Molecular and genetic assessment of selected antiporters and methyl-accepting chemotaxis proteins in Vibrio cholerae Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/tb09j827k

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • The pathogen Vibrio cholerae uses cations as a primary currency of virulence and environmental persistence, using gradients of those cations to move, acquire nutrients, and control virulence gene expression. An understanding of the overlapping roles of bioenergetics and chemotaxis in the virulence and environmental survival of V. cholerae issues from a large body of prior work, but the interplay of each component is not yet clearly understood. To this end, the activity of the antiporters Vc-NhaP1, Vc-NhaA, and Vc-NhaB was assayed, as was the sodium transporting respiratory pump NQR, and environmental stimuli were paired with potential motilitylinked sensors. The Vc-NhaP1 antiporter was found to be a K⁺(Na⁺)/H⁺ antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-logarithmic phase, after 4-6 hours of culturing. Using a pH-sensitive GFP protein, cytosolic pH was shown to be dependent on K⁺ in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless E. coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K⁺ or Na⁺ ions for protons within a broad pH range (7.25 to 9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K⁺(Na⁺)/H⁺ antiporter of the CPA- 1 family that is required for bacterial pH homeostasis and growth in an acidic environment. Further, a model system comprised of a V. cholerae strain lacking both the nqr operon and the ORFs of Vc-nhaA or Vc-nhaB was generated and tested with and without lactate. These strains, along with the single mutants of nqr, Vc-nhaA, and Vc-nhaB, were assessed for aerobic growth as a function of media pH and cation concentration (Na⁺, Li⁺, or K⁺). Loss of Vc-NhaA and, to a lesser extent, Vc-NhaB, was better observed when NQR was absent but lactate was added to facilitate replenishment of the quinone pool. Loss of Vc-NhaA in this background inhibited growth most at basic pH under increasing Na⁺ and Li⁺ conditions, and loss of Vc- NhaB in this background inhibited was most severe in acidic conditions in the presence of 0-100 mM Na⁺ or Li⁺. We also observed the growth inhibition of Vc- NhaA in the absence of NQR and in the presence of lactate and 100-450 mM Li⁺, which has not been previously reported. These growth defects were restored upon expression of the cognate antiporter gene on an inducible expression vector. Lastly, potential chemotaxis stimuli were correlated with cognate methyl-accepting chemotaxis protein (MCP) receptors. The homology of MCP sensory domains among Vibrionaceae demonstrated a subset were unique to V. cholerae. Of these unique MCPs, transposon insertion in VC0098 significantly reduced chemotaxis swarm diameter towards Na⁺ and K⁺. Additionally, the MCP VCA0663 was shown, by transposon mutagenesis and complementation, to direct chemotaxis towards N-acetylglucosamine. Additional observations are described concerning the chemotaxis defects incurred by transposon mutagenesis of MCPs in vitro towards mucin, bile, or L-serine. MCP strains were also tested in vivo for 4 and 24 hours in the infant mouse model of infection. None of the observed chemotaxis defects showed complete loss of chemotaxis by transposon mutagenesis, in line with the hypothesis that the large number of MCPs encoded by V. cholerae result in redundant chemotaxis sensory functions. These findings add to the understanding of how bioenergetics and chemotaxis interact within V. cholerae, a foundation from which the bacterium can be understood and, eventually, controlled.
Resource Type
Date Available
Date Copyright
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Committee Member
Academic Affiliation
Non-Academic Affiliation
Keyword
Subject
Rights Statement
Peer Reviewed
Language
Replaces
Additional Information
  • description.provenance : Rejected by Julie Kurtz(julie.kurtz@oregonstate.edu), reason: Rejecting because the title on the Abstract page doesn't match the title on the Title page. If the title is changed on the Abstract page to match the Title page then the revised Abstract would require printing on cotton bond paper, get professor's signature and submit to the grad school. If the Title page title is changed I can print out the revised page and replace the original copy. Once revised, open the item that was rejected. Replace the attached copy with the revised copy and resubmit. Thanks, Julie on 2012-01-11T21:07:55Z (GMT)
  • description.provenance : Submitted by Matthew Quinn (quinnmat@onid.orst.edu) on 2012-01-19T22:51:59Z No. of bitstreams: 3 MJQ Dissertation FINAL revised last.pdf: 830920 bytes, checksum: 6f378a6fda0c91a55da88293a1210fb7 (MD5) license_rdf: 22760 bytes, checksum: bac85fe9b6ed3c96501e045c0f9e0d61 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)
  • description.provenance : Approved for entry into archive by Julie Kurtz(julie.kurtz@oregonstate.edu) on 2012-01-23T17:43:48Z (GMT) No. of bitstreams: 3 MJQ Dissertation FINAL revised last.pdf: 830920 bytes, checksum: 6f378a6fda0c91a55da88293a1210fb7 (MD5) license_rdf: 22760 bytes, checksum: bac85fe9b6ed3c96501e045c0f9e0d61 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)
  • description.provenance : Made available in DSpace on 2012-01-23T22:11:23Z (GMT). No. of bitstreams: 3 MJQ Dissertation FINAL revised last.pdf: 830920 bytes, checksum: 6f378a6fda0c91a55da88293a1210fb7 (MD5) license_rdf: 22760 bytes, checksum: bac85fe9b6ed3c96501e045c0f9e0d61 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2011-12-05
  • description.provenance : Approved for entry into archive by Laura Wilson(laura.wilson@oregonstate.edu) on 2012-01-23T22:11:23Z (GMT) No. of bitstreams: 3 MJQ Dissertation FINAL revised last.pdf: 830920 bytes, checksum: 6f378a6fda0c91a55da88293a1210fb7 (MD5) license_rdf: 22760 bytes, checksum: bac85fe9b6ed3c96501e045c0f9e0d61 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)
  • description.provenance : Submitted by Matthew Quinn (quinnmat@onid.orst.edu) on 2012-01-08T02:04:57Z No. of bitstreams: 3 QuinnMatthewJ2011.pdf: 848727 bytes, checksum: 97fe9665255a53be7cc9dd977777f425 (MD5) license_rdf: 22760 bytes, checksum: bac85fe9b6ed3c96501e045c0f9e0d61 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)

Relationships

In Administrative Set:
Last modified: 08/08/2017

Downloadable Content

Download PDF
Citations:

EndNote | Zotero | Mendeley

Items