Cell size varies greatly among different types of cells, but the range in size that a specific cell type can reach is limited. A longstanding question in biology is how cells control their size. Escherichia coli adjusts size and growth rate according to the availability of nutrients so that it grows larger and faster in nutrient-rich media than in nutrient-poor media. Here, we describe how, using classical genetics, we have isolated a remarkably small E. coli mutant that has undergone a 70% reduction in cell volume with respect to wild type. This mutant lacks FabH, an enzyme involved in fatty acid biosynthesis that previously was thought to be essential for the viability of E. coli. We demonstrate that although FabH is not essential in wild-type E. coli, it is essential in cells that are defective in the production of the small-molecule and global regulator ppGpp. Furthermore, we have found that the loss of FabH causes a reduction in the rate of envelope growth and renders cells unable to regulate cell size properly in response to nutrient excess. Therefore we propose a model in which fatty acid biosynthesis plays a central role in regulating the size of E. coli cells in response to nutrient availability.acteria regulate their size and growth rate in response to nutrient availability. For example, Escherichia coli, Salmonella typhimurium, and Bacillus subtilis cells grow larger and faster in nutrient-rich medium than in nutrient-poor medium (1-6). Changes in temperature can alter growth rate but not size (2). Therefore, the size a cell attains depends on the nutritional composition of the growth medium, suggesting that nutrients affect a rate-limiting step(s) that controls size and the rate of growth.Bacteria must coordinate cell size, growth rate, and division in response to nutrient availability. Indeed, when E. coli changes its size, it also changes its generation time inversely; however, it maintains the cell mass-to-DNA ratio constant because it initiates DNA replication whenever it reaches a particular cell mass or a multiple of that mass (6). Interestingly, recent studies have shown that B. subtilis and E. coli use different regulatory mechanisms to couple cell size and DNA replication (4, 7). In E. coli DNA replication is not initiated until the cell reaches an appropriate size, but size does not affect the timing of replication in B. subtilis. Nevertheless, the amount of active DnaA, which unwinds DNA at the origin and thereby triggers replication (8, 9), is relevant in controlling the initiation of replication in both bacteria (7). Furthermore, a metabolic pathway for glucolipid biosynthesis regulates cell size in B. subtilis in response to nutrients under conditions that promote rapid growth (4). In this pathway, the UDP-glucose transferase UgtP inhibits the assembly of the divisome, the division machinery. The levels and localization of UgtP vary with nutrient availability so that assembly of the divisome is delayed under nutrient-rich conditions, resulting in longer cells.We do not understand how nu...

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.T he cell envelope of most bacteria contains a cell wall exoskeleton composed of peptidoglycan (PG) that surrounds the cytoplasmic membrane (1, 2). The rigid PG structure protects the bacterium from osmotic rupture, serves as a scaffold onto which other envelope components are attached, and defines cell shape. Underscoring the essentiality of the PG cell wall is the fact that many antibiotics target PG biosynthesis (3).Bacteria build their PG sacculus by polymerizing an N-acetylglucosamine-N-acetylmuramic acid (GlcNAc-MurNAc) disaccharide-pentapeptide into long glycan chains that are cross-linked by peptide bonds between stem peptides (2). This GlcNAcMurNAc disaccharide-pentapeptide is synthesized at the cytoplasmic side of the membrane as a polyisoprenyl lipid-linked precursor known as lipid II (Fig. 1A) (4). Because lipid II polymerization occurs at the extracytoplasmic side of the membrane, an obligatory step in PG biosynthesis is the translocation of the lipidlinked disaccharide-pentapeptide across the cytoplasmic membrane.The use of polyisoprenyl lipid-linked precursors in the biogenesis of envelope glycopolymers is widespread in bacteria. Examples include the biogenesis of PG, certain capsules and exopolysaccharides, and O antigens (5, 6). In these systems, bacteria build each precursor ...

The assembly of β-barrel proteins into membranes is mediated by an evolutionarily conserved machine. This process is poorly understood because no stable partially folded barrel substrates have been characterized. Here, we slowed the folding of the Escherichia coli β-barrel protein, LptD, with its lipoprotein plug, LptE. We identified a late-stage intermediate in which LptD is folded around LptE, and both components interact with the two essential β-barrel assembly machine (Bam) components, BamA and BamD. We propose a model in which BamA and BamD act in concert to catalyze folding, with the final step in the process involving closure of the ends of the barrel with release from the Bam components. Because BamD and LptE are both soluble proteins, the simplest model consistent with these findings is that barrel folding by the Bam complex begins in the periplasm at the membrane interface.outer membrane | Bam complex | β-barrel | protein folding T he assembly of β-barrel membrane proteins into the outer membrane (OM) of Gram-negative bacteria, mitochondria, and chloroplasts is facilitated by conserved cellular machinery (1-4). The β-barrel assembly machine (Bam) folds and inserts integral membrane proteins into the OM of Gram-negative organisms (5). Bam is a five-protein complex consisting of the essential protein BamA, a β-barrel itself, and four lipoproteins, BamB, -C, -D, and -E, of which only BamD is essential (4-8). The Bam complex recognizes a large number of different substrates, but how each component catalyzes the folding and insertion of such structurally diverse substrates is unclear.How β-barrels are assembled into membranes is not obvious. Where and how folding occurs is unclear because intermediates could contain both exposed polar amides and hydrophobic residues until the barrel has completed its fully hydrophobic exterior. By contrast, α-helical membrane proteins have internally satisfied hydrogen bonds, making stepwise assembly from stable secondary structural elements possible. Although Bam has been shown to accelerate membrane β-barrel assembly (9-11), the transient nature of folding intermediates has made accumulating such discrete species for characterization difficult (12-15). If structurally defined folding intermediates were to exist long enough for characterization, they could reveal crucial aspects of the folding process.Here, we studied the assembly of an essential, slow-folding β-barrel, LptD. LptD is one of two components of the OM translocon that transports lipopolysaccharide to the cell surface (16-18). The other component, LptE, is a lipoprotein that forms a plug inside the LptD barrel (19)(20)(21)(22). LptD also contains two disulfide bonds (23), and its assembly involves the formation of consecutive disulfide bonds that after barrel folding rearrange to form nonconsecutive disulfide bonds (24). The assembly of LptD is orders-ofmagnitude slower (∼20 min versus seconds) than that of other barrel substrates (24-26). Because of the slow rate of folding and our ability to use oxidation sta...

Gram-negative bacteria like Escherichia coli are characterized by having two membranes. Systems required for the biogenesis of the Gram-negative outer membrane have been identified except for that required for the transport of newly synthesized phospholipids from the inner to the outer membrane.

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.