YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

Abstract

Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

An Essential Membrane Protein Modulates the Proteolysis of LpxC to Control Lipopolysaccharide Synthesis in Escherichia coli

“…Our results are therefore consistent with a model in which YejM interferes with LpxC proteolysis through its interaction with LapB. Complementary results that also support this model were reported while this paper was under review (17). We propose that the modulation of LpxC degradation by YejM is likely to be homeostatic and responsive to perturbations in the balance between LPS and PL synthesis.…”

The essential inner membrane protein YejM is a metalloenzyme

“…Therefore, our structural data along with extensive phylogenetic and functional analysis provide a strong evidence that YejM/PbgA can bind divalent metal ions at the conserved active site necessary for its enzymatic activity. Notably, our structures show a less tight coordinated Mg 2+ ion that suggests that the metal binding site is possibly reversible, harboring functional relevance that may regulate functionally distinct properties of YejM [25][26][27][28] . Mg 2+ binding sites are known to have coordination plasticity, especially at binding sites that contribute mostly monodentate coordination 45 .…”

“…More recently YejM has been shown to be involved in other membrane homeostasis pathways. Several recent publications and a crystal structure suggest that YejM regulates LPS biosynthesis by preventing excessive degradation of LpxC, a key enzyme in that pathway [25][26][27][28][29] , and the absence of YejMPD results in increase in lipid A core molecules in IM 25 . LpxC is a zinc-dependent metallo-amidase that performs the first committed step in LPS biosynthesis 30 , and is constantly degraded by the FtsH protease 31 .…”

“Asymmetry Is the Rhythmic Expression of Functional Design,” a Quotation from Jan Tschichold

“…1A). Recent papers, one of which is in this issue of the journal (3), find a regulatory role for one of the last essential genes (yejM) in Escherichia coli that lacks a clear function (4,5). The papers provide evidence that YejM senses an intermediate in these biosynthetic pathways to regulate the stability of LpxC, a key enzyme in the biosynthesis of LPS (6,7), to balance the synthesis of PL with LPS and maintain OM asymmetry.…”

“…The Silhavy lab obtained suppressors of a truncated YejM mutant by selecting for growth at 42°C in the presence of SDS and EDTA (EDTA weakens the OM barrier by chelating Mg 2ϩ , which helps to stabilize lateral interactions between LPS molecules) (5). WGS revealed that the suppressor mutations were in yciM and lpxC.…”