scholarly journals Overlapping Repressor Binding Sites Result in Additive Regulation of Escherichia coli FadH by FadR and ArcA

2010 ◽  
Vol 192 (17) ◽  
pp. 4289-4299 ◽  
Author(s):  
Youjun Feng ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Cyclic Amp ◽  
Unsaturated Fatty Acids ◽  
Genetic Regulation ◽  
Receptor Protein ◽  
Anaerobic Conditions ◽  
Mobility Shift ◽  
Long Chain

ABSTRACT Escherichia coli fadH encodes a 2,4-dienoyl reductase that plays an auxiliary role in β-oxidation of certain unsaturated fatty acids. In the 2 decades since its discovery, FadH biochemistry has been studied extensively. However, the genetic regulation of FadH has been explored only partially. Here we report mapping of the fadH promoter and document its complex regulation by three independent regulators, the fatty acid degradation FadR repressor, the oxygen-responsive ArcA-ArcB two-component system, and the cyclic AMP receptor protein-cyclic AMP (CRP-cAMP) complex. Electrophoretic mobility shift assays demonstrated that FadR binds to the fadH promoter region and that this binding can be specifically reversed by long-chain acyl-coenzyme A (CoA) thioesters. In vivo data combining transcriptional lacZ fusion and real-time quantitative PCR (qPCR) analyses indicated that fadH is strongly repressed by FadR, in agreement with induction of fadH by long-chain fatty acids. Inactivation of arcA increased fadH transcription by >3-fold under anaerobic conditions. Moreover, fadH expression was increased 8- to 10-fold under anaerobic conditions upon deletion of both the fadR and the arcA gene, indicating that anaerobic expression is additively repressed by FadR and ArcA-ArcB. Unlike fadM, a newly reported member of the E. coli fad regulon that encodes another auxiliary β-oxidation enzyme, fadH was activated by the CRP-cAMP complex in a manner similar to those of the prototypical fad genes. In the absence of the CRP-cAMP complex, repression of fadH expression by both FadR and ArcA-ArcB was very weak, suggesting a possible interplay with other DNA binding proteins.

scholarly journals A New Member of the Escherichia coli fad Regulon: Transcriptional Regulation of fadM (ybaW)

2009 ◽  
Vol 191 (20) ◽  
pp. 6320-6328 ◽  
Author(s):  
Youjun Feng ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Unsaturated Fatty Acids ◽  
Negative Regulator ◽  
Receptor Protein ◽  
Conjugated Linoleic Acid Isomer ◽  
Linoleic Acid Isomer

ABSTRACT Recently, Nie and coworkers (L. Nie, Y. Ren, A. Janakiraman, S. Smith, and H. Schulz, Biochemistry 47:9618-9626, 2008) reported a new Escherichia coli thioesterase encoded by the ybaW gene that cleaves the thioester bonds of inhibitory acyl-coenzyme A (CoA) by-products generated during β-oxidation of certain unsaturated fatty acids. These authors suggested that ybaW expression might be regulated by FadR, the repressor of the fad (fatty acid degradation) regulon. We report mapping of the ybaW promoter and show that ybaW transcription responded to FadR in vivo. Moreover, purified FadR bound to a DNA sequence similar to the canonical FadR binding site located upstream of the ybaW coding sequence and was released from the promoter upon the addition of long-chain acyl-CoA thioesters. We therefore propose the designation fadM in place of ybaW. Although FadR regulation of fadM expression had the pattern typical of fad regulon genes, its modulation by the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) global regulator was the opposite of that normally observed. CRP-cAMP generally acts as an activator of fad gene expression, consistent with the low status of fatty acids as carbon sources. However, glucose growth stimulated fadM expression relative to acetate growth, as did inactivation of CRP-cAMP, indicating that the complex acts as a negative regulator of this gene. The stimulation of fadM expression seen upon deletion of the gene encoding adenylate cyclase (Δcya) was reversed by supplementation of the growth medium with cAMP. Nie and coworkers also reported that growth on a conjugated linoleic acid isomer yields much higher levels of FadM thioesterase activity than does growth on oleic acid. In contrast, we found that the conjugated linoleic acid isomer was only a weak inducer of fadM expression. Although the gene is not essential for growth, the high basal level of fadM expression under diverse growth conditions suggests that the encoded thioesterase has functions in addition to β-oxidation.

