Bacteria have evolved multiple strategies to sense and rapidly adapt to challenging and ever-changing environmental conditions. The ability to alter membrane lipid composition, a key component of the cellular envelope, is crucial for bacterial survival and adaptation in response to environmental stress. However, the precise roles played by membrane phospholipids in bacterial physiology and stress adaptation are not fully elucidated. The goal of this study was to define the role of membrane phospholipids in adaptation to stress and maintenance of bacterial cell fitness. By using genetically modified strains in which the membrane phospholipid composition can be systematically manipulated, we show that alterations in major Escherichia coli phospholipids transform these cells globally. We found that alterations in phospholipids impair the cellular envelope structure and function, the ability to form biofilms, and bacterial fitness and cause phospholipid-dependent susceptibility to environmental stresses. This study provides an unprecedented view of the structural, signaling, and metabolic pathways in which bacterial phospholipids participate, allowing the design of new approaches in the investigation of lipiddependent processes involved in bacterial physiology and adaptation.IMPORTANCE In order to cope with and adapt to a wide range of environmental conditions, bacteria have to sense and quickly respond to fluctuating conditions. In this study, we investigated the effects of systematic and controlled alterations in bacterial phospholipids on cell shape, physiology, and stress adaptation. We provide new evidence that alterations of specific phospholipids in Escherichia coli have detrimental effects on cellular shape, envelope integrity, and cell physiology that impair biofilm formation, cellular envelope remodeling, and adaptability to environmental stresses. These findings hold promise for future antibacterial therapies that target bacterial lipid biosynthesis.KEYWORDS membranes, metabolism, phospholipids, physiology, stress adaptation, stress response B acteria have to cope with and adapt to a wide range of environmental conditions, such as nutrient limitation or exposure to antibiotics. Therefore, the ability to sense and quickly respond to fluctuating conditions is key for bacterial survival (1-3). Bacterial stress adaptation often requires major metabolic reprogramming that involves coordinated changes in the cell transcriptome, proteome, and metabolome, along with cellular envelope remodeling (4, 5). Escherichia coli cells consist of four compartments: the cytoplasm, the inner membrane, the periplasm, and the outer membrane. The inner and outer membranes exhibit different makeups. The inner membrane is a bilayer containing ␣-helical proteins, and more than 95% of the total lipids are phospholipids; the outer membrane is an asymmetric bilayer made of both phospholipids and

Most bacteria divide using a protein machine called the divisome that spans the cytoplasmic membrane. Key divisome proteins on the membrane’s cytoplasmic side include tubulin-like FtsZ, which forms GTP-dependent protofilaments, and actin-like FtsA, which tethers FtsZ to the membrane. Here we present genetic evidence that in Escherichia coli, FtsA antagonizes FtsZ protofilament bundling in vivo. We then show that purified FtsA does not form straight polymers on lipid monolayers as expected, but instead assembles into dodecameric minirings, often in hexameric arrays. When coassembled with FtsZ on lipid monolayers, these FtsA minirings appear to guide FtsZ to form long, often parallel, but unbundled protofilaments, whereas a mutant of FtsZ (FtsZ*) with stronger lateral interactions remains bundled. In contrast, a hypermorphic mutant of FtsA (FtsA*) forms mainly arcs instead of minirings and enhances lateral interactions between FtsZ protofilaments. Based on these results, we propose that FtsA antagonizes lateral interactions between FtsZ protofilaments, and that the oligomeric state of FtsA may influence FtsZ higher-order structure and divisome function.

Rod-shaped bacteria such as E. coli have mechanisms to position their cell division plane at the precise center of the cell, to ensure that the daughter cells are equal in size. The two main mechanisms are the Min system and nucleoid occlusion (NO), both of which work by inhibiting assembly of FtsZ, the tubulin-like scaffold that forms the cytokinetic Z ring. Whereas NO prevents Z rings from constricting over unsegregated nucleoids, the Min system is nucleoid-independent and even functions in cells lacking nucleoids and thus NO. The Min proteins of E. coli and B. subtilis form bipolar gradients that inhibit Z ring formation most at the cell poles and least at the nascent division plane. This article will outline the molecular mechanisms behind Min function in E. coli and B. subtilis, and discuss distinct Z ring positioning systems in other bacterial species.

A mother cell giving rise to offspring usually needs to choose the site of cytokinesis carefully, as this will determine the size and shape of the daughter cells. Rod-shaped bacteria that divide by binary fission, such as Escherichia coli, often mark their cell division sites at their cell midpoint so that daughter cells are roughly equivalent in size and shape. So how does E. coli know where its middle is? Its cell poles are defined by the previous cell division, but, because E. coli grows by incorporating new cell wall and membrane uniformly along its length, the future cell division site at mid-cell is newly made and has no known pre-existing markers. One way to select the new mid-cell site would be to measure the distance from the two opposing cell poles, using a system that could recognize markers at those poles and define the spot furthest from both markers. This would require that both polar markers act negatively on cell division at equivalent intensities. The result would be a concentration gradient, with the lowest concentration of the negative regulator at the cell midpoint, the greatest distance from both cell poles. It turns out that E. coli and some other rod-shaped bacteria select their cell midpoint using such a negatively acting morphogen gradient, set up by the Min system, which is the focus of this Primer. As is true for many fascinating molecular mechanisms, the first inkling came from the behavior of cells in which this system was broken.

FtsZ, a bacterial homolog of eukaryotic tubulin, assembles into the Z ring required for cytokinesis. In Escherichia coli, FtsZ interacts directly with FtsA and ZipA, which tether the Z ring to the membrane. We used three-dimensional structured illumination microscopy to compare the localization patterns of FtsZ, FtsA, and ZipA at high resolution in Escherichia coli cells. We found that FtsZ localizes in patches within a ring structure, similar to the pattern observed in other species, and discovered that FtsA and ZipA mostly colocalize in similar patches. Finally, we observed similar punctate and short polymeric structures of FtsZ distributed throughout the cell after Z rings were disassembled, either as a consequence of normal cytokinesis or upon induction of an endogenous cell division inhibitor.