| Summary: | Porins are pore forming proteins found in the outer membranes of gram
negative bacteria. They are involved in the transport of small, hydrophilic
molecules across the membrane, and are thus important in the uptake of a variety of
substances from the external environment. An attempt was made to identify porin(s)
in the outer membrane of the gastric pathogen Helicobacter pylori in order to gain
insight into the physiology of this organism. Growth studies were done in order to
optimize large scale growth conditions for H. pylori, and a novel growth media was
developed such that large masses of membrane proteins could be obtained. Five
porin proteins, (HopA, B, C, D and E) were identified and purified. N-terminal
sequence analysis revealed that all five shared a strong degree of homology with
each other which indicated that this was a group of related proteins. Model
membrane analyses showed that the HopA, B, C, and D proteins functioned as
channels. They all were functional as monomers, and they possessed similar
channel forming properties in that they formed channels with conductances between
0.24 and 0.36 nS in 1.0 M KC1 pH 7.0. HopE also formed channels, but it showed
different pore forming characteristics, as even though it still appeared to function as
a monomer, it formed large channels with conductances of 1.5 nS in 1.0 M KC1.
Since most porins previously isolated function as trimers, a series of major outer
membrane proteins which were isolated as monomers from a variety of organisms
were examined for pore forming ability in order to further show precedence for the
existence of functional monomeric porins. Six of these proteins functioned as
porins, indicating that porins which are functional as monomers may be more
common than previously thought. Growth studies utilizing a defined medium
demonstrated that the expression of HopA, B, and C was regulated by the amount
of iron in the medium, and the different expression levels of the porins appeared to
affect the uptake of a number of different antibiotics. Degenerate PCR primers
created from N-terminal and internal protein sequences allowed amplification of
DNA sequences encoding the N-terminal portions of two of the H. pylori porin
proteins, and these DNA sequences were used to identify hopA and hopB clones
from a H. pylori chromosomal DNA library that was created. Hybridization with H.
pylori chromosomal DNA indicated that the hopA and hopB genes are located at
different loci on the chromosome. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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