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J. Bacteriol. doi:10.1128/JB.02017-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Expression and Porin Activity of P28 and OMP-1F during Intracellular Ehrlichia chaffeensis Development

Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210

^* To whom correspondence should be addressed. Email: rikihisa.1{at}osu.edu.

	Abstract

Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by gram-negative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activity as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel -strands. The Sodium dodecyl sulfate stability of the two proteins was consistent with a -barrel structure. Isolated native P28 and OMP-1F exhibited porin activity with the pore size similar to and larger, respectively than OprF, which is the largest pore size porin known to date. E. chaffeensis experiences temperature changes during tick transmission. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed most in the mid-exponential growth phase at 37°C and in the late-exponential growth phase at 28°C. Porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid- and late-exponential growth phases at 28°C and 37°C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as large pore size porins, suggesting that differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.