scholarly journals Posttranscriptional Regulation of the Yersinia pestis Cyclic AMP Receptor Protein Crp and Impact on Virulence

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Wyndham W. Lathem ◽  
Jay A. Schroeder ◽  
Lauren E. Bellows ◽  
Jeremy T. Ritzert ◽  
Jovanka T. Koo ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Yersinia Pestis ◽  
Posttranscriptional Regulation ◽  
Transcriptional Regulator ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Content Type ◽  
Cyclic Amp Receptor Protein ◽  
Protein Levels ◽  
Posttranscriptional Level

ABSTRACTThe cyclic AMP receptor protein (Crp) is a transcriptional regulator that controls the expression of numerous bacterial genes, usually in response to environmental conditions and particularly by sensing the availability of carbon. In the plague pathogenYersinia pestis, Crp regulates the expression of multiple virulence factors, including components of the type III secretion system and the plasminogen activator protease Pla. The regulation of Crp itself, however, is distinctly different from that found in the well-studiedEscherichia colisystem. Here, we show that at physiological temperatures, the synthesis of Crp inY. pestisis positively regulated at the posttranscriptional level. The loss of the small RNA chaperone Hfq results in decreased Crp protein levels but not in steady-state Crp transcript levels, and this regulatory effect occurs within the 5′ untranslated region (UTR) of the Crp mRNA. The posttranscriptional activation of Crp synthesis is required for the expression ofpla, and decouplingcrpfrom Hfq through the use of an exogenously controlled promoter and 5′ UTR increases Pla protein levels as well as partially rescues the growth defect associated with the loss of Hfq. Finally, we show that both Hfq and the posttranscriptional regulation of Crp contribute to the virulence ofY. pestisduring pneumonic plague. The Hfq-dependent, posttranscriptional regulation of Crp may be specific toYersiniaspecies, and thus our data help explain the dramatic growth and virulence defects associated with the loss of Hfq inY. pestis.IMPORTANCEThe Crp protein is a major transcriptional regulator in bacteria, and its synthesis is tightly controlled to avoid inappropriate induction of the Crp regulon. In this report, we provide the first evidence of Crp regulation in an Hfq-dependent manner at the posttranscriptional level. Our discovery that the synthesis of Crp inYersinia pestisis Hfq dependent adds an additional layer of regulation to catabolite repression in this bacterium. Our work provides a mechanism by which the plague pathogen links not just the sensing of glucose or other carbon sources but also other signals that influence Crp abundance via the expression of small RNAs to the induction of the Crp regulon. In turn, this allowsY. pestisto fine-tune Crp levels to optimize virulence gene expression during plague infection and may allow the bacterium to adapt to its unique environmental niches.

scholarly journals The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Jeremy T. Ritzert ◽  
George Minasov ◽  
Ryan Embry ◽  
Matthew J. Schipma ◽  
Karla J. F. Satchell
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Yersinia Pestis ◽  
Carbon Sources ◽  
Transcriptional Regulator ◽  
Receptor Protein ◽  
Content Type ◽  
Bacterial Physiology

ABSTRACT Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis. Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection. IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

scholarly journals The Cyclic AMP Receptor Protein, CRP, Is Required for Both Virulence and Expression of the Minimal CRP Regulon in Yersinia pestis Biovar microtus

2008 ◽  
Vol 76 (11) ◽  
pp. 5028-5037 ◽  
Author(s):  
Lingjun Zhan ◽  
Yanping Han ◽  
Lei Yang ◽  
Jing Geng ◽  
Yingli Li ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Yersinia Pestis ◽  
Core Promoter ◽  
Lethal Dose ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein ◽  
Subcutaneous Infection ◽  
Growth Phenotype ◽  
In Vivo Growth

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.

Cyclic AMP receptor protein is a repressor of adenylyl cyclase gene cyaA in Yersinia pestis

2013 ◽  
Vol 59 (5) ◽  
pp. 304-310 ◽  
Author(s):  
Shi Qu ◽  
Yiquan Zhang ◽  
Lei Liu ◽  
Li Wang ◽  
Yanping Han ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Yersinia Pestis ◽  
Core Promoter ◽  
Growth Conditions ◽  
Receptor Protein ◽  
Camp Production ◽  
Mobility Shift ◽  
Cyclic Amp Receptor Protein ◽  
Mobility Shift Assay

Yersinia pestis is one of the most dangerous pathogens. The cyclic AMP receptor protein (CRP) is required for the full virulence of Y. pestis, and it acts as a transcriptional regulator to control a large regulon, which includes several virulence-associated genes. The regulatory action of CRP is triggered only by binding to the small molecule cofactor cyclic AMP (cAMP). cAMP is synthesized from adenosine triphosphate by the adenylyl cyclase encoded by cyaA. In the present work, the regulation of crp and cyaA by CRP was investigated by primer extension, LacZ fusion, electrophoretic mobility shift assay, and DNase I footprinting. No transcriptional regulatory association between CRP and its own gene could be detected under the growth conditions tested. In contrast, CRP bound to a DNA site overlapping the core promoter −10 region of cyaA to repress the cyaA transcription. The determination of cellular cAMP levels further verified that CRP negatively controlled cAMP production. Repression of cAMP production by CRP through acting on the cAMP synthesase gene cyaA would represent a mechanism of negative automodulation of cellular CRP function.

