Global Transcription Factor Research Articles

Role of the Synechococcus PCC 7942 nitrogen regulator protein PipX in NtcA-controlled processes

The Synechococcus sp. PCC 7942 nitrogen regulator PipX interacts in a 2-oxoglutarate-dependent manner with the global nitrogen transcription factor NtcA and the signal transduction protein P(II). In vivo, PipX is involved in the NtcA-dependent induction of glnB and glnN genes. To further investigate the extent to which PipX is involved in global nitrogen control, the effect of pipX inactivation on various nitrogen-regulated processes was determined. The PipX-deficient mutant was able to use nitrate as a nitrogen source and to efficiently inhibit the nitrate transport upon ammonium addition but showed decreased nitrate and nitrite reductase activities and a delay in the induction of nitrate utilization after transfer of cultures from ammonium- to nitrate-containing media. In contrast to the wild-type, glutamine synthetase activity was not upregulated upon depletion of combined nitrogen from cultures of the mutant strain. Inactivation of pipX impaired induction of nblA and delayed phycobilisome degradation, but did not affect recovery of nitrogen-deprived cultures. Taken together, the results indicate that PipX interacts with NtcA to facilitate efficient acclimation of cyanobacteria to conditions of nitrogen limitation.

LexA Represses CTXΦ Transcription by Blocking Access of the α C-terminal Domain of RNA Polymerase to Promoter DNA

CTXPhi is a Vibrio cholerae-specific temperate filamentous phage that encodes cholera toxin. CTXPhi lysogens can be induced with DNA damage-inducing agents such as UV light, leading to the release of CTXPhi virions and the rapid dissemination of cholera toxin genes to new V. cholerae hosts. This environmental regulation is directly mediated by LexA, the host-encoded global SOS transcription factor. LexA and a phage-encoded repressor, RstR, both repress transcription from P(rstA), the primary CTXPhi promoter. Because the LexA binding site is located upstream of the core P(rstA) promoter and overlaps with A-tract sequences, we speculated that LexA represses P(rstA) by occluding a promoter UP element, a binding site for the C-terminal domain of the alpha subunit of RNA polymerase (RNAP) (alphaCTD). Using in vitro transcription assays, we have shown that the LexA binding site stimulates maximal rstA transcription in the absence of any added factors. The alphaCTD of RNAP is required for this stimulation, demonstrating that the LexA site contains, or overlaps with, a promoter UP element. LexA represses rstA transcription by normal RNAP but fails to repress rstA transcription catalyzed by RNAP lacking the alphaCTD. DNase I footprint analysis mapped the alphaCTD binding site to the upstream promoter region that includes the LexA binding site. The addition of free alpha subunits blocked the binding of LexA to rstA promoter DNA, indicating that LexA and the alphaCTD directly compete for binding to their respective sites. To our knowledge, this is the first report of a repressor blocking transcription initiation by occluding a promoter UP element.

Regulation of FNR Dimerization by Subunit Charge Repulsion

Dimerization of the global anaerobic transcription factor FNR is essential for FNR activity. Under aerobic conditions FNR is an inactive monomeric species because it lacks the oxygen labile [4Fe-4S] cluster required for dimerization. In this study, we investigated the protein side chains that inhibit FNR dimerization under aerobic conditions. Substitution of Asp(154) within the predicted dimerization helix with residues containing neutral or positively charged side chains increased FNR activity under aerobic conditions, whereas replacement of Asp(154) with Glu inhibited FNR activity similar to WT-FNR. Similar results were obtained when making analogous substitutions of Glu(150). In vitro analysis of representative FNR mutant proteins indicated that their increased activity under aerobic conditions resulted from an [4Fe-4S] independent mechanism of dimerization. In addition, simultaneous substitution of residues 150 and 154 with Lys restored inhibition of FNR activity under aerobic growth conditions. Collectively, these data indicate that charge repulsion by side chains at positions 150 and 154 is necessary to inhibit dimerization under aerobic conditions. They also suggest that a [4Fe-4S]-dependent conformational change overcomes charge repulsion between subunits under anaerobic conditions. Comparison of the trypsin sensitivity of [4Fe-4S]-FNR and apoFNR indicated that there are no major differences in protease sensitivity between these forms, whereas circular dichroism suggested that small changes in secondary structure occur between the cluster-containing FNR and apoFNR. Thus, the [4Fe-4S]-dependent conformational change necessary to overcome inter-subunit charge repulsion and create a subunit interface more favorable for dimerization must be small.

