Genome-Wide Identification of In Vivo Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum

ABSTRACT The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a >15,000-fold loss of virulence after subcutaneous infection but a <40-fold increase in the 50% lethal dose by intravenous inoculation. Therefore, CRP is required for the virulence of Y. pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.

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Genome-Wide Mapping of Collier In Vivo Binding Sites Highlights Its Hierarchical Position in Different Transcription Regulatory Networks

PLoS ONE ◽

10.1371/journal.pone.0133387 ◽

2015 ◽

Vol 10 (7) ◽

pp. e0133387 ◽

Cited By ~ 7

Author(s):

Mathilde de Taffin ◽

Yannick Carrier ◽

Laurence Dubois ◽

Laetitia Bataillé ◽

Anaïs Painset ◽

...

Keyword(s):

Binding Sites ◽

Regulatory Networks ◽

In Vivo Binding ◽

Genome Wide ◽

Hierarchical Position

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

Biochemical Journal ◽

10.1042/bj2530809 ◽

1988 ◽

Vol 253 (3) ◽

pp. 809-818 ◽

Cited By ~ 10

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.

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Evidence in vivo for autogenous control of the cyclic AMP receptor protein gene (crp) in Escherichia coli by divergent RNA.

Journal of Bacteriology ◽

10.1128/jb.170.11.5076-5079.1988 ◽

1988 ◽

Vol 170 (11) ◽

pp. 5076-5079 ◽

Cited By ~ 17

Author(s):

K Okamoto ◽

S Hara ◽

R Bhasin ◽

M Freundlich

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Protein Gene ◽

Receptor Protein ◽

Cyclic Amp Receptor Protein ◽

Autogenous Control

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Genome-Wide Identification of Transcription Start Sites Yields a Novel Thermosensing RNA and New Cyclic AMP Receptor Protein-Regulated Genes in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00398-11 ◽

2011 ◽

Vol 193 (11) ◽

pp. 2871-2874 ◽

Cited By ~ 19

Author(s):

R. Raghavan ◽

A. Sage ◽

H. Ochman

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Receptor Protein ◽

Transcription Start ◽

Cyclic Amp Receptor Protein ◽

Transcription Start Sites ◽

Genome Wide

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Downregulation of the Escherichia coli guaB Promoter by Upstream-Bound Cyclic AMP Receptor Protein

Journal of Bacteriology ◽

10.1128/jb.00672-09 ◽

2009 ◽

Vol 191 (19) ◽

pp. 6094-6104 ◽

Cited By ~ 6

Author(s):

Seyyed I. Husnain ◽

Stephen J. W. Busby ◽

Mark S. Thomas

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Cyclic Amp ◽

Binding Sites ◽

Progressive Increase ◽

Receptor Protein ◽

Lower Growth ◽

Cyclic Amp Receptor Protein ◽

Rate Dependent ◽

A Site

ABSTRACT The Escherichia coli guaB promoter (P guaB ) is responsible for directing transcription of the guaB and guaA genes, which specify the biosynthesis of the nucleotide GMP. P guaB is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P guaB contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the guaB transcription start site, where it serves to downregulate P guaB . The CRP-mediated repression of P guaB activity increases in media that support lower growth rates. Inactivation of the crp or cyaA gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P guaB . Thus, GRDC of P guaB involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P guaB and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.

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Cyclic AMP Receptor Protein-Dependent Repression of Heat-Labile Enterotoxin

Infection and Immunity ◽

10.1128/iai.00928-08 ◽

2008 ◽

Vol 77 (2) ◽

pp. 791-798 ◽

Cited By ~ 30

Author(s):

Maria D. Bodero ◽

George P. Munson

Keyword(s):

Cyclic Amp ◽

Binding Sites ◽

The Other ◽

Receptor Protein ◽

Heat Stable ◽

Heat Labile ◽

Dnase I Footprinting ◽

Camp Receptor

ABSTRACT Enterotoxigenic Escherichia coli is a major cause of acute diarrheal illness worldwide and is responsible for high infant and child mortality rates in developing nations. Two types of enterotoxins, one heat labile and the other heat stable, are known to cause diarrhea. The expression of soluble heat-labile toxin is subject to catabolite (glucose) activation, and three binding sites for cAMP receptor protein (CRP or CAP) were identified upstream and within the toxin promoter by DNase I footprinting. One CRP operator is centered at −31.5, thus encompassing the promoter's −35 hexamer. Potassium permanganate footprinting revealed that the occupancy of this operator prevents RNA polymerase from forming an open complex in vitro. However, the operator centered at −31.5 is not sufficient for full repression in vivo because the deletion of the other two CRP binding sites partially relieved the CRP-dependent repression of the heat-labile toxin promoter. In contrast to heat-labile toxin, CRP positively regulates the expression of heat-stable toxin. Thus, the conditions for the optimal expression of one enterotoxin limit the expression of the other. Since glucose inhibits the activity of CRP by suppressing the pathogen's synthesis of cyclic AMP (cAMP), the concentration of glucose in the lumen of the small intestine may determine which enterotoxin is maximally expressed. In addition, our results suggest that the host may also modulate enterotoxin expression because cells intoxicated with heat-labile toxin overproduce and release cAMP.

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The AraC/XylS Family Activator RhaS Negatively Autoregulates rhaSR Expression by Preventing Cyclic AMP Receptor Protein Activation

Journal of Bacteriology ◽

10.1128/jb.00829-08 ◽

2009 ◽

Vol 192 (1) ◽

pp. 225-232 ◽

Cited By ~ 11

Author(s):

Jason R. Wickstrum ◽

Jeff M. Skredenske ◽

Vinitha Balasubramaniam ◽

Kyle Jones ◽

Susan M. Egan

Keyword(s):

Cyclic Amp ◽

Binding Site ◽

Binding Sites ◽

Protein Concentration ◽

Receptor Protein ◽

Cyclic Amp Receptor Protein ◽

Protein Activation ◽

High Protein Concentration ◽

Negative Autoregulation ◽

The Difference

ABSTRACT The Escherichia coli RhaR protein activates expression of the rhaSR operon in the presence of its effector, l-rhamnose. The resulting RhaS protein (plus l-rhamnose) activates expression of the l-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively. Here, we further investigated our previous finding that rhaS deletion resulted in a threefold increase in rhaSR promoter activity, suggesting RhaS negative autoregulation of rhaSR. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at rhaSR and that RhaS was able to bind to the RhaR binding site at rhaSR. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the rhaSR promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at rhaSR. Although RhaS and RhaR activate rhaSR transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in rhaSR transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.

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