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 ...

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