Abstract
The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.
Highlights
A role for the MsbA protein in PL transport was suggested [2, 3]
The NMB1919 gene was cloned, and we investigated whether this Neisserial gene could complement an E. coli msbA mutation
Whereas de novo synthesized phospholipids (Fig. 2A) and LPS (Fig. 2B) accumulated at 44 °C in the IM of strain WD2 carrying the empty vector, consistent with previous results [2], the localization of these compounds to the outer membrane (OM) was restored when the strain was complemented with the Neisserial msbA gene (Fig. 2A, B) and did not deviate from that observed in the wild-type strain grown at 44 °C
Summary
A role for the MsbA protein in PL transport was suggested [2, 3]. The msbA gene was first identified in E. coli as a multicopy suppressor of a mutation in the htrB (lpxL) gene, which encodes an enzyme involved in a late step of the biosynthesis of lipid A [4, 5], a structural component of LPS [6]. It was observed that in the temperature-sensitive msbA mutant, newly synthesized PL accumulated in the IM at the restrictive temperature [2] and that they were poorly accessible to membrane-impermeable reagents under those conditions [3]. These results strongly suggest that MsbA is involved in LPS transport and in PL transport. We already exploited this property of N. meningitidis to demonstrate that the OM protein designated Imp, which is essential in E. coli [10], has a role in LPS transport [11]. We have described an msbA mutant in N. meningitidis
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