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Infection and Immunity, November 2008, p. 5028-5037, Vol. 76, No. 11
0019-9567/08/$08.00+0 doi:10.1128/IAI.00370-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
The Cyclic AMP Receptor Protein, CRP, Is Required for Both Virulence and Expression of the Minimal CRP Regulon in Yersinia pestis Biovar microtus
,
Lingjun Zhan,1,2,
Yanping Han,2,
Lei Yang,1,2,
Jing Geng,2
Yingli Li,2
He Gao,2
Zhaobiao Guo,2
Wei Fan,3
Gang Li,3
Lianfeng Zhang,1
Chuan Qin,1
Dongsheng Zhou,2* and
Ruifu Yang2*
Institute of Laboratory Animal Sciences, Chinese Academy of Medicine Peking Union Medical College, Beijing 100021, China,1 State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071, China,2 Laboratory Animal Center, Academy of Military Medical Sciences, Beijing 100071, China3
Received 23 March 2008/ Returned for modification 5 May 2008/ Accepted 6 August 2008
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.
* Corresponding author. Mailing address: State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071, China. Phone for Ruifu Yang: 86-10-669-48595. Fax: 86-10-638-15689. E-mail: ruifuyang{at}gmail.com. Phone for Dongsheng Zhou: 86-10-669-48594. Fax: 86-10-638-15689. E-mail: dongshengzhou1977{at}gmail.com
Published ahead of print on 18 August 2008.
Supplemental material for this article may be found at http://iai.asm.org/.
L.Z., Y.H., and L.Y. contributed equally to this work.
Infection and Immunity, November 2008, p. 5028-5037, Vol. 76, No. 11
0019-9567/08/$08.00+0 doi:10.1128/IAI.00370-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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