RovA, a global regulator of Yersinia pestis , specifically required for bubonic plague

The pathogenic species of Yersinia contain the transcriptional regulator RovA. In Yersinia pseudotuberculosis and Yersinia enterocolitica, RovA regulates expression of the invasion factor invasin (inv), which mediates translocation across the intestinal epithelium. A Y. enterocolitica rovA mutant has a significant decrease in virulence by LD(50) analysis and an altered rate of dissemination compared with either wild type or an inv mutant, suggesting that RovA regulates multiple virulence factors. Here, we show the involvement of RovA in the virulence of Yersinia pestis, which naturally lacks a functional inv gene. A Y. pestis DeltarovA mutant is attenuated approximately 80-fold by LD(50) and is defective in dissemination/colonization of spleens and lungs after s.c. inoculation. However, the DeltarovA mutant is only slightly attenuated when given via an intranasal or i.p. route, indicating a more important role for RovA in bubonic plague than pneumonic plague or systemic infection. Microarray analysis was used to define the RovA regulon. The psa locus was among the most highly down-regulated loci in the DeltarovA mutant. A DeltapsaA mutant had a significant dissemination defect after s.c. infection but only slight attenuation by the pneumonic-disease model, closely mimicking the virulence defect seen with the DeltarovA mutant. DNA-binding studies revealed that RovA specifically interacts with the psaE and psaA promoter regions, indicating a direct role for RovA in regulating this locus. Thus, RovA appears to be a global transcription factor in Y. pestis and, through its regulatory influence on genes such as psaEFABC, contributes to the virulence of Y. pestis.

Transcriptional regulation of the fad regulon genes of Escherichia coli by ArcA

ArcA is a global transcription factor required for optimal growth of Escherichia coli during anaerobic growth. In this study, the role of ArcA on the transcriptional regulatory subnetwork of the fad regulon was investigated. Gene expression profiles of deletion mutants (Delta arcA, Delta fadR and Delta arcA/Delta fadR) indicated that (i) ArcA is a major transcription factor for the transcriptional regulation of fatty acid metabolism in the absence of oxygen, and (ii) ArcA and FadR cooperatively regulate the fad regulon under anaerobic conditions. To determine the direct interaction between ArcA and the promoters of the fad regulon genes, chromatin immunoprecipitation (ChIP) analysis was performed. ChIP analysis suggested that ArcA directly binds to the promoter regions of the fad regulon genes in vivo. An ArcA-binding motif was identified from known binding sequences and predicted putative binding sites in the promoter regions of the fad regulon genes. These results indicate that ArcA directly represses the expression of fad regulon genes during anaerobic growth.

Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2‐oxoglutarate dependent manner with PII and NtcA

Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. The global nitrogen transcription factor NtcA and the signal transduction protein PII are both involved in 2-oxoglutarate sensing. PII proteins, probably the most conserved signal transduction proteins in nature, are remarkable for their ability to interact with very diverse protein targets in different systems. Despite widespread efforts to understand nitrogen signalling in cyanobacteria, the involvement of PII in the regulation of transcription activation by NtcA remains enigmatic. Here we show that PipX, a protein only present in cyanobacteria, interacts with both PII and NtcA and provides a mechanistic link between these two factors. A variety of in vivo and in vitro approaches were used to study PipX and its interactions with PII and NtcA. 2-Oxoglutarate favours complex formation between PipX and NtcA, but impairs binding to PII, suggesting that partner swapping between these nitrogen regulators is driven by the 2-oxoglutarate concentration. PipX is required for NtcA-dependent transcriptional activation in vivo, thus implying that PipX may function as a prokaryotic transcriptional coactivator.

