PolyP levels during exponential growth and the stationary phase
In M. xanthus, polyP was not synthesized during the exponential growth phase, but was synthesized during the stationary phase (Fig. 1A). This result was consistent with previously reported results for M. xanthus, Bacillus cereus, and Escherichia coli (Rao et al., 1998; Shi et al., 2004; Zhang et al., 2005). PolyPs in M. xanthus cells were gradually produced from 20 h of culture (early stationary phase) and increased rapidly at 36 to 40 h of culture (mid-stationary phase). Thereafter, the amount of polyPs was maintained even during the death period of 50 to 60 h of culture. PolyP synthetic activity was also absent during the exponential growth phase but was induced during the stationary phase (24 to 60 h of culture) (Fig. 1B). We previously reported that M. xanthus Ppk1 synthesizes polyPs using high concentrations of phosphate (0.5 to 10 mM) and pyrophosphate (0.5 to 5 mM) as primers (Harita et al., 2023). Therefore, it was presumed that polyP in stationary-phase cells of M. xanthus may be synthesized by transferring the phosphate at the γ-position of ATP to intracellular phosphate and pyrophosphate as an initial primer.
Urea-PAGE analysis of the extracted polyPs revealed that those with chains longer than 1,000 residues were synthesized at 32 h of culture (Fig. 1C). At 57 h of culture, the long-chain polyPs disappeared and appeared at the same position as the polyP60–70 standard. Since degradation activity was consistently observed from the exponential growth phase to the stationary phase (Fig. 1B), Ppx2 likely digested the accumulated long-chain polyPs. These results suggest that polyPs in M. xanthus are synthesized from the early to mid-stationary phases but are degraded from the late-stationary phase to the death phase.
The ppk1 and ppx2 mutants, as well as the wild-type strain, grew during the exponential growth phase (data not shown), but the ppk1 mutant died earlier than the wild-type strain and the ppx2 mutant after the late stationary phase (Fig. 1D). These results suggest that polyP plays an important role in the survival of M. xanthus from the late stationary phase to the death phase.
PolyP synthesis under various stress conditions
We measured polyP levels in M. xanthus cells under various stress conditions (Fig. 2A). PolyP synthesis was induced by high-temperature, oxidative, and phosphate starvation stresses. When 0.07 mM H2O2 was added to vegetative cells in the CTT liquid medium, polyp levels increased approximately eight-fold after 6 h of incubation (Fig. 2B). After the addition of 0.07 mM H2O2, polyP synthetic activity increased from 2 h and peaked at 4 h. During this period, the degradation activity for polyP3 and polyP60 − 70 was observed and remained unchanged. Meanwhile, the addition of 0.35 mM H2O2 increased intracellular polyP levels by only 1.2-fold. This may be because the addition of 0.35 mM H2O2 killed most of the cells.
The E. coli ppk mutant showed a similar degree of sensitivity to low H2O2 concentrations (2.5 to 10 mM) but the mutant was much more sensitive to higher H2O2 concentrations of (15 to 45 mM) than the wild-type strain (Rao NN, Kornberg A 1996). Furthermore, Acinetobacter baumannii Ppk1 was essential for its persistence maintenance to resist stimuli of 0.05 mM H2O2 stress (Lv et al., 2022). However, no significant difference in sensitivity to 0.07 or 0.35 mM H2O2 was observed between the M. xanthus wild-type, ppk1 mutant, and ppx2 mutant cells (data not shown). In E. coli, catalase activity in ppk1 mutant cells was 35–60% of that of the wild-type cells (Rao NN, Kornberg A 1996). Catalase activity in M. xanthus cells is induced by the addition of H2O2 (Kimura et al., 2022). When catalase activity was measured by adding 0.07 mM H2O2 to cultured cells in the CTT medium, the catalase activity of ppk1 mutant cells was 1.1–1.4 times higher than that of wild-type cells (Fig. 2C), which was different from that of the ppk mutant strain of E. coli. In contrast, the catalase activity of ppx2 mutant cells was 1.2–1.6-fold lower than that of wild-type cells (Fig. 2C). Moreover, 0.01 mM polyP700 − 1000 inhibited catalase activity in M. xanthus by 15% (data not shown), which may explain why the catalase activity of the ppk1 or ppx2 mutant was higher or lower than that of the wild-type, respectively.
The optimal temperature for M. xanthus growth is 30°C, and it grows but dies quickly at 37°C (heat stress). When M. xanthus cells cultured at 30°C in the CTT liquid medium were incubated at 37°C for 6 h, polyP levels in M. xanthus cells increased approximately three-fold (Fig. 2A). However, there was no difference in the growth and death at 37°C among the wild-type, ppk1 mutant, and ppx2 mutant strains (data not shown). No increase in polyPs was observed under osmotic stress of 0.25M NaCl. Meanwhile, it was reported that the E. coli ppk mutant showed greater sensitivity to heat at 55°C and to an osmotic challenge with 2.5 M NaCl (Rao and Kornberg 1996). These results suggest that polyP synthesis is induced by oxidative and heat stresses in M. xanthus, but polyP is not involved in their stress tolerance.
