Triggering sporulation in Bacillus subtilis with artificial two‐component systems reveals the importance of proper Spo0A activation dynamics

Abstract

SummarySporulation initiation in Bacillus subtilis is controlled by the phosphorylated form of the master regulator Spo0A which controls transcription of a multitude of sporulation genes. In this study, we investigated the importance of temporal dynamics of phosphorylated Spo0A (Spo0A∼P) accumulation by rewiring the network controlling its phosphorylation. We showed that simultaneous induction of KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself from separately controlled inducible promoters can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency in this artificial two-component system was significantly impaired when KinC and/or Spo0A induction was too high. Using mathematical modelling, we showed that gradual accumulation of Spo0A∼P is essential for the proper temporal order of the Spo0A regulon expression, and that reduction in sporulation efficiency results from the reversal of that order. These insights led us to identify premature repression of DivIVA as one possible explanation for the adverse effects of accelerated accumulation of Spo0A∼P on sporulation. Moreover, we found that positive feedback resulting from autoregulation of the native spo0A promoter leads to robust control of Spo0A∼P accumulation kinetics. Thus we propose that a major function of the conserved architecture of the sporulation network is controlling Spo0A activation dynamics.

In vivo functional characterization of the transmembrane histidine kinase KinC in Bacillus subtilis

“…S3B, C). These results were similar to those in our previous reports (Eswaramoorthy et al, 2010;Vishnoi et al, 2013). Based on these results, the optimum concentration of IPTG to induce efficient sporulation (~10 8 ml 21 , a level similar to the WT sporulating cells) with the synthesis of KinC was determined to be 10 mM.…”

“…A WT strain harbouring mutations in both kinA and kinB is not able to sporulate, indicating that KinC activity is not sufficient for triggering sporulation. However, when the KinC protein level is increased, it is sufficient to induce sporulation (Vishnoi et al, 2013). Based on these facts and observations, we speculate that two different levels (low and high) of Spo0A~P are achieved through two distinct pathways regulated by KinC and KinA, respectively, as follows: (1) when KinC is fully activated during the early phase of starvation, cells produce cannibalism toxin proteins and form biofilm by expressing only a low level of Spo0A~P, mainly through KinC, resulting in avoidance of sporulation decision-making; (2) however, the level of fully active KinC is not sufficient to achieve the threshold of Spo0AyP needed for entry into sporulation during this period; and (3) when KinA activity increases gradually later on, cells ultimately end up forming spores with high levels of Spo0A~P (Ló pez et al, 2009a).…”

“…To overcome these technical issues and to provide an improved method for the in vivo quantitative assay of KinC activity, we took advantage of the fact that sporulation is efficiently induced when the levels of KinC are increased by the induction of KinC synthesis using the IPTG-inducible construct (Fig. S3A) Vishnoi et al, 2013). First, to exclude the possible involvement of the sporulation kinases KinA and KinB, we constructed a strain harbouring the IPTG-inducible KinC in the kinA and kinB double knockout strain.…”

Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

“…While many of the bacterial two-component systems control rapid and transient gene expression in response to various and specific stimuli (3), the phosphorelay system is involved in the control of the crucial and highly coordinated cell fate decision, which ultimately leads to the development of spores (2,4,(9)(10)(11). Accumulated evidence indicates that a temporal and spatial increase in the level and activity of the master regulator Spo0A is required for sporulation to proceed properly (12)(13)(14). Under nutrient-rich conditions, the DNA-binding protein AbrB represses genes involved in the transition from vegetative growth to stationary phase, and only a basal level of Spo0A is expressed from the relatively weak, constitutive A -controlled promoter Pv (15).…”

“…In addition, the transcription of genes for Spo0F and Spo0A is positively regulated by Spo0A (16). Through this feedback regulation of the phosphorelay, a gradual increase in the cellular level and activity of Spo0A is achieved (12,13).…”