Temperature-sensitive lesions in the Francisella novicida valA gene cloned into an Escherichia coli msbA lpxK mutant affecting deoxycholate resistance and lipopolysaccharide assembly at the restrictive temperature

Abstract

The valAB locus of Francisella novicida has previously been found to be highly similar at the deduced amino acid level to msbA lpxK of Escherichia coli. Both ValA and MsbA are members of the superfamily of ABC transporters, and they appear to have similar functions. In this study we describe the isolation of a temperature-sensitive valAB locus. DNA sequence analysis indicates that the only changes to the ValAB deduced amino acid sequence are changes of S453 to an F and T458 to an I in ValA. E. coli strains defective in msbA and expressing temperature-sensitive ValA rapidly ceased growth when shifted from a permissive temperature to a restrictive temperature. After 1 h at the restrictive temperature, cells were much more sensitive to deoxycholate treatment. To test the hypothesis that ValA is responsible for the transport or assembly of lipopolysaccharide, we introduced gseA, a Kdo (3-deoxy-D-manno-octulosonic acid) transferase from Chlamydia trachomatis, into a strain with a temperature-sensitive valA allele and a nonfunctional msbA locus. These recombinants were defective in cell surface expression of the chlamydial genus-specific epitope within 15 min of a shift to the nonpermissive temperature. Also, there was enhanced association of the epitope with the inner membrane after a shift to the nonpermissive temperature. Thus, we propose that ValA is involved in the transport of lipopolysaccharide to the outer membrane.We have recently discovered a genetic locus in Francisella novicida that contains an apparent operon consisting of two open reading frames, valAB (13). F. novicida strains thought to harbor mutations in valAB are defective in growth in macrophages and exhibit increased sensitivity to serum and deoxycholate, thus suggesting compromised outer membrane integrity. These two genes show high identity at the deduced amino acid level to the Escherichia coli genes msbA and lpxK, respectively.Interestingly, the msbA gene was originally identified as a multicopy suppressor of the E. coli htrB gene (8). Mutants having lesions in htrB exhibit an unusual phenotype whereby growth and viability of mutants at temperatures above 32.5°C in rich media are impaired. The gene product of htrB has been shown to be a Kdo (3-deoxy-D-manno-octulosonic acid)-dependent acyl transferase involved in lipopolysaccharide (LPS) biosynthesis (3). We have previously shown that, similar to msbA, valAB can suppress mutations in htrB, which suggests that both gene products may have similar functions related to LPS expression. Recent work has demonstrated that lpxK (previously called orfE) encodes a lipid A 4Ј-kinase (6).The presumptive gene products ValA and MsbA are members of the superfamily of ATP binding cassette (ABC) transporters. ABC transporters are a large family of integral membrane proteins that are responsible for the uptake or efflux of a wide variety of both proteinaceous and nonproteinaceous substrates (4). In several bacterial systems, the involvement of ABC transporters in the export of capsular polysaccharide and the tra...

Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis

“…By using a quantitative new 32 P i labeling assay, we demonstrate that htrB-deficient cells accumulate tetra-acylated lipid A molecular species and glycerophospholipids within their inner membranes at 42°C, whereas msbA-deficient strains accumulate hexa-acylated lipid A and glycerophospholipids in their inner membranes, even when extra copies of orfE/lpxK are provided in trans. Our data strengthen the view that MsbA is involved in the transport of newly synthesized core-lipid A molecules across the inner membrane (24,25) and suggest that MsbA may also transport glycerophospholipids.…”

“…11). Taken together, these findings lend additional support to the idea that MsbA may function as a transporter of newly synthesized lipid A-core molecules in E. coli (24,25).…”

“…The lack of the msbA covering plasmid during the period of 32 A problem with the published MsbA depletion experiments, as well as the experiment in Fig. 10, is that msbA is an upstream gene in an operon (24,25). The essential downstream gene (orfE/lpxK), now known to encode the 4Ј-kinase of lipid A biosynthesis (Fig.…”

“…(a) Tetra-acylated lipid A species accumulate in the inner membranes of htrB mutants at 42°C, but htrB mutants grown at 30°C are able to make penta-and hexa-acylated lipid A molecules, presumably using alternative acyltransferases. LPS had recently been shown to accumulate in the inner membranes of mutants with disrupted htrB and msbA genes, as judged by gel electrophoresis, pulse labeling with N-acetyl[ 3 H]glucosamine (24), or antibody binding (25). However, no unambiguous way to quantify the amount of lipid A and to determine the extent of its acylation in isolated membrane vesicles had been described.…”

Francisella tularensis:Taxonomy, Genetics, and Immunopathogenesis of a Potential Agent of Biowarfare

“…LVS lipid A is not phosphorylated at either the 1-or 4′-position (125). Dephosphorylation of the 1-position is catalyzed by LpxE and requires prior LPS transport across the inner membrane by ValA, a homolog of the ABC transporter MsbA (89,90,127). In contrast to LVS, F. tularensis subsp.…”