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 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.

scholarly journals Interdependence of Activation at rhaSR by Cyclic AMP Receptor Protein, the RNA Polymerase Alpha Subunit C-Terminal Domain, and RhaR

2000 ◽  
Vol 182 (23) ◽  
pp. 6774-6782 ◽  
Author(s):  
Carolyn C. Holcroft ◽  
Susan M. Egan
Keyword(s):  
Cyclic Amp ◽  
Rna Polymerase ◽  
Synergistic Effects ◽  
Receptor Protein ◽  
Mobility Shift ◽  
Α Subunit ◽  
Subunit C ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain ◽  
Full Activation

ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of l-rhamnose. β-Galactosidase assays of various rhaS-lacZ promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at −111.5 is also required for full activation of rhaSR expression. To address the mechanisms of activation by CRP and the RNA polymerase α-subunit C-terminal domain (α-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of α (α-Δ235), and alanine substitutions throughout α-CTD. We found that DNA-contacting residues in α-CTD are required for full activation, and for simplicity, we discuss α-CTD as a third activator of rhaSR. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and α-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an α-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and α-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the α-CTD–CRP and α-CTD–RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, α-CTD, and RhaR.

Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer

1980 ◽  
Vol 26 (12) ◽  
pp. 1508-1511 ◽  
Author(s):  
Ann D. E. Fraser ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Sole Carbon Source ◽  
Receptor Protein ◽  
Deficient Mutant ◽  
Phosphotransferase System ◽  
Cyclic Amp Receptor Protein ◽  
Cyclic Monophosphate

It has not been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP–CRP complex. 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM). Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases im mediately upon addition of DG. These results suggest that the formation of PTSM is dependent on cyclic AMP. In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred. These results suggest that the formation of PTSM does not require an exogenous inducer.

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 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

Extracellular accumulation of L-glutamate in adenylyl cyclase deficient or cyclic AMP receptor protein deficient mutants of Escherichia coli

1980 ◽  
Vol 26 (6) ◽  
pp. 718-721
Author(s):  
Kwok-Luen Leung ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Cell Membrane ◽  
Stationary Phase ◽  
Adenylyl Cyclase ◽  
Cell Envelope ◽  
Coli Cell ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein ◽  
E Coli

An Escherichia coli cya mutant deficient in adenylyl cyclase and an E. coli crp mutant deficient in cyclic AMP receptor protein (CRP) accumulate substantial amounts of L-glutamate extracellularly when entering stationary phase of growth. The cya mutant grown in the presence of cyclic AMP accumulates little glutamate whereas the addition of cyclic AMP has no effect on glutamate accumulation in the crp mutant. It is proposed that an E. coli cell entering stationary phase requires a change in cell envelope structure which involves a cyclic AMP – CRP dependent process, and without this process the permeability of the cell membrane increases.

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