Affiliation:
1. Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688
Abstract
ABSTRACT
Rickettsia prowazekii
is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to
sn
-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is
R. prowazekii
open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P
in vitro
. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the
R. prowazekii gpsA
gene functioned to complement an
Escherichia coli gpsA
mutant. Furthermore,
gpsA
mRNA was detected in
R. prowazekii
purified from hen egg yolk sacs, and G3PDH activity was assayable in
R. prowazekii
lysed-cell extracts. Together, these data strongly suggested that
R. prowazekii
encodes and synthesizes a functional GpsA enzyme, yet
R. prowazekii
is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that
R. prowazekii
transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified
R. prowazekii
transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA
in vivo
as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
17 articles.
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