Regulatory elements in aflatoxin biosynthesis

This review provides a synopsis of factors involved in the regulation of aflatoxin inAspergillus species at the molecular level. Much of the knowledge available today on the regulation of secondary metabolite production in fungi has been gleaned from studies of the aflatoxin gene cluster inA. flavus andA. parasiticus and the sterigmatocystin gene cluster inA. nidulans. Regulation of these two gene clusters is under the control of both pathway-specific transcription factors such as AflR and AflJ and global or broad-domain transcription factors such as AreA and PacC. Study of secondary metabolite (sec-) mutants inA. parasiticus first identified an association between mycotoxin production and fungal development. This linkage has been extended at the molecular level by the characterization of a G-protein/cAMP/Protein kinase A signaling pathway that regulates sporulation via the transcription factor BrlA and aflatoxin/sterigmatocystin production via AflR. Another global regulator of mycotoxin production, VeA, mediates a developmental light-response inA. nidulans andA. flavus. Though not similar to any known fungal transcriptional regulators, VeA controls aflatoxin/sterigmatocystin production via transcriptional control of AflR and it also regulates development of sexual structures such as cleistothecia inA. nidulans and sclerotia inA. flavus.

Promoter Shuffling at a Nuclear Gene for Mitochondrial RPL27. Involvement of Interchromosome and Subsequent Intrachromosome Recombinations

The Reclinomonas americana mitochondrial genome contains a mitochondrial ribosomal protein L27 (rpl27) gene, whereas the rpl27 gene is absent from all plant mitochondrial genomes examined to date. This suggests that plant mitochondrial rpl27 genes have been transferred previously from the mitochondrial genome to the nuclear genome. A nuclear cDNA encoding mitochondrial RPL27 was identified in rice (Oryza sativa). Three similar sequences were identified: rpl27-1 and rpl27-2 on chromosome 8 and rpl27-3 on chromosome 4. Harr plot analysis suggests that they were generated by inter- and intrachromosomal duplications. Interestingly, the transcribed rpl27 gene (rpl27-1) acquired a promoter sequence that was derived from the rice spt16 (Osspt16) gene, the homolog of a global transcription factor in yeast (Saccharomyces cerevisiae) located downstream from the rpl27-3 sequence on chromosome 4, after inter- and intrachromosomal recombination. Reverse transcription-PCR and promoter assay revealed that the rpl27 mRNAs were mainly transcribed from rpl27-1. A repeat of seven nucleotides (AATAGTT) was identified at the junction of rpl27-1 and rpl27-2 on chromosome 8, and the same repeat was also identified at the 5' end of rpl27-2 and the 3' end of rpl27-1. This repeat (AATAGTT) contains the hot-spot sequence AGTT, which is preferentially recognized by topoisomerase I in wheat (Triticum aestivum) germ, suggesting the involvement of topoisomerase I in this recombination. We here report the example of promoter shuffling and show that this promoter shuffling resulted from a recent segmental duplication through inter- and intrachromosomal recombination events.

Genome analysis of Escherichia coli promoter sequences evidences that DNA static curvature plays a more important role in gene transcription than has previously been anticipated

We have performed a computer analysis to study the prevalence of DNA static curvature in the regulatory regions of Escherichia coli, detecting a large number of operons with curved DNA fragments in their 5′ upstream regions. A statistical analysis reveals that all the global transcription factors identified so far in E. coli have a tendency to regulate operons with curved DNA sequences in their upstream regions. In addition to these global regulators, we also found that the PurR, ArgR, FruR, TyrR, and CytR specific regulators present a similar propensity. Interestingly, for these cases we found no previous reference describing a possible relationship with curved DNA regions. To validate our theoretical results, we performed site-directed mutagenesis to reduce the degree of DNA curvature in the regulatory sequences of the aroG, pyrC, and argCBH operons. The effects of these changes were measured by polyacrylamide gel electrophoresis assays and further evaluated in vivo by transcriptional fusions to a reporter gene. All our results point toward a more widespread role of curved DNA in gene transcription, a fact that has previously been underestimated.

