Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

The Gram-negative bacterial periplasm; Architecture significance (#126)

Eric JL Mandela 1 , Von L Torres 2 , Christopher J Stubenrauch 1 , Chaille T Webb 1 , Cheng Huang 3 , Ralf Schittenhelm 3 , JC Gumbart 4 , Iain D Hay 5 , Trevor Lithgow 1
  1. Infection and Immunity Program , Biomedicine Discovery Institute and Department of Microbiology, Monash University, , Clayton, Victoria, Australia
  2. Diamantina Institute, University of Queensland , Brisbane, Queensland, Australia
  3. Monash Proteomic & Metabolomic Facility,, Clayton, 3800, Australia , Monash University, Clayton, VIC, Australia
  4. School of Physics, , Georgia Institute of Technology,, Atlanta, , Georgia, USA
  5. Biological sciences , University of Auckland, , Auckland, New Zealand

Here we investigated the key player in the bacterial cell envelope organisation; a small, alpha-helical lipoprotein protein Lpp, AKA Braun’s lipoprotein. Lpp provides the only covalent connection between the outer membrane (OM) and the peptidoglycan (PG). The N-terminus is attached to the OM while the C-terminus is attached to the PG. Recent work has shown that Lpp primarily controls the width of the periplasm. Lengthening of Lpp allowed the expansion of the periplasm relative to the number of heptad repeats inserted within the Lpp sequence.

The bacterial cell envelope is the interface through which bacteria interact with their environment, everything going into or out of the cell must transit it, and many important cellular functions take place within its enclosed periplasm: cell division regulation, osmoregulation, peptidoglycan synthesis, multidrug efflux systems, and many others. We sought to use a synthetic lethal genetic screening approach to identify periplasmic processes that are dependent on the periplasmic architecture. In addition, we performed RNA-seq and relative quantitative proteomics on the strain with an enlarged periplasm, to investigate its adaptive response to the architectural change.  Here we report the effect of a widened periplasm on peptidoglycan assembly. The activation of the major penicillin-binding proteins (PBP1A and PBP1B) by their cognate lipoproteins (LpoA and LpoB) is compromised which leads to synthetic lethal phenotype when either of the redundant pathways is removed. We also show the significance of OmpA, Pal,  YiaD and TolC in the non-covalent binding of the PG. These genetic screen results are further studied to better understand the role of periplasmic architecture in Gram-negative bacteria.

  1. Asmar, A. T., Ferreira, J. L., Cohen, E. J., Cho, S. H., Beeby, M., Hughes, K. T., & Collet, J. F. (2017). Communication across the bacterial cell envelope depends on the size of the periplasm. PLoS Biol, 15(12), e2004303. doi:10.1371/journal.pbio.2004303
  2. Cohen, E. J., Ferreira, J. L., Ladinsky, M. S., Beeby, M., & Hughes, K. T. (2017). Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane. Science, 356(6334), 197-200. doi:10.1126/science.aam6512