scholarly journals Alterations in the binding site of the cyclic AMP receptor protein at the Escherichia coli galactose operon regulatory region

1988 ◽  
Vol 253 (3) ◽  
pp. 809-818 ◽  
Author(s):  
K Gaston ◽  
B Chan ◽  
A Kolb ◽  
J Fox ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Binding Assays ◽  
Cyclic Amp Receptor Protein ◽  
Stimulation Of

Gene manipulation techniques have been used to alter the binding site for the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) at the regulatory region of the Escherichia coli galactose (gal) operon. The effects of these changes on CRP-dependent stimulation of expression from the galP1 promoter in vivo have been measured, and gel binding assays have been used to measure the affinity of cAMP-CRP for the modified sites. Firstly we have deleted progressively longer sequences from upstream of the gal CRP site in order to locate the functional limit of the site. A deletion to -49, removing the first base that corresponds to the consensus sequence for a CRP binding site, is sufficient to reduce CRP binding and block CRP-dependent stimulation of P1. Secondly, we used synthetic oligonucleotides to invert the asymmetric nucleotide sequence at the gal CRP binding site or to make the sequence symmetric. Inversion of the site has little effect on CRP binding, the architecture of open complexes at P1 revealed by DNAase I footprinting, or the stimulation of transcription from P1. Making the site symmetric increases the affinity for CRP by over 50-fold and leads to increased transcription from P1, whilst hardly altering the DNAase I footprint of open complexes. Our results confirm that the strength of binding of CRP depends on the nature of the site and show that it is this that principally accounts for differences in CRP-dependent stimulation of transcription.

scholarly journals Binding of the Escherichia coli cyclic AMP receptor protein to DNA fragments containing consensus nucleotide sequences

1989 ◽  
Vol 261 (2) ◽  
pp. 649-653 ◽  
Author(s):  
K Gaston ◽  
A Kolb ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Inverse Correlation ◽  
Nucleotide Sequences ◽  
Receptor Protein ◽  
Dna Fragments ◽  
Naturally Occurring ◽  
The Stability

Binding of the Escherichia coli CRP protein to DNA fragments carrying nucleotide sequences closely corresponding to the consensus is very tight with a dissociation time of over 2 h in our conditions. The concentration of cyclic AMP required for this binding is below the physiological range of intracellular cyclic AMP concentrations. Changes in nucleotide sequence at positions that are not well-conserved between different naturally-occurring CRP sites allow a more rapid dissociation of CRP-DNA complexes. There is an inverse correlation between the stability of CRP binding to sites in vitro and the repression by glucose of expression dependent on these sites in vivo: expression that is dependent on the tighter binding sites cannot be repressed by the inclusion of glucose in the growth medium.

scholarly journals Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

2005 ◽  
Vol 187 (19) ◽  
pp. 6708-6718 ◽  
Author(s):  
Jason R. Wickstrum ◽  
Thomas J. Santangelo ◽  
Susan M. Egan
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
In Vitro Transcription ◽  
Dnase I ◽  
Receptor Protein ◽  
Transcription Activator ◽  
Transcription Start ◽  
Dnase I Footprinting

ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position −111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

scholarly journals The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the traJ Promoter of the Escherichia coli Conjugative Plasmid pRK100

2003 ◽  
Vol 185 (5) ◽  
pp. 1616-1623 ◽  
Author(s):  
Marjanca Starčič ◽  
Darja Žgur-Bertok ◽  
Bart J. A. M. Jordi ◽  
Marc M. S. M. Wösten ◽  
Wim Gaastra ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Promoter Region ◽  
Targeted Mutagenesis ◽  
Conjugal Transfer ◽  
Conjugative Plasmid ◽  
Receptor Protein ◽  
Wild Type ◽  
Mobility Shift

