Understanding the linked roles of polyphosphate and the stringent response in bacterial physiology - Project Summary Bacteria must be able to sense and coordinate an appropriate response quickly to ensure their continued survival in their constantly changing environments. Bacteria must be able to respond to a wide variety of stressors including DNA damage, nutrient starvation, heat, cold, acid, toxins, and others. To survive, bacteria have developed several conserved stress response pathways such as the well-known stringent response triggering production of (p)ppGpp in response to amino acid starvation and other stressors. Another stress sensing system found in nearly all living cells, from bacteria to eukaryotic cells, is inorganic polyphosphate (polyP), which contributes to the survival and virulence (ability to cause infection or harm) of bacterial cells. In E. coli, there was a long-standing hypothesis that that (p)ppGpp directly stimulated the production of polyP, based on the observation that (p)ppGpp inhibits exopolyphosphotase (PPX), reducing the degradation of polyP. Recent work from our lab, however, has found that polyP production is not dependent on (p)ppGpp, but now suggests previously unknown interactions between polyP and the stringent response. Both ∆relA ∆spoT mutants lacking (p)ppGpp and ∆ppk mutants lacking polyP are well-known for their multiple amino acid requirements. We were therefore surprised to find that an E. coli triple ∆ppk ∆relA ∆spoT mutant fails to grow on MOPS minimal media even when supplemented with casamino acids (CAA). Growth of the ∆ppk ∆relA ∆spoT strain is rescued by a currently unknown small molecule component of yeast extract, but under these conditions, our preliminary data shows severe and unexpected morphological defects in the bacterial cells, including filamentous and branching cells, and a strikingly high rate of mis-localized FtsZ rings. These data suggest unexpected and possibly redundant roles for polyP and (p)ppGpp outside of their canonical roles in stress response, survival and virulence. This suggests these two disparate, yet fundamentally conserved stress response pathways may be linked together and play a novel role in controlling the fundamental processes of cell division, elongation, and cell wall synthesis. Notably, these core biological functions tend to be highly conserved among bacterial species and essential for cell survival and are the primary targets of antibiotic therapies. The essentiality of polyP, SpoT and the RelA/SpoT homologs found in other bacteria make these attractive targets for antibiotic development, as an interruption of the stringent response or polyP makes cells at least unable to respond properly to cell stress and host defenses and can, under some circumstances, kill bacterial cells outright. Our goal is to elucidate the molecular mechanism of the interactions between (p)ppGpp and polyP and how they affect the growth, division, and metabolism of the bacterial cell.
