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.

Patrycja Gorzelak,Gracjana Klein,Satish Raina

doi:10.3390/ijms22105099

Patrycja Gorzelak, Gracjana Klein + Show 1 more

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https://doi.org/10.3390/ijms22105099

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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.

Highlights

The defining and most conserved feature of Gram-negative bacteria is the presence of an asymmetric outer membrane (OM), which is essential for their viability [1]
In E. coli K-12, most of the genes whose products are involved in LPS core biosynthesis, including various glycosyl/heptosyltransferases and sugar nucleotide kinases, are organized as three operons in the waa locus, spanning from gmhD to waaA (Figure 1)
In our previous studies describing the LPS composition of a suppressor-free ∆waaA strain, we showed that lipid IVA can serve as an acceptor for the incorporation of secondary acyl lauroyl, myristoyl, and palmitoleate chains under slow-growth conditions when bacteria are grown at 21 ◦ C, without the prior incorporation of Kdo residues [19]

Summary

Introduction

The defining and most conserved feature of Gram-negative bacteria is the presence of an asymmetric outer membrane (OM), which is essential for their viability [1]. Lipopolysaccharide (LPS) constitutes the major component of the OM, and is one of the main virulence factors of pathogenic Gram-negative bacteria [1,2,3]. LPS is a complex glycolipid comprised of a hydrophobic membrane-anchored lipid A and a core oligosaccharide, which is linked to the O-antigen in smooth-type bacteria [2]. In Escherichia coli, the core oligosaccharide can be subdivided into the inner and outer cores. The inner core generally contains a conserved structural element of 3-deoxy-α-D-manno-oct-2-ulopyranosonic acid (Kdo), L-glycero-α-D-manno-heptopyranose (Hep), and phosphate residues [5]

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