ABSTRACT The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system. A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100. The defect in the cya mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region. DNase I footprint experiments mapped the CRP binding site around position −67.5 upstream of the putative traJ promoter. Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100.

scholarly journals Expression and Regulation of a Silent Operon, hyf, Coding for Hydrogenase 4 Isoenzyme in Escherichia coli

2004 ◽  
Vol 186 (2) ◽  
pp. 580-587 ◽  
Author(s):  
William T. Self ◽  
Adnan Hasona ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Type Strain ◽  
Wild Type Strain ◽  
Receptor Protein ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Heterologous Promoter ◽  
Cyclic Amp Receptor Protein ◽  
Hydrogenase 3

ABSTRACT On the basis of hyf-lacZ fusion studies, the hyf operon of Escherichia coli, noted for encoding the fourth hydrogenase isoenzyme (HYD4), is not expressed at a significant level in a wild-type strain. However, mutant FhlA proteins (constitutive activators of the hyc-encoded hydrogenase 3 isoenzyme) activated hyf-lacZ. HyfR, an FhlA homolog encoded by the hyfR gene present at the end of the hyf operon, also activated transcription of hyf-lacZ but did so only when hyfR was expressed from a heterologous promoter. The HYD4 isoenzyme did not substitute for HYD3 in H2 production. Optimum expression of hyf-lacZ required the presence of cyclic AMP receptor protein-cyclic AMP complex and anaerobic conditions when HyfR was the activator.

scholarly journals Functional Roles of arcA, etrA, Cyclic AMP (cAMP)-cAMP Receptor Protein, and cya in the Arsenate Respiration Pathway in Shewanella sp. Strain ANA-3

2008 ◽  
Vol 191 (3) ◽  
pp. 1035-1043 ◽  
Author(s):  
Julie N. Murphy ◽  
K. James Durbin ◽  
Chad W. Saltikov
Keyword(s):  
Cyclic Amp ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Receptor Protein ◽  
Binding Motif ◽  
Anaerobic Conditions ◽  
Mobility Shift ◽  
Terminal Electron Acceptor ◽  
Moderate Growth ◽  
Arsenate Respiration

ABSTRACT Microbial arsenate respiration can enhance arsenic release from arsenic-bearing minerals—a process that can cause arsenic contamination of water. In Shewanella sp. strain ANA-3, the arsenate respiration genes (arrAB) are induced under anaerobic conditions with arsenate and arsenite. Here we report how genes that encode anaerobic regulator (arcA and etrA [fnr homolog]) and carbon catabolite repression (crp and cya) proteins affect arsenate respiration in ANA-3. Transcription of arcA, etrA, and crp in ANA-3 was similar in cells grown on arsenate and cells grown under aerobic conditions. ANA-3 strains lacking arcA and etrA showed minor to moderate growth defects, respectively, with arsenate. However, crp was essential for growth on arsenate. In contrast to the wild-type strain, arrA was not induced in the crp mutant in cultures shifted from aerobic to anaerobic conditions containing arsenate. This indicated that cyclic AMP (cAMP)-cyclic AMP receptor (CRP) activates arr operon transcription. Computation analysis for genome-wide CRP binding motifs identified a putative binding motif within the arr promoter region. This was verified by electrophoretic mobility shift assays with cAMP-CRP and several DNA probes. Lastly, four putative adenylate cyclase (cya) genes were identified in the genome. One particular cya-like gene was differentially expressed under aerobic versus arsenate respiration conditions. Moreover, a double mutant lacking two of the cya-like genes could not grow with arsenate as a terminal electron acceptor; exogenous cAMP could complement growth of the double cya mutant. It is concluded that the components of the carbon catabolite repression system are essential to regulating arsenate respiratory reduction in Shewanella sp. strain ANA-3.

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