Periodic Reporting for period 4 - OMPorg (Spatiotemporal organisation of bacterial outer membrane proteins)
Reporting period: 2022-03-01 to 2023-08-31
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 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?
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?
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. We opted to abandon Objective three due to the greater progress made in other objectives.
Objective 1 – Molecular basis of OMP island formation. 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. We went on to use super-resolution microscopy to probe this organisation further. 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 developed tools for labelling OMPs in Pseudomonas aeruginosa and Klebsiella pneumoniae. The two most important discoveries from OMPorg relate to this objective.
First, we discovered that the spatiotemporal behaviour of OMPs is actually governed by the cell wall, which dictates the functionality of the BamA protein. This was a collaboration with Waldemar Vollmer's lab (Newcastle/Brisbane). This work demonstrated that Gram-negative bacteria coordinate growth of their cell wall with the outer membrane by rendering BamA responsive to the maturation state of the peptidoglycan.
Second, we discovered that the outer membrane is not simply an asymmetric lipid bilayer as discovered by Nikaido over 50 years ago but an asymmetric proteolipid membrane. OMPs are interlaced with LPS to form OMP-LPS-OMP networks that span the cell surface.
Objective 2 – Do OMPs influence the functions of proteins in the inner membrane of Gram-negative bacteria? We answered to this question. The two membranes of Gram-negative bacteria are separated by a 30 nm gap known as the periplasm. We showed 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 OMPs dictating how inner membrane proteins behave. Such connections have since been seen in the transenvelope LPS biogenesis machine by the Kahne lab in Harvard.
Objective 4 - Do OMP islands coordinate processes in the outer membrane? As a first step in addressing this fundamental problem, we developed strategies to simultaneously image (by super-resolution fluorescence microscopy) the location of LPS (lipid) in the outer membrane and OMPs. Through this work, we have discovered that LPS and OMPs exhibit very different spatiotemporal behaviour. This work is currently being prepared for publication.
Objective 1 – Molecular basis of OMP island formation. 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. We went on to use super-resolution microscopy to probe this organisation further. 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 developed tools for labelling OMPs in Pseudomonas aeruginosa and Klebsiella pneumoniae. The two most important discoveries from OMPorg relate to this objective.
First, we discovered that the spatiotemporal behaviour of OMPs is actually governed by the cell wall, which dictates the functionality of the BamA protein. This was a collaboration with Waldemar Vollmer's lab (Newcastle/Brisbane). This work demonstrated that Gram-negative bacteria coordinate growth of their cell wall with the outer membrane by rendering BamA responsive to the maturation state of the peptidoglycan.
Second, we discovered that the outer membrane is not simply an asymmetric lipid bilayer as discovered by Nikaido over 50 years ago but an asymmetric proteolipid membrane. OMPs are interlaced with LPS to form OMP-LPS-OMP networks that span the cell surface.
Objective 2 – Do OMPs influence the functions of proteins in the inner membrane of Gram-negative bacteria? We answered to this question. The two membranes of Gram-negative bacteria are separated by a 30 nm gap known as the periplasm. We showed 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 OMPs dictating how inner membrane proteins behave. Such connections have since been seen in the transenvelope LPS biogenesis machine by the Kahne lab in Harvard.
Objective 4 - Do OMP islands coordinate processes in the outer membrane? As a first step in addressing this fundamental problem, we developed strategies to simultaneously image (by super-resolution fluorescence microscopy) the location of LPS (lipid) in the outer membrane and OMPs. Through this work, we have discovered that LPS and OMPs exhibit very different spatiotemporal behaviour. This work is currently being prepared for publication.
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.
Our use of photoactivatable crosslinking into the outer membrane from OMPs was the means by which we discovered OMP-LPS-OMP networks in Gram-negative bacteria.
Our use of photoactivatable crosslinking into the outer membrane from OMPs was the means by which we discovered OMP-LPS-OMP networks in Gram-negative bacteria.