Prokaryotic Transcriptional Activators Research Articles

Engineering Prokaryotic Transcriptional Activator XylR as a Xylose-Inducible Biosensor for Transcription Activation in Yeast.

Biosensors regulated by specific substrates are needed to develop genetic tools to meet the needs of engineering microbial cell factories. Here, a xylose-inducible biosensor (xylbiosensor), comprising the Escherichia coli activation factor XylR, fusion activation domain (AD) VPRH, and a hybrid promoter with operator xylO, was established in Yarrowia lipolytica. The addition of xylose to an engineered Y. lipolytica strain harboring the xylbiosensor could trigger significant transcriptional activation of target genes, such as mcherry and the xylose utilization gene. Furthermore, a novel promoter Pleu-Pxo-Ptef was developed to construct a bidirectional expression system. The xylbiosensor showed good portability in Saccharomyces cerevisiae, suggesting its potential value in other eukaryotic cells. This study is the first to construct a "turn-on" xylbiosensor induced by xylose addition based on a prokaryotic activator XylR and eukaryotic universal AD. The xylbiosensor exhibits potential in pathway engineering for xylose utilization and xylose-derived product biosynthesis in yeast.

Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast.

In cell factory development, screening procedures, often relying on low-throughput analytical methods, are lagging far behind diversity generation methods. This renders the identification and selection of the best cell factory designs tiresome and costly, conclusively hindering the manufacturing process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast by means of flow cytometry.

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR.

The activities of many prokaryotic sigma54-dependent transcriptional activators are controlled by the N-terminal A-domain of the protein, which is linked to the central transcriptional activation domain via a short B-linker. It used to be thought that these B-linkers simply serve as flexible tethers. Here we show that the B-linker of the aromatic-responsive regulator DmpR and many other regulators of the family contain signature heptad repeats with regularly spaced hydrophobic amino acids. Mutant analysis of this region of DmpR demonstrates that B-linker function is dependent on the heptad repeats and is critical for activation of the protein by aromatic effectors. The phenotypes of DmpR mutants refute the existing model that the level of ATPase activity directly controls the level of transcription it promotes. The mutant analysis also shows that the B-linker is involved in repression of ATPase activity and that allosteric changes upon effector binding are transduced to alleviate both B-linker repression of ATP hydrolysis and A-domain repression of transcriptional activation. The mechanistic implications of these findings for DmpR and other family members are discussed.

HbpR, a new member of the XylR/DmpR subclass within the NtrC family of bacterial transcriptional activators, regulates expression of 2-hydroxybiphenyl metabolism in Pseudomonas azelaica HBP1.

The regulation of 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl degradation in Pseudomonas azelaica is mediated by the regulatory gene, hbpR. The hbpR gene encodes a 63-kDa protein belonging to the NtrC family of prokaryotic transcriptional activators and having the highest homology to members of the XylR/DmpR subclass. Disruption of the hbpR gene in P. azelaica and complementation in trans showed that the HbpR protein was the key regulator for 2-hydroxybiphenyl metabolism. Induction experiments with P. azelaica and Escherichia coli containing luxAB-based transcriptional fusions revealed that HbpR activates transcription from a promoter (P(hbpC)) in front of the first gene for 2-hydroxybiphenyl degradation, hbpC, and that 2-hydroxybiphenyl itself is the direct effector for HbpR-mediated activation. Of several compounds tested, only the pathway substrates 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl and structural analogs like 2-aminobiphenyl and 2-hydroxybiphenylmethane were effectors for HbpR activation. HbpR is therefore, to our knowledge, the first regulator of the XylR/DmpR class that recognizes biaromatic but not monoaromatic structures. Analysis of a spontaneously occurring mutant, P. azelaica HBP1 Prp, which can grow with the non-wild-type effector 2-propylphenol, revealed a single mutation in the hbpR gene (T613C) leading to a Trp-->Arg substitution at amino acid residue 205. P. azelaica HBP1 derivative strains without a functional hbpR gene constitutively expressed the genes for 2-hydroxybiphenyl degradation when complemented in trans with the hbpR-T613C gene. This suggests the importance of this residue, which is conserved among all members of the XylR/DmpR subclass, for interdomain repression.

A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator.

A crystal structure for a member of the AraC prokaryotic transcriptional activator family, MarA, in complex with its cognate DNA-binding site is described. MarA consists of two similar subdomains, each containing a helix-turn-helix DNA-binding motif. The two recognition helices of the motifs are inserted into adjacent major groove segments on the same face of the DNA but are separated by only 27 A thereby bending the DNA by approximately 35 degrees. Extensive interactions between the recognition helices and the DNA major groove provide the sequence specificity.

DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site.

We present evidence that Escherichia coli RNA polymerase beta subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage lambda. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of lambda plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the beta subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed.

FIS activates sequential steps during transcription initiation at a stable RNA promoter.

FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.

Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters.

ArcA protein bearing an amino-terminal, oligohistidine extension has been purified, and its DNA binding activity has been characterized with or without prior incubation with carbamoyl phosphate. Electrophoretic mobility shift assays and DNase I protection assays indicate that where the phosphorylated form of the ArcA protein (ArcA-P) is expected to act as a transcriptional repressor (e.g., of lctPRD and gltA-sdhCDAB), the effect is likely to be mediated by sequestration of cis-controlling transcriptional regulatory elements. In contrast, in the case of cydAB, for which ArcA-P is expected to function as a transcriptional activator, two discrete binding sites have been identified upstream of a known promoter, and activation from these sites is likely to be mediated by a mechanism typical of the type I class of prokaryotic transcriptional activators. An additional ArcA-P binding site has also been located downstream of the known promoter, and a distinct role for this site in the regulation of the cydAB operon during anoxic growth transitions is suggested. These results are discussed within the framework of an overall model of signaling by the Arc two-component signal transduction system in response to changes in aerobiosis.

