| Summary: | An OprF-deficient mutant of P. aeruginosa strain M-2 was
constructed by Ω mutagenesis. This strain was unable to grow in low
osmolarity media and was 70% the length of the parental strain. These
results confirmed that these phenotypes were not strain specific.
Consistent with the appearance of OprF-deficient strains in clinical
situations, an OprF-deficient strain was shown to not have a major
growth disadvantage in an in vivo chamber model.
Plasmids encoding truncated and cysteine-to-serine mutants of
OprF were constructed by linker-insertion mutagenesis, PCR and
subcloning of a previously characterized mutant. Analysis of the
resulting proteins indicated that between 102 and 163 N-terminal amino
acids of OprF was required for protein expression as determined by
Western immunoblotting. All of the truncated mutants expressed were
associated with the outer membrane, indicating that the N-terminal 163
amino acids of OprF were sufficient for this association. None of the
truncated-OprF mutants tested were associated with the peptidoglycan,
indicating that more than 215 N-terminal amino acids of OprF are
required for this association. Strains that encoded at least two cysteines
were 2-mercaptoethanol modifiable. The full-length cloned OprF and the
truncated mutant expressing the N-terminal 290 amino acids were
normally heat modifiable. When denatured prior to electrophoresis, the
remaining truncated mutants had apparent molecular weights lower
than those of untreated samples indicating that between 215 and 290
amino acids were required for the protein to be normally heat modified.
Although the truncated-OprF mutants were able to grow in low
osmolarity media and were significantly longer than the OprF-deficient
strain, the C-terminal half of OprF appeared to be required for the wild-type
growth and length.
The cysteine-to-serine OprF mutants were associated with the
outer membrane and were heat modifiable. Analysis of these mutants
with and without 2-mercaptoethanol was consistent with the hypothesis
that wild-type OprF has two disulphide bonds
Monoclonal antibody reactivity with overlapping octapeptides
equivalent to the primary sequence of OprF localized three epitopes.
Indirect immunofluorescence and opsonic phagocytosis studies identified
the surface location of epitopes binding nine OprF-specific monoclonal
antibodies. This information was incorporated into an updated
secondary structure model of OprF. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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