Periodic Reporting for period 3 - OMPorg (Spatiotemporal organisation of bacterial outer membrane proteins) Reporting period: 2020-09-01 to 2022-02-28 Summary of the context and overall objectives of the project OMPorg aims to discover how bacteria organise proteins embedded in their outer membrane and the consequences of this organisation to the organism. Bacteria are an integral part of our bodily physiology – it is estimated they outnumber our own cells around ten-fold – where they play important roles in, for example, digestion. Bacteria can also be harmful, causing a variety of diseases such as typhoid and cholera, a problem that is greatly exacerbated if the organism is resistant to antibiotics. Bacteria can be broadly classified as Gram-positive or Gram-negative; meaning they either do or do not stain with the classic Gram stain. The absence of staining is the hallmark of Gram-negative bacteria, which harbour a second membrane not found in Gram-positive bacteria. This robust ‘outer membrane’ serves many essential functions in Gram-negative bacteria such as protecting the organism against harsh environments, including our immune system, providing a means of adhering to surfaces where bacteria often accumulate, permitting the diffusion and import of nutrients into the cell and excluding major classes of antibiotics that kill Gram-positive bacteria. Consequently, the outer membrane is a complex and adaptable cellular structure that is important for the viability of the organism in different ecological niches and represents a target for new antibiotics. We know much about the composition of the outer membrane and the many proteins and lipids within it since the membrane was discovered over half a century ago. The dogma up until recently was that these components were randomly mixed in the membrane. In 2015 my laboratory discovered that the proteins in the outer membrane are organised into large supramolecular assemblies we call outer membrane protein (OMP) islands, which can be >0.5 µm in diameter. This discovery immediately answered a major question in outer membrane biology; how are OMPs, which drive most aspects of outer membrane function, changed from one generation to the next? In Escherichia coli - our model organism - the answer is that they are pushed outwards towards the two poles of the cell, somewhat like tectonic plates, during growth and replaced by centrally-located new OMP islands. The resulting binary partitioning results in half of bacterial cells having new OMPs after just two divisions. Organisation of proteins in bacteria is not a new concept but previous examples centred on the cytoplasm and the cytoplasmic (inner) membrane. The discovery of OMP islands as an organising principle in the outer membrane of Gram-negative bacteria raises many new questions about this remarkable cellular structure, questions that lie at the heart of OMPorg.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? Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The main focus thus far by the staff supported by OMPorg has been on objectives one, two and four, with additional work on a new objective linked to a fortuitous discovery we made while addressing these objectives. Objective three is still in development and will not be described further here. 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) As part of our investigations into OMP organisation we made an unexpected discovery regarding how Gram-negative bacteria stabilise their outer membrane during cell division. This discovery was based on a new method we developed in collaboration with Seán Murray (Max Planck, Marburg) for analysing fluorescence microscopy data (SpatialFRAP) for very slowly diffusing molecules in the outer membrane of live E. coli cells. The method proved crucial for interpreting the mobility of the outer membrane lipoprotein Pal (labelled with the fluorescent protein mCherry) because the diffusion of Pal varies both temporally and spatially during the E. coli cell cycle. Because Pal is simultaneously bound to the outer membrane (by a lipoyl tether) and the underlying cell wall (peptidoglycan) this reduces its mobility in the outer membrane. A requirement for cell division to proceed however is that Pal relocate en masse to stabilise the link between the outer membrane and cell walls of daughter cells. We found that it is the role of the Tol-Pal assembly, through its connection to the proton gradient across the inner membrane, to catalyse the accumulation of Pal at cell division sites. Tol-Pal exploits the energy of the cell to mobilise Pal in the outer membrane and then captures these mobilised molecules at the division site. This new mechanism, which we call mobilisation-and-capture, has solved a 60-year old problem in microbiology. 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. 3D-Structured illumination data (false colour) for BamA containing OMP islands in E. coli