scholarly journals Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis

2009 ◽  
Vol 9 (1) ◽  
pp. 178 ◽  
Author(s):  
Lingjun Zhan ◽  
Lei Yang ◽  
Lei Zhou ◽  
Yingli Li ◽  
He Gao ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Yersinia Pestis ◽  
Negative Regulation ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein

scholarly journals Involvement of a Putative Cyclic AMP Receptor Protein (CRP)-Like Binding Sequence and a CRP-Like Protein in Glucose-Mediated Catabolite Repression ofthnGenes in Rhodococcus sp. Strain TFB

2012 ◽  
Vol 78 (15) ◽  
pp. 5460-5462 ◽  
Author(s):  
Laura Tomás-Gallardo ◽  
Eduardo Santero ◽  
Belén Floriano
Keyword(s):  
Cyclic Amp ◽  
Catabolite Repression ◽  
Transcriptional Regulators ◽  
Receptor Protein ◽  
Content Type ◽  
Cyclic Amp Receptor Protein ◽  
Catabolic Genes ◽  
Binding Sequence ◽  
Rhodococcus Sp

ABSTRACTGlucose catabolite repression of tetralin catabolic genes inRhodococcussp. strain TFB was shown to be exerted by a protein homologous to transcriptional regulators of the cyclic AMP receptor (CRP)-FNR family. The protein was detected bound to putative CRP-like boxes localized at the promoters of thethnA1andthnSgenes.

scholarly journals Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase

1988 ◽  
Vol 250 (3) ◽  
pp. 897-902 ◽  
Author(s):  
M Pinkney ◽  
J G Hoggett
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Native Protein ◽  
Cyclic Amp Receptor Protein ◽  
Dimeric Form ◽  
Polarization Studies ◽  
Rna Polymerase Holoenzyme

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

scholarly journals Repression by Cyclic AMP Receptor Protein at a Distance

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
David J. Lee ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Cyclic Amp ◽  
Promoter Activity ◽  
Bacterial Genome ◽  
Receptor Protein ◽  
Transcription Start ◽  
Content Type ◽  
Cyclic Amp Receptor Protein ◽  
Α Subunits

ABSTRACT In a previous study of promoters dependent on the Escherichia coli cyclic AMP receptor protein (CRP), carrying tandem DNA sites for CRP, we found that the upstream-bound CRP could either enhance or repress transcription, depending on its location. Here, we have analyzed the interactions between CRP and the C-terminal domains of the RNA polymerase α subunits at some of these promoters. We report that the upstream-bound CRP interacts with these domains irrespective of whether it up- or downregulates promoter activity. Hence, disruption of this interaction can lead to either down- or upregulation, depending on its location. IMPORTANCE Many bacterial promoters carry multiple DNA sites for transcription factors. While most factors that downregulate promoter activity bind to targets that overlap or are downstream of the transcription start and −10 element, very few cases of repression from upstream locations have been reported. Since more Escherichia coli promoters are regulated by cyclic AMP receptor protein (CRP) than by any other transcription factor, and since multiple DNA sites for CRP are commonplace at promoters, our results suggest that promoter downregulation by transcription factors may be more prevalent than hitherto thought, and this will have implications for the annotation of promoters from new bacterial genome sequences.

scholarly journals Cyclic AMP Receptor Protein RegulatescspE, an Early Cold-Inducible Gene, in Escherichia coli

2011 ◽  
Vol 193 (22) ◽  
pp. 6142-6151 ◽  
Author(s):  
Sheetal Uppal ◽  
Svetlana R. Maurya ◽  
Ramesh S. Hire ◽  
Narendra Jawali
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Cyclic Amp ◽  
Target Site ◽  
Class I ◽  
Receptor Protein ◽  
Upstream Region ◽  
Core Motif ◽  
Content Type ◽  
Cyclic Amp Receptor Protein

cspE, a member of thecspAfamily of cold shock proteins inEscherichia coli, is an early cold-inducible protein. The nucleic acid melting ability and transcription antiterminator activity of CspE have been reported to be critical for growth at low temperature. Here, we show that the cyclic AMP receptor protein (CRP), a global regulator involved in sugar metabolism, upregulatescspEinE. coli. Sequence analysis of thecspEupstream region revealed a putative CRP target site centered at −61.5 relative to the transcription start. The binding of CRP to this target site was demonstrated using electrophoretic mobility shift assays. The presence of this site was shown to be essential for PcspEactivation by CRP. Mutational analysis of the binding site indicated that the presence of an intact second core motif is more important than the first core motif for CRP-PcspEinteraction. Based on the promoter architecture, we classified PcspEas a class I CRP-dependent promoter. This was further substantiated by our data demonstrating the involvement of the AR1 domain of CRP in PcspEtranscription. Furthermore, the substitutions in the key residues of the RNA polymerase α-subunit C-terminal domain (α-CTD), which are important for class I CRP-dependent transcription, showed the involvement of 265 and 287 determinants in PcspEtranscription. In addition, the deletion ofcrpled to a growth defect at low temperature, suggesting that CRP plays an important role in cold adaptation.

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