Ababneh, Qutaiba O (2015-08). Elucidating the Role of Small Molecule Signals in Bacterial Development. Doctoral Dissertation. Thesis uri icon

abstract

  • The nucleotide second messengers pppGpp and ppGpp ((p)ppGpp) are responsible for the global down-regulation of transcription, translation, DNA replication, and growth rate during stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many non-stringent processes, including virulence, persister cell formation, and biofilm production. In Bacillus subtilis, (p)ppGpp production is controlled by bifunctional synthetase/hydrolase, RelA, and two monofunctional synthetases, YwaC and YjbM. We observed that a relA mutant grows exclusively as unchained, motile cells in contrast to the wildtype (PY79), which grows as both motile, unchained and nonmotile, chained cells. The phenotypic switch from non-motile, chained cells to unchained, motile cells is promoted by the alternative sigma factor, SigD. We found that the relA mutant is trapped in a SigD ON state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility in B. subtilis. Furthermore, we showed that this cell chaining and motility is directly regulated by the GTP-sensing protein, CodY. (p)ppGpp accumulation inhibits GTP synthesis and CodY has three putative binding sites in the sigD-containing fla/che operon. Our current model is that (p)ppGpp synthesis leads to decreased levels of GTP and a concomitant decrease in CodY-GTP mediated repression of fla/che operon transcription. This work highlighted the critical role of basal (p)ppGpp levels in significantly skewing developmental decision-making outcomes. Entry into sporulation is governed by Spo0A, which accumulates as cells enter stationary phase and is activated by a phosphorylation cascade initiated by sensor kinases in response to environmental cues. Environmental cues are critical to successful sporulation, as prior studies have shown that expression of constitutively active Spo0A is not sufficient to induce sporulation during exponential growth. However, previous reports indicate that a gradual increase in Spo0A-P, mediated through artificial expression of the kinase KinA, is sufficient to trigger sporulation during exponential growth. We report here that sporulation via KinA induction requires an extracellular factor or factors (FacX) that only accumulates to sufficient levels during post-exponential growth. FacX is retained by dialysis with a cutoff smaller than 500 Dalton, can be concentrated, and is susceptible to proteinase K digestion, similar to quorum-sensing peptides shown to be involved in promoting sporulation. However, unlike previously characterized peptides, FacX activity is not dependent on SigH or Spo0A, and does not require the oligopeptide transporters Opp and App. We also find that B. subtilis can be induced to sporulate following the acute induction of active Spo0A in media containing FacX. These results indicate that there is no formal requirement for gradual Spo0A-P accumulation in successful sporulation and instead support the idea that sporulation requires both sufficient levels of active Spo0A and at least one other signal or condition.
  • The nucleotide second messengers pppGpp and ppGpp ((p)ppGpp) are responsible for the global down-regulation of transcription, translation, DNA replication, and growth rate during stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many non-stringent processes, including virulence, persister cell formation, and biofilm production. In Bacillus subtilis, (p)ppGpp production is controlled by bifunctional synthetase/hydrolase, RelA, and two monofunctional synthetases, YwaC and YjbM. We observed that a relA mutant grows exclusively as unchained, motile cells in contrast to the wildtype (PY79), which grows as both motile, unchained and nonmotile, chained cells. The phenotypic switch from non-motile, chained cells to unchained, motile cells is promoted by the alternative sigma factor, SigD. We found that the relA mutant is trapped in a SigD ON state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility in B. subtilis. Furthermore, we showed that this cell chaining and motility is directly regulated by the GTP-sensing protein, CodY. (p)ppGpp accumulation inhibits GTP synthesis and CodY has three putative binding sites in the sigD-containing fla/che operon. Our current model is that (p)ppGpp synthesis leads to decreased levels of GTP and a concomitant decrease in CodY-GTP mediated repression of fla/che operon transcription. This work highlighted the critical role of basal (p)ppGpp levels in significantly skewing developmental decision-making outcomes.

    Entry into sporulation is governed by Spo0A, which accumulates as cells enter stationary phase and is activated by a phosphorylation cascade initiated by sensor kinases in response to environmental cues. Environmental cues are critical to successful sporulation, as prior studies have shown that expression of constitutively active Spo0A is not sufficient to induce sporulation during exponential growth. However, previous reports indicate that a gradual increase in Spo0A-P, mediated through artificial expression of the kinase KinA, is sufficient to trigger sporulation during exponential growth. We report here that sporulation via KinA induction requires an extracellular factor or factors (FacX) that only accumulates to sufficient levels during post-exponential growth. FacX is retained by dialysis with a cutoff smaller than 500 Dalton, can be concentrated, and is susceptible to proteinase K digestion, similar to quorum-sensing peptides shown to be involved in promoting sporulation. However, unlike previously characterized peptides, FacX activity is not dependent on SigH or Spo0A, and does not require the oligopeptide transporters Opp and App. We also find that B. subtilis can be induced to sporulate following the acute induction of active Spo0A in media containing FacX. These results indicate that there is no formal requirement for gradual Spo0A-P accumulation in successful sporulation and instead support the idea that sporulation requires both sufficient levels of active Spo0A and at least one other signal or condition.

publication date

  • August 2015