Incubation of exponentially growing M. xanthus cells cultured for 6 h in phosphate-free CTT liquid medium resulted in an approximately eight-fold increase in the amount of intracellular polyPs. When the wild-type, ppk1 mutant, and ppx2 mutant strains were grown in phosphate-free CTT liquid medium, there was no difference in the growth of these strains during the exponential growth phase, whereas the ppk1 mutant died early (Fig. 2D). However, even in the CTT medium containing phosphate, the ppk1 mutant died early in the stationary phase (Fig. 1D), suggesting that polyP production induced by phosphate deficiency does not play a role in the growth of M. xanthus under phosphate starvation.
PolyP levels during developmental phases
On starvation-induced agar medium (CF agar), approximately 100,000 cells of M. xanthus aggregate to form a fruiting body by gliding, within which individual cells develop into myxospores. We previously reported that polyPs were rapidly synthesized in developmental M. xanthus cells on CF agar but were degraded by Ppx1 and Ppx2 during the fruiting body formation and sporulation stage, suggesting that mature spore formation of M. xanthus may not require long-chain polyPs (Fig. 3). The ppk1 mutant did not synthesize polyP, and the amount of polyPs synthesized by the ppx1 mutant did not significantly differ from that of the wild-type strain (Fig. 3). Since M. xanthus had a polyP3-degrading enzyme other than Ppx1 (Harita et al, 2023), and Ppx2 can degrade polyPs longer than polyP4, the polyP levels of the ppx1 mutant strain should not differ from the wild-type strain. Meanwhile, the ppx2 mutant showed an accumulation of long-chain polyPs in the exponential growth phase (0 h), which further increased during development. At 72 h of development, the wild-type strain contained more short-chain polyPs than the polyP60 − 70 standard, whereas the ppx2 mutant strain accumulated short- to long-chain polyPs. The amount of polyP synthesized by the ppx2 mutant strain was approximately four-fold higher than those of the wild-type strain.
Although there were differences in polyP accumulation between the wild-type, ppk1, and ppx2 mutant strains during development, there were no clear differences in fruiting body and spore formation between these strains (Fig. 4). In addition, there was no clear difference in sporulation between the ppx1 and pap mutant strains and the wild-type strain. This result suggested that polyP synthesis and accumulation did not affect starvation-induced fruiting body and sporulation in M. xanthus. Zhang et al., (2005) reported that the M. xanthus ppk1 mutant on the TPM (10 mM Tris-HCl, pH 8.0, 8 mM MgSO4, and 1 mM KPO4, pH 7.6) agar plate was delayed in aggregation and altered in fruiting body morphology; the number of spores formed by the ppk1 mutant was 32% of the wild-type strain. Spore count measurements in M. xanthus are prone to error, and we could not find clear differences between the wild-type and these mutants. The Bacillus cereus ppk mutant also showed similar sporulation efficiency as the wild-type strain (Shi et al., 2004).
The amount of Mg 2+ that chelated with polyP during development.
Since polyP chelates divalent metal ions, we measured the amount of intracellular Mg2+ in M. xanthus and the amount of Mg2+ bound to polyP (Fig. 5A). In the wild-type strain, Mg2+ bound to polyP was detected on day 1 of starvation-induced development, but the amount of Mg2+ bound to polyP was about one-fifteenth that of intracellular Mg2+ in the cells. Mn2+ was not detected in this experiment because the concentration was too low. Commercial polyP700 − 1000 and polyP60 − 70 chelated Mn2+ to the same extent as Mg2+ (data not shown). Although Mn2+ may also be chelated by polyP produced during development, the amount of Mn2+ chelated by polyP is estimated to be about 15-fold less than the amount of intracellular Mn2+, similar to Mg2+. This result suggests that some enzymes of M. xanthus require Mg2+ and/or Mn2+ for their activity, but the binding of starvation-generated polyP to Mg2+ and Mn2+ may not affect the activity of those enzymes (Okada and Kimura, 2022). Mg2+ chelated by polyP was not detected in the ppk1 mutant but was approximately three-fold higher in the ppx2 mutant than in the wild-type strain on day 1 of development (Fig. 5A).
Effect of biofilm formation and polysaccharide production by polyP synthesis
PolyP has been reported to be involved in biofilm formation in Bacillus cereus, Pseudomonas aeruginosa, and Porphyromonas gingivalis. (Chen et al., 2002; Rashid et al., 2000; Shi et al., 2004). In M. xanthus, the secreted polysaccharide is a structural component of biofilm and is also important for type IV pilus-dependent motility and fruiting body formation (Pérez-Burgos et al., 2020). Polysaccharide production and biofilm formation in the ppx2 mutant during development were 1.2 to 1.9 times higher than those in the wild-type strain, but no significant difference was observed between the ppk1 mutant and the wild-type strain (Fig. 5B&C), suggesting that polyP synthesis is not necessary for polysaccharide production and biofilm formation in M. xanthus.
