Abstract
The lpcA locus has been identified in Escherichia coli K12 novobiocin-supersensitive mutants that produce a short lipopolysaccharide (LPS) core which lacks glyceromannoheptose and terminal hexoses. We have characterized lpcA as a single gene mapping around 5.3 min (246 kilobases) on the E. coli K12 chromosome and encoding a 22.6-kDa cytosolic protein. Recombinant plasmids containing only lpcA restored a complete core LPS in the E. coli strain chi711. We show that this strain has an IS5-mediated chromosomal deletion of 35 kilobases that eliminates lpcA. The LpcA protein showed discrete similarities with a family of aldose/ketose isomerases and other proteins of unknown function. The isomerization of sedoheptulose 7-phosphate, into a phosphosugar presumed to be D-glycero-D-mannoheptose 7-phosphate, was detected in enzyme reactions with cell extracts of E. coli lpcA+ and of lpcA mutants containing the recombinant lpcA gene. We concluded that LpcA is the phosphoheptose isomerase used in the first step of glyceromannoheptose synthesis. We also demonstrated that lpcA is conserved among enteric bacteria, all of which contain glyceromannoheptose in the inner core LPS, indicating that LpcA is an essential component in a conserved biosynthetic pathway of inner core LPS.
Highlights
LPS,1 an integral component of the outer membrane of Gram-negative bacteria, consists of lipid A attached to a core oligosaccharide, and in some microorganisms, contains an Ospecific surface polysaccharide which is subsequently attached to the terminal residues of the core [1,2]
Since the lpcA locus has been mapped near the proAB genes [6], we examined strain 711 for characteristics of inner core LPS defects
This gene is physically unlinked to the majority of genes used in core biosynthesis that are found within the rfa cluster at 81 min on the chromosomal map
Summary
LPS,1 an integral component of the outer membrane of Gram-negative bacteria, consists of lipid A attached to a core oligosaccharide, and in some microorganisms, contains an Ospecific surface polysaccharide which is subsequently attached to the terminal residues of the core [1,2]. Identification of the lpcA Locus—To investigate if sequences near the proAB genes include the gene(s) responsible for the LPS defect in 711, 711 cells were transformed with the cosmid pE4021 which contains a DNA segment spanning the proAB region [33] (Fig. 2).
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