Probing the Cell Growth-Dependent Spatial Distribution and Dynamics of Proteins Inserted in the Bacterial Outer Membrane

Abstract

The Gram-negative cell envelope incorporates two lipid bilayers: an inner membrane (IM) with a protein and phospholipid composition characteristic of a typical cell membrane and an atypical, asymmetric outer membrane (OM). Protein insertion and time-dependent re-distribution in the IM is fairly well understood, but these processes remain poorly characterised in the OM. The pattern of insertion for an endogenous OM porin over-produced in Escherichia coli and its rearrangement during cell growth has recently been visualised. In this published work, distinct fluorescent puncta coincident with newly inserted proteins appeared randomly in the OM as discrete bursts, and they did not re-distribute appreciably during cell growth. We build on this previous work by addressing whether protein production levels characteristic of normal exponential cell growth alter the protein insertion process in E. coli JM83. To specifically label low-abundance outer membrane receptors (BtuB and Cir), and avoid the problems of over-expression, we have utilised organic dye-labelled colicin molecules (colicins E9 and Ia) engineered to retain high-affinity receptor binding (Kd ≈ pM) and prevent OM translocation. This allowed us to follow insertion of BtuB and Cir in live cells and their re-distribution during cell growth and binary fission using both fluorescence recovery after photo-bleaching (FRAP) confocal microscopy and single-molecule total internal reflection fluorescence (TIRF) microscopy. Single particle tracking and FRAP microscopy of colicin-receptor complexes revealed that BtuB and Cir are sequestered to randomly positioned patches in the OM, which persist during cell growth, and through multiple rounds of binary fission. Work is underway to correlate the random sites of receptor protein insertion with the periplasmic/OM molecular machinery necessary to mediate this process.