Periodic Reporting for period 2 - OMPorg (Spatiotemporal organisation of bacterial outer membrane proteins)
Reporting period: 2019-03-01 to 2020-08-31
OMPorg is important to society for three reasons. First, it will provide fundamental new knowledge about an aspect of bacterial membrane biology about which little is known. Since Gram-negative bacteria are evolutionary relatives of eukaryotic organelles such as mitochondria what we learn in OMPorg may be transferable to eukaryotes. Second, by furnishing important information on how the outer membrane is built and maintained, OMPorg will identify potential avenues for the pursuit of novel antibiotics aimed at its disruption. Gram-negative bacteria currently account for the majority of priority multidrug resistant bacterial pathogens identified by the world health organisation. Third, by understanding how OMPs are assembled into supramolecular assemblies in the outer membrane OMPorg will expedite biotechnological innovations aimed at exploiting bacteria as ‘cell factories’ for the production and secretion of important biomolecules.
OMPorg has four major objectives, each subdivided into multiple smaller components. The major objectives are as follows: First, what is the molecular basis of OMP island formation? Second, do OMPs influence the functions of proteins in the inner membrane of Gram-negative bacteria? Third, do repository cells endow bacterial populations with OMP memory? Repository cells are those cells that house the majority of old OMPs following cell division. Fourth, do OMP islands coordinate processes in the outer membrane?
Objective 1 – Molecular basis of OMP island formation. This was largest of the four aims with most resource (and hence manpower) given over to this objective. The reason for this is because we know so little about the composition of OMP islands; for example, do they also contain the major lipid in the outer membrane, lipopolysaccharide (LPS)? What is the diversity of OMPs within them? Do OMPs engage in promiscuous interactions with each other? Towards this end, we have developed several new fluorescent labels for OMPs, in addition to those we had originally used in our 2015 paper, to determine if our initial observations were correct. In particular, we were interested to find out if very abundant OMPs showed similar organisation to those we had originally observed for low abundance OMPs. While many OMPs behave exactly as we had previously observed we find that other OMPs show profound differences. We are currently using super-resolution microscopy methods to probe further into this organisation. Most of our work has been on the workhorse bacterium Escherichia coli. To validate if the behaviours we observe are also relevant for other bacteria, especially pathogenic bacteria, we have been developing new tools for labelling OMPs in Pseudomonas aeruginosa (one of the main causes of lung infections in cystic fibrosis patients) and Klebsiella pneumoniae (a major cause of sepsis in hospitals). This work is still ongoing. We have made major advances in our understanding of how the OMP biogenesis machine Bam is distributed in the outer membrane of E. coli. Using very specific, fluorescently-labelled antibodies we have been tracking the key biogenesis protein, BamA. BamA is the universally conserved biogenesis machine found in the outer membranes of all Gram-negative bacteria. We have made surprising discoveries about the distribution of BamA that run counter to the prevailing view in the literature-this work is currently being prepared for publication. We have made great progress in understanding the molecular composition of an OMP island. We’ve tackled this difficult problem by incorporating specific chemical entities into OMPs at defined locations within the membrane that become crosslinked to their neighbours when activated by UV light. The site of crosslinking is then identified by isolating the specific OMP (following isolation of the outer membrane fraction) and using liquid chromatography coupled to tandem mass spectrometry. This methodology took 18 months to develop but is now working well. We see important differences in how OMPs interact with one another in the membrane depending on the type of OMP. Further work is needed to understand these architectural differences.
Objective 2 – Do OMPs influence the functions of proteins in the inner membrane of Gram-negative bacteria? We have now demonstrated that the answer to this question is a definitive yes. The two membranes of Gram-negative bacteria are separated by a 30 nm gap known as the periplasm. We have shown that the highly restricted mobility of OMPs becomes imprinted on inner membrane proteins when the two become connected through the periplasm by energized protein bridges, which in turn leads to the OMP dictating how inner membrane proteins behave. Such connections are essential in Gram-negative bacteria; for example, it is the means by which energy-dependent uptake of essential vitamins and nutrients occurs. Up to now, there has been little exploration of the crosstalk between the two membranes in live bacteria. We plan on further work in this exciting area to see if the lessons we have learnt are applicable to different systems.
Objective 4 - Do OMP islands coordinate processes in the outer membrane? As a first step in addressing this fundamental problem, we are developing strategies to simultaneously image (by super-resolution fluorescence microscopy) the location of LPS (lipid) in the outer membrane and OMPs. This work is still at a preliminary stage but we have already obtained intriguing data which is being followed up.
Many of the approaches we are developing through OMPorg can already be described as ‘beyond the state of the art’ or least soon to be so. Defining the close neighbours of OMPs within OMP islands by photocrosslinking will for the first time explain the detailed connections of OMPs within large supramolecular complexes. It will also allow us to stabilise these platforms to enable their purification and subsequent analysis. The methods we are developing to simultaneously map LPS and OMPs in the outer membrane of E. coli live cells are at the forefront of bacterial imaging technology and promise to reveal for the first time how bacteria organise their outer membrane.