Purification and characterization of the Proteus vulgaris BlaA protein, the activator of the beta-lactamase gene.

Induction of the expression of the beta-lactamase gene, blaB, is regulated by the blaA gene located just upstream of blaB in the opposite direction in Proteus vulgaris. The expression of the blaA gene is negatively autoregulated by its own product BlaA, the activator of the blaB gene. The P. vulgaris BlaA protein shares high amino acid homology with the LysR family members, which are prokaryotic transcriptional activators that possess a putative helix-turn-helix DNA binding motif. To characterize its function, we purified the BlaA protein to homogeneity from Escherichia coli carrying the expression plasmid of the blaA gene driven by the tac promoter. The gel shift assay and DNaseI footprinting showed that purified BlaA specifically bound to the blaA promoter region, which resides immediately upstream of that of blaB. The binding region contained an inverted repeat, including a T-N11-T sequence which is similar to the LysR motif (T-N11-A) that is conserved in some LysR family members [Goethals et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1646-1650]. We also showed that the BlaA protein forms a dimer in solution, using glycerol gradient centrifugation and glutaraldehyde crosslinking.

A small C-terminal region of the Escherichia coli MalT protein contains the DNA-binding domain.

MalT, the transcriptional activator of the Escherichia coli maltose regulon, is a 901-amino acid-long protein that specifically binds to short, asymmetric nucleotide sequences present in several copies in the promoters of the regulon. We report that the protein structure involved in this specific binding is carried by a small C-terminal part of MalT encompassing the last 95 amino acid residues. This was demonstrated by fusing the last 95 codons of malT to the gene that encodes glutathione S-transferase, purifying the hybrid protein by affinity chromatography, and comparing the DNase I and dimethyl sulfate footprints of the hybrid and of wild-type MalT on different MalT-binding sites. MalT belongs to a large family of prokaryotic transcriptional activators, which share significant homology in their approximately 60-amino acid C-terminal regions. Our result strongly supports the suggestion that the region of homology corresponds to the DNA-binding domain of the proteins in this family.

Identification of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH.

Expression of the lysine decarboxylase gene (cadA) of Escherichia coli is induced upon external acidification. To dissect the molecular mechanisms responsible for this regulation, we analyzed a 4.2-kbp region upstream from cadA. DNA sequencing revealed two long open reading frames upstream of and on the same strand as cadA. One of these, cadB, is 444 codons long and is situated immediately upstream of cadA. Transcriptional fusions between fragments upstream of cadA and lacZ, Northern (RNA) hybridization, primer extension, and site-directed mutagenesis experiments defined a promoter, Pcad, upstream of cadB that was responsible for pH-regulated expression of cadA. Upstream of Pcad is an open reading frame, cadC, consisting of 512 codons. The predicted amino terminal region of the cadC gene product (CadC) resembles the carboxy-terminal domain of prokaryotic transcriptional activators involved in environmental sensing. Tn10 insertions within or immediately upstream of cadC abolished Pcad activity, suggesting that cadC encodes a positive transcription factor. Expression of plasmid-borne cadC in the Tn10 mutants restored Pcad activity, while introduction of a plasmid expressing truncated CadC resulted in the inability to complement. The presence of Pcad on a multicopy plasmid was found to lower expression arising from chromosomal Pcad, suggesting that a positive-acting factor is limiting. Our data suggests that cadA, cadB, and the acid-inducible Pcad comprise, at least in part, the cad operon which is under control of the cadC product.

Purification and properties of the bacteriophage P2 ogr gene product. A prokaryotic zinc-binding transcriptional activator.

The bacteriophage P2 ogr gene product, a 72-residue basic protein rich in cysteine and histidine, is a positive regulatory factor for phage late gene transcription in both P2 and satellite phage P4. We have developed a simple purification procedure for Ogr protein synthesized from an overproducing plasmid. Inclusion bodies formed upon overproduction were denatured using 8 M urea, and the overproduced protein was purified by gel filtration. The purified Ogr was allowed to refold under optimized conditions and was subsequently shown to be able to transactivate the phage P4 late promoter Psid in an in vitro coupled transcription-translation system. Using a 65Zn blotting method and absorption spectroscopy, we show that Ogr is a zinc-binding protein and that the conserved cysteine residues are involved in forming a complex with Zn(II). The purification procedure described allows Ogr to be obtained in both high purity and yield.

Characterization of a new prokaryotic transcriptional activator and its DNA recognition site

The expression of the Bacillus subtilis phage φ29 DNA is controlled by the viral gene 4 product, which is required for the initiation of transcription at the unique late promoter A3. Protein p4 binds specifically to a φ29 DNA fragment containing the A3 promoter. DNase I footprinting analysis has shown that the DNA binding region for protein p4 is located between nucleotides −50 and −100 relative to the transcription start site. Methylation interference assays suggest that two eight base-pair long inverted repeats located within this binding region are the protein p4 recognition sequence. These results, together with the fact that the protein p4-dependent in vitro transcription requires the B. subtilis σ 43 -RNA polymerase, indicate that protein p4 is a transcriptional activator. The protein p4 DNA recognition region is statically bent as suggested by gel retardation and chemical cleavage assays. A model of protein p4 binding to its DNA target site is proposed.