Tentative model integrating the termination factor Rho into the regulatory networks controlling the adaptation to stationary phase and the programs of genetic competence and sporulation in <i>B</i>. <i>subtilis</i>.

For simplicity, only the key elements relevant to this study are shown, and the discussion of the regulatory activities of CodY and AbrB is limited to the comK gene. The arrowhead lines and bar-headed lines indicate positive and negative regulation, respectively. Solid and dashed headed lines indicate strong and weakened regulation, respectively, and the dashed lines without extremities stand for the loss of positive regulation. Red and blue bar-headed lines depict the Rho-mediated negative regulatory events identified previously (red) and in this study (blue). (A) In B. subtilis cells grown under starvation conditions, the increased (p)ppGpp synthesis linked to the activation of stringent response leads to lowering of GTP levels, thus decreasing the activity of CodY and stimulating the expression of the kinA and kinB genes, which are under positive stringent control [112]. The major sensor protein kinases KinA and KinB initiate transfer of the phosphoryl groups to Spo0A. KbaA protein stimulates KinB activity. The gradual increase in Spo0A∼P activates expression of comK directly and indirectly by relieving the Rok- and AbrB-mediated repression of comK. By repressing AbrB, Spo0A~P activates the expression of sigH, thus creating a positive feed-back loop of autoregulation [37,79]. When concentration of Spo0A~P reaches a high threshold level, it represses comK and activates expression of early sporulation genes. Synthesis of (p)ppGpp is necessary throughout the entire sporulation pathway [126]. In starving cells, Rho levels decrease due to the negative autoregulation [72] and some unknown regulatory circuits. Lowering of the Rho levels decreases the efficiency of the Rho-controlled transcription terminators thus activating, directly and indirectly, transcription of a set of genes and ncRNAs involved in adaptation to stationary phase. In particular, the lowering of Rho weakens the activity of the kinB intragenic Rho-dependent transcription terminator; as a result, the intensified read-through at the terminator stimulates synthesis of the full-length kinB transcript and consequently the activity of Spo0A~P [26]. (B) Counteracting the natural decrease of Rho levels by rho over-expression alters the normal progression to stationary phase as depicted. Stabilization of the intracellular levels of Rho in the stationary Rho+ cells improves the Rho-dependent transcription termination and consequently alters the expression of the Rho-controlled genes and ncRNAs. A high Rho inhibits read-through at the intragenic transcription terminators of kinB and the newly identified Rho target kbaA gene thus decreasing the full-gene transcription of kinB and kbaA. A decreased flux of the phosphate alters the Spo0A~P-mediated inhibition of AbrB and thus disrupts its positive autoregulatory loop. As a result, Spo0A~P does not reach the threshold levels necessary neither for the de-repression of comk, nor for the efficient activation of early sporulation genes. The increase in Rho levels reinforces a still unknown mechanism by which Rho modulates the synthesis of (p)ppGpp and regulates the activation of stringent response. Altering the balance between p(p)ppGpp and GTP levels leads to an increased repression of comK by CodY and a weak activation of the positively-regulated stringent genes kinA and kinB, which additionally contributes to decreasing Spo0A~P activity. In addition, the analysis of the Rho+ strain pinpoints the existence of yet non-identified targets of Rho-mediated regulation among the competence and sporulation genes. Overall, the increased control of Rho over its multiple targets provokes the loss of competence and sporulation in Rho+ cells.