The cell envelope of Gram-negative bacteria is composed of the inner and outer membranes (IM and OM, respectively), an aqueous compartment known as the periplasm, and a cell wall composed of peptidoglycan. Proper envelope biogenesis is crucial for the bacterium and it requires the coordinated synthesis, transport and assembly of all its components. In our laboratory, we use genetic and biochemical approaches to understand envelope biogenesis in the Gram-negative bacterium Escherichia coli.
Understanding peptidoglycan biogenesis
The peptidoglycan cell wall is a glycopeptide polymer composed of glycan chains that are cross-linked through stem peptides. The peptidoglycan cell wall is essential for viability as it protects bacteria from osmotic lysis. In addition, the peptidoglycan mesh determines cell shape and serves as a scaffold for other envelope structures.
As cells grow and divide, they must incorporate newly synthesized peptidoglycan into the preexisting cell wall. To accomplish this, the cell first synthesizes a lipid-linked peptidoglycan precursor, known as lipid II, in the cytoplasmic leaflet of the IM. Then, lipid II is transported across the IM so that its disaccharide-peptide moiety is incorporated into the preexisting periplasmic peptidoglycan structure. The addition of the new material into the cell wall involves the actions of transglycosylases, transpeptidases, and hydrolases. Our laboratory studies the function of MurJ, the flippase that transports lipid II across the IM.
Understanding LPS biogenesis
In Gram-negative bacteria, the OM serves as the main protective barrier against toxic molecules present in the environment. Because of the impermeability of its OM, E. coli and other Gram negatives are naturally resistant to many antibiotics. The main component of the OM responsible for providing this barrier-like quality to the OM is LPS, a large lipopolysaccharide that is located at the cell surface.
LPS is synthesized in the IM, so it must be transported across the cell envelope before it is assembled at the cell surface. The ATP-binding cassette (ABC) transporter MsbA flips LPS across the IM. Then, the Lpt trans-envelope complex mediates the extraction of LPS from the IM and its transport across the aqueous periplasmic compartment and the OM. The Lpt complex is composed of seven different proteins that are essential for LPS transport and viability of E. coli. In our laboratory, we investigate how the Lpt machine assembles and functions to transport LPS across the cell envelope.
Natividad Ruiz's Curriculum Vitae
- Bertani, B.R. Taylor, R.J, Nagy, E., Kahne, D*. and Ruiz, N*. A cluster of residues in the lipopolysaccharide exporter that selects substrate variants for transport to the outer membrane. Mol Microbiol (in press) doi: 10.1111/mmi.14059. PMID: 29995974.
- Bertani, B. R. and Ruiz, N. (2018) Function and biogenesis of lipopolysaccharides. Eco Sal Plus 2018; doi:10.1128/ecosalplus.esp-0001-2018 (in press). PMID: 30066669.
- Rubino, F.A., Kumar, S., Ruiz, N., Walker, S., Kahne, D. (2018) Membrane potential is required for MurJ function. J Am Chem Soc. 140(13):4481-4484. PMID: 29558128.
- May, J.M., Owens, T., Mandler, M., Simpson, B.W., Lazarus, M., Sherman, D.J., Davis, R.M., Okuda, S., Massefski, W., Ruiz, N., Kahne, D. (2017) The antibiotic novobiocin binds and activates the ATPase that powers lipopolysaccharide transport. J Am Chem Soc 139(48):17221-17224. PMID: 29135241
- Chamakura, K.R., Sham, L.T., Davis, R.M., Min, L., Cho, H., Ruiz, N., Bernhardt, T.G., Young, R. (2017) A viral protein antibiotic inhibits lipid II flippase activity. Nature Microbiol 2(11):1480-1484. PMID: 28894177.
- Elhenawy, W., Davis, R.M., Fero, J, Salama, N.R., Feldman, M.F., Ruiz, N. (2016) The O-antigen flippase Wzk can substitute for MurJ in peptidoglycan synthesis in Helicobacter pylori and Escherichia coli. PLoS ONE. 11(8):e0161587. PMID: 27537185.
- Simpson, B.W., Owens, T.W., Orabella, M.J., Davis, R.M., May, J.M., Trauger, S.A., Kahne, D., Ruiz, N. (2016) Identification of residues in the lipopolysaccharide ABC transporter that coordinate ATPase activity with extractor function. mBio 7(5): e01729-16. PMID: 27795402.
- Ruiz, N. (2016) Filling holes in peptidoglycan biogenesis of Escherichia coli. Curr Opin Microbiol 34:1-6. PMID: 27449418.
- Okuda, S., Sherman, D.J., Silhavy, T.J., Ruiz, N., Kahne, D. (2016) Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model. Nat Rev Microbiol 14:337-45. PMID: 27026255.
- Ruiz, N. (2016) Lipid flippases for bacterial peptidoglycan biosynthesis. Lipid Insights 8(s1) 21–31. PMID: 26792999.
- Simpson, B.W., May, J.M., Sherman, D.J., Kahne, D., Ruiz, N. (2015) Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane. Phil Trans R Soc B 370:20150029. PMID: 26370941.
- May, J.M., Sherman, D.J., Simpson, B.W., Ruiz, N., Kahne, D. (2015) Lipopolysaccharide transport to the cell surface periplasmic transport and assembly into the outer membrane. Phil Trans R Soc B 370:20150027. PMID: 26370939.
- Butler, E.K., Tan, W.B., Joseph, H., and Ruiz, N. (2014) Charge requirements of lipid II flippase activity in Escherichia coli. J Bacteriol 196:4111-4119. PMID: 25225268.
- Sham, L. T., Butler, E. K., Lebar, M. D., Kahne, D., Bernhardt, T.G., and Ruiz, N. (2014) MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis. Science 345:220-222. PMID: 25013077.
- Malojčića, G., Andresa, D., Grabowicz, M., Ruiz, N., Silhavy, T. J., and Kahne, D. (2014) LptE binds to and alters the physical state of LPS to catalyze its assembly at the cell surface. Proc Natl Acad Sci U S A 111:9467-9472. PMID: 24938785.
- Sherman, D.J., Lazarus, M.B., Murphy, L., Liu, C., Walker, S. Ruiz, N., and Kahne, D. (2014) Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport. Proc Natl Acad Sci U S A 111:4982-4987. PMID:24639492
- Butler, E.K., Davis, R.M., Bari, V., Nicholson, P.A., and Ruiz, N. (2013) Structure-function analysis of MurJ reveals a solvent-exposed cavity containing residues essential for peptidoglycan biogenesis in Escherichia coli. J Bacteriol 195:4639-4649. PMID:23935042.
- Yao, Z., Davis, R.M., Kishony, R., Kahne, D., and Ruiz, N. (2012) Regulation of cell size in response to nutrient availability by fatty acid biosynthesis in Escherichia coli. Proc Natl Acad Sci U S A 109:E2561-E2568. PMID:22908292.
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