Biology subjects

Fivenson, E. M.

Publications and source records attributed to Fivenson, E. M..

4 recordsLinked to original sources

bioRxiv2026

A potential role for acyl-phosphate in the coordination of phospholipid and lipopolysaccharide synthesis in Escherichia coli

The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli. This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced. IMPORTANCEOver the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

microbiology↗

bioRxiv2024

MreC-MreD structure reveals a multifaceted interface that controls MreC conformation

The peptidoglycan (PG) cell wall is critical for bacterial growth and survival and is a primary antibiotic target. MreD is an essential accessory factor of the Rod complex, which carries out PG synthesis during elongation, yet little is known about how MreD facilitates this process. Here, we present the cryo-electron microscopy structure of Thermus thermophilus MreD in complex with another essential Rod complex component, MreC. The structure reveals that a periplasmic-facing pocket of MreD interacts with multiple membrane-proximal regions of MreC. We use single-molecule FRET to show that MreD controls the conformation of MreC through these contacts, inducing a state primed for Rod complex activation. Using E. coli as a model, we demonstrate that disrupting these interactions abolishes Rod complex activity in vivo. Our findings reveal the role of MreD in bacterial cell shape determination and highlight its potential as an antibiotic target.

microbiology↗

bioRxiv2023

A role for the Gram-negative outer membrane in bacterial shape determination

The cell envelope of Gram-negative bacteria consists of three distinct layers: the cytoplasmic membrane, a cell wall made of peptidoglycan (PG), and an asymmetric outer membrane (OM) composed of phospholipid in the inner leaflet and lipopolysaccharide (LPS) glycolipid in the outer leaflet. The PG layer has long been thought to be the major structural component of the envelope protecting cells from osmotic lysis and providing them with their characteristic shape. In recent years, the OM has also been shown to be a load-bearing layer of the cell surface that fortifies cells against internal turgor pressure. However, whether the OM also plays a role in morphogenesis has remained unclear. Here, we report that changes in LPS synthesis or modification predicted to strengthen the OM can suppress the growth and shape defects of Escherichia coli mutants with reduced activity in a conserved PG synthesis machine called the Rod system (elongasome) that is responsible for cell elongation and shape determination. Evidence is presented that OM fortification in the shape mutants restores the ability of MreB cytoskeletal filaments to properly orient the synthesis of new cell wall material by the Rod system. Our results are therefore consistent with a role for the OM in the propagation of rod shape during growth in addition to its well-known function as a diffusion barrier promoting the intrinsic antibiotic resistance of Gram-negative bacteria. SIGNIFICANCEThe cell wall has traditionally been thought to be the main structural determinant of the bacterial cell envelope that resists internal turgor and determines cell shape. However, the outer membrane (OM) has recently been shown to contribute to the mechanical strength of Gram-negative bacterial envelopes. Here, we demonstrate that changes to OM composition predicted to increase its load bearing capacity rescue the growth and shape defects of Escherichia coli mutants defective in the major cell wall synthesis machinery that determines rod shape. Our results therefore reveal a previously unappreciated role for the OM in bacterial shape determination in addition to its well-known function as a diffusion barrier that protects Gram-negative bacteria from external insults like antibiotics.

microbiology↗

bioRxiv2020

An essential membrane protein modulates the proteolysis of LpxC to control lipopolysaccharide synthesis in Escherichia coli

Gram-negative bacteria are surrounded by a complex cell envelope that includes two membranes. The outer membrane prevents many drugs from entering these cells and is thus a major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share a common intermediate. Proper membrane biogenesis therefore requires that the flux through each pathway be balanced. In Escherichia coli, a major control point in establishing this balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are controlled through its degradation by the inner membrane protease FtsH and its presumed adaptor protein LapB(YciM). How turnover of LpxC is controlled has remained unclear for many years. Here, we demonstrate that the essential protein of unknown function YejM(PbgA), which we have renamed ClxD (control of LpxC degradation), participates in this regulatory pathway. Suppressors of ClxD essentiality were identified in lpxC and lapB, and LpxC overproduction was shown to be sufficient to allow survival of {Delta}clxD mutants. Furthermore, the half-life of LpxC was shown to be reduced in cells lacking ClxD, and genetic and physical interactions between LapB and ClxD were detected. Taken together, our results are consistent with a model in which ClxD directly modulates LpxC turnover by FtsH-LapB to regulate LPS synthesis and maintain membrane homeostasis. SIGNIFICANCEThe outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the synthesis of these lipid species must be balanced for the membrane to maintain its barrier function in blocking drug entry. In this report, we identify an essential protein of unknown function as a key new factor in maintaining LPS/phospholipid balance in the model bacterium Escherichia coli. Our results provide novel insight into how this organism and most likely other Gram-negative bacteria maintain membrane homeostasis and their intrinsic resistance to antibiotics.

microbiology↗