Molecular Basis of Essentiality of Early Critical Steps in the Lipopolysaccharide Biogenesis in Escherichia coli K-12: Requirement of MsbA, Cardiolipin, LpxL, LpxM and GcvB

Abstract

To identify the physiological factors that limit the growth of Escherichia coli K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire waa locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IVA derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ(gmhD-waaA) identified two independent single-amino-acid substitutions—P50S and R310S—in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene clsA was found to be essential for the growth of ΔlpxLMP, ΔlpxL, ΔwaaA, and Δ(gmhD-waaA) bacteria, with a conditional lethal phenotype of Δ(clsA lpxM), which could be overcome by suppressor mutations in MsbA. Suppressor mutations basS A20D or basR G53V, causing a constitutive incorporation of phosphoethanolamine (P-EtN) in the lipid A, could abolish the Ca++ sensitivity of Δ(waaC eptB), thereby compensating for P-EtN absence on the second Kdo. A single-amino-acid OppA S273G substitution is shown to overcome the synthetic lethality of Δ(waaC surA) bacteria, consistent with the chaperone-like function of the OppA oligopeptide-binding protein. Furthermore, overexpression of GcvB sRNA was found to repress the accumulation of LpxC and suppress the lethality of LapAB absence. Thus, this study identifies new and limiting factors in regulating LPS biosynthesis.

Checkpoints That Regulate Balanced Biosynthesis of Lipopolysaccharide and Its Essentiality in Escherichia coli

“…To ensure that primarily LPS with hexa-acylated lipid A is translocated, MsbA provides an essential checkpoint as it selects species that are hexa-acylated by several orders of magnitude [ 19 , 20 ]. At this point, the viability of bacteria with either tetra- or penta-acylated lipid A requires the presence of cardiolipin (CL), which presumably aids in the transport of such LPS, constituting another checkpoint [ 21 , 22 ]. Once LPS is flipped by MsbA, it is delivered to the Lpt machinery for its final assembly in the OM.…”

Lipopolysaccharide of the Yersinia pseudotuberculosis Complex

“…Their expression level increases as the cultivation temperature decreases from 37 to 21 • C. The mutant at both genes synthesizes tetraacyl lipid A, which is similar to lipid IV A (Figure 1C) and identical to that synthesized by wild-type Y. pestis strains at 37 • C [37]. Kdo-independent late acylation in the lipid A has been shown in E. coli under slow growth conditions at low temperatures or upon the overexpression of genes encoding late-acyl transferases [39][40][41]. The outer membrane palmitoyl transferase protein PagP was originally identified in Salmonella and is responsible for the addition of the secondary 16:0 acyl group (palmitate) to lipid A [42].…”

“…While the tetraacylated form may help to overcome warmblooded-host immunity, it makes Y. pestis more susceptible to the AMP cecropin A [37]. Resistance to AMP, dependent on the structure of lipid A, is not uncommon in other bacterial species as well [39,42]. Y. pestis AMP resistance is not only dependent on LPS structural changes, since mutants with an intact LPS structure, but susceptible to AMPs and reduced survival rates in fleas are described [73].…”

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology

“…LPS to be tetraacylated as opposed to the pentaacylated structure seen in Lcr. Notably, in E. coli , tetraacylated lipid A precursors are not as efficiently translocated to the outer membrane as are penta- or hexa-acylated lipid A species leading to temperature-dependent growth defects [ 36 , 37 ].…”

“…Avirulent bacteria have either reduced size or completely lack the O -polysaccharide. For example, in Salmonella enteritidis , it was shown that virulent bacteria had a higher number of O -polysaccharide repeating units, and thus a larger molecular size, compared to avirulent strains [ 37 ]. Similarly, it has been shown that the rough LPS mutants of the intracellular Brucella spp.…”