Adenylate energy charge during development
Organisms use ATP as their major energy source for metabolic reactions, and the levels of ATP, ADP and AMP reflect roughly the energetic status of the cell (Knowles 1980). The ratio of ATP, ADP, and AMP is more functionally crucial than the absolute concentration of ATP and is used as an indicator of the cell’s energy state, determined from the formula adenylate energy charge (AEC) = ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]) (Atkinson and Walton 1967; De La Fuente et al., 2014). Many organisms under optimal growth conditions maintain their AEC between 0.70 and 0.95.
Intracellular AMP, ADP, and ATP levels in the M. xanthus wild-type, and ppk1, ppx2, and pap mutant cells were measured during the exponential growth phase and starvation-induced development, and AEC values were determined (Fig. 6A). The AEC values of these strains at the exponential growth phase were 0.80 to 0.81.
M. xanthus cells sporulated on CF medium from day 2 and the number of mature spores increased until day 5 (Fig. 4B). The AEC values of wild-type cells cultured on CF medium for three days were maintained at roughly 0.80 (average ATP:ADP:AMP ratio = 1.00:0.48:0.08), and then decreased to 0.77 (ATP:ADP:AMP = 1.00:0.50:0.12). We previously reported that M. xanthus Ppk1 synthesizes polyPs at high ATP concentrations but begins to generate ATP at an ATP:ADP ratio (mM) of 2:1 and ATP synthesis becomes more active as the ADP ratio is further increased. In late development, Ppk1 may carry out ATP synthesis using ADP and polyPs rather than polyP synthesis.
Meanwhile, the AEC values of ppk1 mutant cells were maintained at approximately 0.79 (average ATP:ADP:AMP ratio = 1.00:0.51:0.08) for up to two days in culture and then decreased to 0.74 (ATP:ADP:AMP = 1.00:0.39:0.22). The AEC values of the pap mutant cells were altered similarly to the ppk1 mutant cells, and the average ATP:ADP:AMP ratio of the pap mutant cells cultured for three to seven days was 1.00:0.34:0.24. ppk1 and pap mutant cells at spore maturation (three to seven days in culture) had a lower ADP ratio and a higher AMP ratio compared to wild-type cells. This result suggests that Pap may generate ADP from AMP and polyP during spore maturation. The concentration of polyPs (average chain length 850 residues) at 24 h of development was calculated to be 6 µM (Harita et al., 2023). Since the Km values of AMP, polyP60-70, and polyP700-1000 for M. xanthus Pap were 0.74 mM, 0.13 mM, and 0.01 mM, respectively (Kimura and Kamatani 2021), Pap may function with increasing concentrations of AMP and medium to long-chain polyPs.
In contrast, the AEC values of the ppx2 mutant during development were approximately 0.2 higher than those of the wild-type strain, with an average ATP:ADP:AMP ratio of 1.00:0.34:0.10. Since the ppx2 mutant cells contain a large amount of polyPs, Ppk1 and Pap may produce more ATP and ADP from ADP, and AMP and ATP, respectively, in ppx2 mutant cells than in the wild-type cells.
Adenylate kinase (Adk) is a ubiquitous enzyme that catalyzes a reversible phosphoryl transfer reaction between ATP and AMP to produce two ADP molecules, thus regulating the equilibrium between adenosine phosphorylation states and AMP recovery and playing a fundamental role in cellular energy homeostasis (Atkinson 1977). M. xanthus has two Adks, AdkA and AdkB, and Ppk1 also has Adk activity in the presence of low polyPs concentrations (Kimura et al., 2019; Harita et al., 2023). Adk activity in M. xanthus was highest in cells at the exponential growth phase and on day 1 of development, and then rapidly decreased to 15% by day 3 of development (Fig. 6B). Meanwhile, the specific activity of Pap in M. xanthus during development increased by 2.3–2.4-fold on days 1 and 2 of incubation (Kimura and Kamatani 2021). These results suggest that the conversion of AMP to ADP in M. xanthus cells is performed by Adk using ATP during the growth phase, by Adk and Pap on the first day of starvation-induced development, and mainly by Pap using polyPs after the second day of development.
After 10 days of spore formation on CF agar medium at 30°C, spores were sonicated twice for 30 s, treated at 60°C for 15 min, and inoculated into CTT liquid medium. After 20 h, spores of the wild-type strain began to germinate. In contrast, spores of the ppx2, pap and ppk1 mutants began to germinate approximately 2, 8, and 12 h later than spores of the wild-type strain, respectively (data not shown). The spores of the ppk1 and pap mutants had more AMP and less ADP than the wild-type spores, therefore the spores of these mutants may have taken longer to germinate. Meanwhile, Zhang et al. (2005) reported that the spores of the M. xanthus wild-type and the pap mutant strains germinated at the same time, while ppk1 germinated later.
In conclusion, Myxococcus xanthus synthesized polyPs during the stationary phase, starvation-induced development, oxidative stress, and phosphate deficiency. The ppk1 mutant showed reduced survival from the stationary to the death phases compared to the wild-type strain. During the mature sporulation stage, the AEC values of the ppk1 and pap mutants were lower than that of the wild-type strain, and spore germination of ppk1 and pap mutants was delayed compared to that of the wild-type strain. This result may be due to the inability of Pap to generate ADP from AMP using polyPs.