## 4 Roles of PPK2s in Bacterial Physiology and Virulence
### Bacterial Homeostasis and Stress Response  
_P. aeruginosa_ has among the best-characterized PPK2 physiology to date. The Kornberg lab's initial studies of _P. aeruginosa_ PPK2A (PA0141) revealed that expression of this enzyme was induced over 100-fold at stationary phase [34], suggesting its importance for stress survival. It is important to note that _P. aeruginosa_ encodes three PPK2 enzymes: PPK2A (PA0141), PPK2B (PA2428), and PPK2C (PA33455), in addition to PPK1 (PA5242). Work by [PERSON] et al. demonstrated that a quadruple knockout strain of _P. aeruginosa_ PA14 lacking all four PPK enzymes (\(\Delta polyP\)) was defective in cell cycle exit, which is dependent on the formation of organized polyP granules [39]. Importantly, there was no significant difference in growth kinetics between WT and \(\Delta polyP\) PA14 [57]. As expected, the \(\Delta polyP\) strain did not produce any detectable polyP, but a strain that retained functional PPK2A (\(\Delta ppk\Delta ppk2B\Delta ppk2C\)) still formed polyP granules and exhibited near-normal cell cycle exit. In contrast, individual presence of chromosomally encoded \(ppk2B\) or \(ppk2C\) was insufficient for polyP granule formation. Interestingly, when \(ppk2B\) was re-introduced on a plasmid, it was sufficient to generate polyP granules [39], suggesting that PPK2B could also compensate for the loss of PPK1, provided that conditions are suitable for expression or otherwise full induction of enzymatic activity. In _Campylobacter jejuni_, \(ppk2\) deletion resulted in decreased polyP-dependent GTP synthesis. This yielded an increased intracellular ATP:GTP ratio but had no effect on  
Figure 3: Schematic of bacterial virulence phenotypes linked to PPK2 enzymes. Figure created using BioRender. polyP or guanosine tetraphosphate (ppGpp) alarmone levels. _ppk2_ deletion was also associated with significant variations in transcriptional abundance. For example, expression of the gene encoding SpoT--an enzyme which synthesizes and hydrolyzes ppGpp--was upregulated over 25-fold in the \(\Delta\)_ppk2_ mutant, which the authors hypothesize may serve as a compensatory mechanism to keep ppGpp levels low. The _C. jejuni ppk2_ mutant was also defective in survival during stationary phase nutrient starvation, osmotic stress, and aerobic stress [48]. A _ppk2_ mutant strain of _Mycobacterium smegmatis_ displayed a similarly elevated ATP:GTP ratio, indicating that PPK2 modulates nucleotide pools in vivo. _M. smegmatis_ PPK2 has been shown to directly bind to nucleoside diphosphate kinase (Ndk) to direct its activity towards synthesis of GTP as opposed to CTP or UTP, which may contribute to the elevated ATP:GTP ratio. The _M. smegmatis ppk2_ mutant was also compromised in survival following thermal, acidic, and hypoxic stress. Double knockout of both _ppk2_ and _ppk1_ in _M. smegmatis_ appears to be lethal [54], and repeated attempts to construct a _C. jejuni ppk1-ppk2_ double knockout were also unsuccessful [48], suggesting a critical role of polyP in these species. In _Mycobacterium tuberculosis_, a _ppk2_ transposon mutant accumulated significantly more intracellular polyP despite also exhibiting significantly reduced _ppk1_ gene expression [41]. The _M. tuberculosis ppk2_ mutant also accumulated less glycerol-3-phosphate, an important phospholipid precursor, and 1-deoxy-xylulose-5-phosphate, a peptidoglycan biosynthetic metabolite [74]. Remarkably, _R. eutropha_--an environmental bacterium known for its ability to grow using hydrogen as the electron donor--encodes seven _ppk_ genes: two _ppk1_ and five _ppk2_ genes. At least three of these enzymes (PPK1a, PPK2b, and PPK2c) contribute to polyP synthesis and at least four of them (PPK1a, PPK2c, PPK2d, and PPK2e) colocalize with polyP granules in vivo [40]. Overexpression of plasmid-encoded _ppk2c_ in an _R. eutropha_ polyP-free background (\(\Delta\)_ppk-all_) lacking all seven PPKs was sufficient to generate large polyP granules [75]. To study the contribution of these enzymes to _R. eutropha_ physiology, [PERSON] et al.