PCR-Based Tandem Epitope Tagging System for Escherichia Coli Genome Engineering

Biological discovery in the postgenomic era requires a systematic and high-throughput experimental approach. To this end, a versatile PCR-based tandem epitope tagging system is described, which inserts a tandem epitope coding sequence into any desired position of the Escherichia coli chromosome. Template plasmids were constructed that carry tandem copies of the epitope encoding sequence, Flp recombinase target (FRT) sites, and antibiotic resistance genes. The linear DNA fragment, amplified from the template plasmid with extensions homologous to the end of the target gene and part of its downstream region, was transformed into E. coli K-12 MG1655 harboring the bacteriophage gamma Red recombination system. The antibiotic resistance gene was then removed from the inserted heterologous PCR fragment using Flp recombinase. This epitope tagging system was applied to global transcription factors of E. coli to obtain proteins fused with tandem c-myc epitope tags. The tandem myc epitope-fused transcription factors were successfully detected by Western blot analysis and chromatin immunoprecipitation with increased detection sensitivity and higher yield. Higher copy numbers of the epitope molecule allowed the use of more stringent experimental conditions to increase the signal-to-noise ratio in subsequent experimental applications. Furthermore, judging from the measurement of gene expression using reverse transcription PCR (RT-PCR), the epitope-fused transcription factors retained their normal function for gene regulation in vivo.

Analysis of a non-canonical NtcA-dependent promoter in Synechococcus elongatus and its regulation by NtcA and PII

In this communication, we present a genetic analysis of the glnN promoter region of Synechococcus elongatus PCC 7942. luxAB reporter fusions were used to characterize the glnN promoter by deletion and site-directed mutational analysis. Reporter gene expression analysis was performed in S. elongatus wild-type and mutant strains to reveal the role of the global nitrogen responsive transcription factor NtcA and of the P(II) signalling protein on regulation of glnN gene expression. A non-canonical NtcA-binding motif is responsible for NtcA-dependent, nitrogen-responsive regulation of glnN. The P(II) signalling protein has opposing effects on NtcA-dependent glnN expression. Under conditions of nitrate-growth, it depresses expression, whereas under conditions of combined nitrogen starvation, it is required for full induction. Furthermore, sequences upstream of the NtcA-binding site have repressive effect on glnN promoter activity.

Stationary-Phase Regulation of RpoS Translation in Escherichia coli

In enteric bacteria, adaptation to a number of different stresses is mediated by the RpoS protein, one of several sigma factors that collectively allow a tailored transcriptional response to environmental cues. Stress stimuli including low temperature, osmotic shock, nutrient limitation, and growth to stationary phase (SP) all result in a substantial increase in RpoS abundance and activity. The mechanism of regulation depends on the specific signal but may occur at the level of transcription, translation, protein activity, or targeted proteolysis. In both Escherichia coli and Salmonella enterica, SP induction of RpoS in rich medium is >30 fold and includes effects on both transcription and translation. Recently, we found that SP control of rpoS transcription in S. enterica involves repression of the major rpoS promoter during exponential phase by the global transcription factor Fis. Working primarily with E. coli, we now show that 24 nucleotides of the rpoS ribosome-binding site (RBS) are necessary and sufficient for a large part of the increase in rpoS translation as cells grow to SP. Genetic evidence points to an essential role for the leader nucleotides just upstream of the Shine-Dalgarno sequence but is conflicted on the question of whether sequence or structure is important. SP regulation of rpoS is conserved between E. coli and S. enterica. When combined with an fis mutation to block transcriptional effects, replacement of the rpoS RBS sequence by the lacZ RBS eliminates nearly all SP induction of RpoS.

DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus

The level of DnaA, a key bacterial DNA replication initiation factor, increases during the Caulobacter swarmer-to-stalked transition just before the G1/S transition. We show that DnaA coordinates DNA replication initiation with cell cycle progression by acting as a global transcription factor. Using DnaA depletion and induction in synchronized cell populations, we have analysed global transcription patterns to identify the differential regulation of normally co-expressed genes. The DnaA regulon includes genes encoding several replisome components, the GcrA global cell cycle regulator, the PodJ polar localization protein, the FtsZ cell division protein, and nucleotide biosynthesis enzymes. In cells depleted of DnaA, the G1/S transition is temporally separated from the swarmer-to-stalked cell differentiation, which is normally coincident. In the absence of DnaA, the CtrA master regulator is cleared by proteolysis during the swarmer-to-stalked cell transition as usual, but DNA replication initiation is blocked. In this case, expression of gcrA, which is directly repressed by CtrA, does not increase in conjunction with the disappearance of CtrA until DnaA is subsequently induced, showing that gcrA expression requires DnaA. DnaA boxes are present upstream of many genes whose expression requires DnaA, and His6-DnaA binds to the promoters of gcrA, ftsZ and podJ in vitro. This redundant control of gcrA transcription by DnaA (activation) and CtrA (repression) forms a robust switch controlling the decision to proceed through the cell cycle or to remain in the G1 stage.

Systematic search for the Cra‐binding promoters using genomic SELEX system

Cra (or FruR), a global transcription factor with both repression and activation activities, controls a large number of the genes for glycolysis and gluconeogenesis. To get insights into the entire network of transcription regulation of the E. coli genome by Cra, we isolated a set of Cra-binding sequences using an improved method of genomic SELEX. From the DNA sequences of 97 independently isolated DNA fragments by SELEX, the Cra-binding sequences were identified in a total of ten regions on the E. coli genome, including promoters of six known genes and four hitherto-unidentified genes. All six known promoters are repressed by Cra, but none of the activation-type promoters were cloned after two cyles of SELEX, because the Cra-binding affinity to the repression-type promoters is higher than the activation-type promoters, as determined by the quantitative gel shift assay. Of a total of four newly identified Cra-binding sequences, two are associated with promoter regions of the gapA (glyceraldehyde 3-phosphate dehydrogenase) and eno (enolase) genes, both involved in sugar metabolism. The regulation of newly identified genes by Cra was confirmed by the in vivo promoter strength assay using a newly developed TFP (two-fluorescent protein) vector for promoter assay or by in vitro transcription assay in the presence of Cra protein.

HetR-Dependent and -Independent Expression of Heterocyst-Related Genes in an Anabaena Strain Overproducing the NtcA Transcription Factor

Heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of ntcA and hetR being dependent on each other. In this study we constructed strains that constitutively express the ntcA gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the effects of such NtcA levels on heterocyst differentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as devBCA or the cox2 operon, and NtcA-dependent excision of the 11-kb nifD-intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA overproducing strain could not grow diazotrophically. Overexpression of ntcA in a hetR background promoted expression of devBCA in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR.

Combined Global Localization Analysis and Transcriptome Data Identify Genes That Are Directly Coregulated by Adr1 and Cat8

In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.

