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. / Graduation date: 2012
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/26906 |
| Date | 05 December 2011 |
| Creators | Quinn, Matthew J. |
| Contributors | Hase, Claudia |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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