By Aritha Dornau
In many bacteria the Phosphotransferase System (PTS) is the main route for importing key sugars such as glucose, maltose and mannitol from the environment. Sugars transported via the PTS become phosphorylated by their associated transporter: glucose, for example, is converted to glucose-6-phosphate and can then enter directly into glycolysis. Several key PTS proteins operate upstream of transport, executing a cascade of phosphorylation reactions that ultimately prime transporters for phospho-transfer. PTS activity has also been shown to have substantial downstream affects, regulating major cellular processes such as chemotaxis.
Previous studies have largely focused on characterising individual branches of the PTS network and so far the global architecture of this complex system has been difficult to piece together. In order to gain a more holistic view of the PTS, Victor Sourjvik’s group at the Max Plank Institute for Terrestrial Microbiology are exploring the interactions between a range of proteins within the PTS network. Using Förster resonance energy transfer (FRET), a technique that can detect when fluorescently labelled proteins come into close proximity with one another, they were able to investigate the dynamics of protein interactions within the PTS of Escherichia coli.
Intracellular FRET was used to test interactions between more than sixty protein pairs within the PTS network, revealing nine pairs that responded to PTS sugars, four of which responded in a stimulation-dependent manner. PTS activity was also observed upon stimulation with non-PTS sugars and compounds such as serine, pyruvate, glycerol and oxaloacetate, while compounds that were previously hypothesised to play a role in PTS regulation, such as glucose-6-phosphate, did not elicit a measurable response.
The study also demonstrated that all FRET pairs with a cytoplasmic PTS component were recruited to membrane transporters upon stimulation with PTS sugars. Converging at the site of transport is likely advantageous to the cell as the reduction in diffusion time increases reaction efficiency. Interestingly, recruitment did not require stimulation by a transporter’s cognate sugar, and was even observed with non-PTS sugars. If a cell detects one type of sugar in its environment it is possible that others are close by, so it would make sense to immediately prepare for uptake of other sugars. Furthermore, for several FRET pairs, the amplitude of the measured response was the same regardless of which PTS sugar was used to stimulate the cell, as illustrated in the figure below. This indicates that the PTS is not intrinsically biased toward any particular sugar and that cells use the PTS network to sense sugar influx on a global scale.
All FRET experiments were carried out in buffer using naïve E. coli cells that were initially grown on amino acids and had never been exposed to sugars. The observed FRET responses therefore reflect the basal state of the PTS network. Exposure to a sugar initiates transcription of catabolic enzymes and transporters to maximise exploitation of the new carbon source, resulting in the sugar-specific preferences observed in many microorganisms. Thus the uptake rates measured in these experiments do not necessarily reflect rates observed in actively growing cells. Surprisingly, upon measuring the basal uptake rates of PTS sugars, the group found that sugars with a higher metabolic efficiency had higher basal uptake rates – indicating that E. coli may be evolutionarily optimised for growth on different carbon sources.
To investigate this further the authors used mathematical modelling to explore how the rate of biomass production correlates with the basal rate of sugar uptake. The model demonstrated that, in accordance with the experimental data, the basal sugar uptake rate increases linearly with metabolic efficiency. Energy expended on sugar catabolism must be delicately balanced with energy used for sugar transport, therefore metabolic pre-optimisation may help E. coli to rapidly maximise its growth rate when encountering a mixture of sugars.
The E. coli chemotactic response to glucose is known to be stimulated by both the glucose chemoreceptor and the PTS. To get a better idea of how PTS signals propagate through to the chemotaxis network, the group looked at the FRET response of PTS network proteins and chemotaxis regulatory proteins in an E. coli strain lacking a glucose-inducible chemoreceptor. The measured responses showed a linear correlation between the FRET signals with increasing glucose levels, providing further evidence that the PTS plays a role in chemotaxis and showing that PTS-induced chemotaxis operates autonomously from chemoreceptor-induced responses.
This research has provided significant insights into the dynamic nature of the PTS and its integrated role in bacterial physiology. Further work in this fundamental area will be highly impactful, as a better understanding of the mechanisms that allow hungry bacteria to sense sugars in the environment and respond optimally is valuable for the myriad of biotechnological applications that require efficient sugar exploitation to facilitate industrial scalability.
Source: Somavanshi, R., et al. (2016). “Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli.” PLoS Biol 14(8): e2000074.