Bioenergetic remodeling of heart mitochondria by thyroid hormone

Changes in thyroid status are associated with profound alterations in biochemical and physiological functioning of cardiac muscle impacting metabolic rate, contractility and structural hypertrophy. Using an in vivo model of chronic treatment with thyroid hormone (T4, 0.3 mg/kg/day), we evaluated how mitochondria are regulated in response to T4, and assessed the relationship of T4-induced mitochondrial biogenesis and bioenergetics to overall cardiac hypertrophy. The role of thyroid hormone in cardiac bioenergetic remodeling was addressed in rats treated with T4 for 5, 10 and 15 days. Over that time, myocardial oxygen consumption substantially increased as did cardiac hypertrophy. Myocardial levels of mitochondrial enzyme activities, mitochondrial DNA (mtDNA), specific proteins and transcript were assessed. Activity levels of respiratory complexes I-V and citrate synthase significantly increased with 15 but not with 5 or 10-day T4 treatment. Myocardial levels of mtDNA, mitochondrial proteins (e.g. cytochrome c, cytochrome b, ATPase subunits, MnSOD) and the global transcription factor PPARalpha were significantly elevated with 15-day T4. Transcript analysis revealed increased expression of transcription factors and cofactors involved in mitochondrial biogenesis including PPARalpha, mtTFA, ErbAalpha and PGC-1alpha. Our findings indicate parallel increases in myocardial mitochondrial bioenergetic capacity, oxygen consumption and markers of mitochondrial biogenesis with 15-day T4; these changes were not present with 10-day T4 even with significant cardiac hypertrophy. The marked, parallel increases in PPARalpha levels suggest its potential involvement in mediating myocardial-specific remodeling of mitochondria in response to T4.

Regulation of Escherichia coli hemolysin E expression by H-NS and Salmonella SlyA.

The Escherichia coli hlyE gene (also known as clyA or sheA) codes for a novel pore-forming toxin. Previous work has shown that the global transcription factors FNR and CRP positively regulate hlyE expression by binding at the same site. Here in vivo transcription studies reveal that FNR occupies the hlyE promoter more frequently than CRP, providing a mechanism for the moderate upregulation of hlyE expression in response to two distinct environmental signals (oxygen and glucose starvation). It has been reported that H-NS interacts with two large regions of the hlyE promoter (PhlyE), one upstream of the -35 element and one downstream of the -10 element. Here we identify two high-affinity H-NS sites, H-NS I, located at the 3' end of the extended upstream footprint, and H-NS II, located at the 5' end of the extended downstream footprint. It is suggested that these high-affinity sites initiate the progressive formation of higher order complexes, allowing a range of H-NS-mediated regulatory effects at PhlyE. Finally, the identification of a SlyA binding site that overlaps the H-NS I site in PhlyE suggests a mechanism to explain how SlyA overproduction enhances hlyE expression by antagonizing the negative effects of H-NS.

Open reading frame all0601 from Anabaena sp. strain PCC 7120 represents a novel gene, cnaT, required for expression of the nitrate assimilation nir operon.

Expression of the nitrate assimilation nir operon in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 requires the action of both the global nitrogen control transcription factor NtcA and the pathway-specific transcriptional regulator NtcB. In the genome of this cyanobacterium, the ntcB gene is found in a cluster of genes located in the complementary strand, upstream from the nir operon. Just downstream of ntcB, there is an open reading frame, all0601 (previously designated orf356 and now designated the cnaT gene), that putatively encodes a protein similar to proteins with glycosyl transferase activity and that is also present clustered together with ntcB homologues or nitrate assimilation structural genes in other cyanobacterial genomes. An insertional mutant of cnaT was generated and found to be unable to assimilate nitrate, although it could use ammonium or dinitrogen as a source of nitrogen for growth. In the mutant, under derepression conditions, nir operon mRNA (as determined by RNA-DNA hybridization and primer extension analysis) and enzymes of the nitrate reduction system (i.e., nitrate reductase and nitrite reductase) were expressed at low or undetectable levels. Inactivation of cnaT did not impair expression of ntcB, and expression of cnaT itself was constitutive and regulated by neither NtcA nor NtcB. Regulation of expression of the nir operon in Anabaena sp. strain PCC 7120 by CnaT and the previously described regulatory elements, NtcA and NtcB, is discussed.

Transcriptome Profiling and Deciphering the Regulatory Role of Global Transcription Factor fadR

Transcriptome Profiling and Deciphering the Regulatory Role of Global Transcription Factor fadR