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Two promoters and two translation start sites control the expression of the Shigella flexneri outer membrane protease IcsP

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Abstract

The Shigella flexneri outer membrane protease IcsP proteolytically cleaves the actin-based motility protein IcsA from the bacterial surface. The icsP gene is monocistronic and lies downstream of an unusually large intergenic region on the Shigella virulence plasmid. In silico analysis of this region predicts a second transcription start site 84 bp upstream of the first. Primer extension analyses and beta-galactosidase assays demonstrate that both transcription start sites are used. Both promoters are regulated by the Shigella virulence gene regulator VirB and both respond similarly to conditions known to influence Shigella virulence gene expression (iron concentration, pH, osmotic pressure, and phase of growth). The newly identified promoter lies upstream of a Shine–Dalgarno sequence and second 5′-ATG-3′, which is in frame with the annotated icsP gene. The use of either translation start site leads to the production of IcsP capable of proteolytically cleaving IcsA. A bioinformatic scan of the Shigella genome reveals multiple occurrences of in-frame translation start sites associated with putative Shine–Dalgarno sequences, immediately upstream and downstream of annotated open reading frames. Taken together, our observations support the possibility that the use of in-frame translation start sites may generate different protein isoforms, thereby expanding the proteome encoded by bacterial genomes.

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Acknowledgments

We thank N. Ward and C. Ross for their assistance with this project. This study was supported by the National Institutes of Health (NIH) grant P20 RR-016464 from the IDeA Networks of Biomedical Research Excellence Program of the National Center for Research Resources grant, by the NIH grant R15 AI090573-01 and by UNLV start-up funds to H.J.W. K.M.L. was a 2009 Recipient of a Barry Goldwater Scholarship and C.T.H. is currently supported in part by the Post-9/11 GI Bill.

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Author notes

  1. Karen M. Levy

    Present address: San Diego School of Medicine, University of California, La Jolla, CA, 92093-0621, USA

  2. Stephanie K. Labahn

    Present address: School of Allied Health Sciences, University of Nevada, Las Vegas, NV, 89154-3034, USA

  3. Lia A. Africa

    Present address: University of Nevada School of Medicine, 1664 N. Virginia Street, Reno, NV, 89557, USA

Authors and Affiliations

  1. School of Life Sciences, University of Nevada, 4505 Maryland Parkway, Las Vegas, NV, 89154-4004, USA

    Christopher T. Hensley, Karen M. Levy, Stephanie K. Labahn, Lia A. Africa & Helen J. Wing

  2. Department of Molecular Biology, University of Wyoming, Laramie, WY, 82071, USA

    Olga K. Kamneva

Authors
  1. Christopher T. Hensley
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  2. Olga K. Kamneva
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  3. Karen M. Levy
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Corresponding author

Correspondence to Helen J. Wing.

Additional information

Communicated by John Helmann.

Electronic supplementary material

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Online Resource 1

Position-specific scoring matrix for ribosome binding sites associated with the S. flexneri 2a strain 301 virulence plasmid pCP301 (NC_004851.1) and chromosome (NC_004337.1). Six nucleotide-long motif overrepresented in immediate upstream regions of annotated start codons identified by MEME (http://www.meme.sdsc.edu) and used to search for Shine–Dalgarno sequences upstream of predicted additional start codons. (PPT 142 kb)

Online Resource 2

Distribution of p-values for predicted ribosome binding sites with respect to both the annotated translation start sites (black) and the predicted additional translation start sites (gray). (PPT 122 kb)

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Hensley, C.T., Kamneva, O.K., Levy, K.M. et al. Two promoters and two translation start sites control the expression of the Shigella flexneri outer membrane protease IcsP. Arch Microbiol 193, 263–274 (2011). https://doi.org/10.1007/s00203-010-0669-2

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