Insights into the Function of YciM, a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

Abstract

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.A ntibiotic resistance has become a worldwide problem that threatens the effectiveness of many medicines used today to treat bacterial infections. A particularly serious threat is the emergence of Gram-negative pathogens, including Escherichia coli and Pseudomonas aeruginosa, which are resistant to all of the available antibacterial agents (1). It is therefore urgent to develop new antibiotics active against Gram-negative bacteria, which requires a deep understanding of the biology of these organisms. Proteins playing a role in the assembly of the cell envelope are attractive targets for antibiotic development because this structure is essential for viability and protection against toxic compounds such as antibiotics.The envelope of Gram-negative bacteria is characterized by the presence of two concentric membranes, the inner membrane (IM) and outer membrane (OM), which are separated by the periplasm, a compartment that represents between 10 and 20% of the total cell volume and contains the peptidoglycan layer, or sacculus (2-4). The peptidoglycan sacculus is a heteropolymeric macromolecule composed of relatively short glycan strands and peptide cross-links, which serve to maintain cell morphology and give protection from turgor pressure. Although several proteins that play a key role in the biogenesis of the cell envelope have been identified recently (5-7), we have a poor understanding of how this complex architecture is assembled and of how envelope biogenesis is coordinated with cell growth and division. For instance, we do not know how the phospholipids that ...

Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein

“…The lapB gene was found to be essential at 37°C, and depletion of LapB was shown to cause accumulation of LpxC and increase in LPS amounts, which supports our results. In another study, the lapB mutant was identified in a screen of an E. coli genomic knock-out library (ASKA library), on the basis of its sensitivity to various antibiotics (66). The sensitivity of lapB (yciM) mutants toward antibiotics had already been shown in an earlier study using the same deletion library (68).…”

“…During the revision process of this manuscript, three groups described characterization of the lapB gene (yciM) in E. coli and its homolog in Neisseria meningitidis (65)(66)(67). In one study, the lapB (yciM) gene was identified by serendipity, whereby the lapB deletion strain in the ASKA library carried a suppressor mutation in the lpxC gene (65).…”

Genomic Characterization of Colistin Heteroresistance in Klebsiella pneumoniae during a Nosocomial Outbreak

“…In the heteroresistant strain from patient E, a mutation leading to an amino acid substitution encoded within the yciM gene was found. In Escherichia coli, yciM contributes to cell wall integrity by regulating LPS biosynthesis (46,47), and a deletion in yciM leads to decreased susceptibility to colistin (48). It is possible that the mutation in yciM in K. pneumoniae increases LPS production, leading to higher levels of LPS in the outer membrane, which could titrate out the destabilizing effect of colistin binding to LPS.…”

An Unexpected Duo: Rubredoxin Binds Nine TPR Motifs to Form LapB, an Essential Regulator of Lipopolysaccharide Synthesis

“…LapB contains three major structural motifs: the N-terminal transmembrane helix, several tetratricopeptide repeats (TPR), and a C-terminal rubredoxin metal binding domain (Nicolaes et al, 2014). The N-terminal transmembrane helix anchors LapB to the inner membrane of Gram-negative bacteria with the soluble domain in the cytoplasm (Nicolaes et al, 2014).…”