactopyranoside, and the first strand cDNA synthesis kit for reverse transcription-PCR (Avian Myeloblastosis Virus) were purchased from Boehringer Mannheim. [␥-32 P]ATP (3000Ci/mmol) and [ 14 C]mannitol (50 -62 mCi/mmol) were obtained from Amersham, Ni-NTA agarose from Qiagen, and PET15b from Novagen. RNase-free RQ1-DNase I was obtained from the Promega core footprinting system. Thrombin, with a specific activity of 8165 units/mg of protein, was from ICN Bio...
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