Chapter 5 : Chance and Necessity in Bacillus subtilis Development

Developmental modules in B. subtilis and their major components. All of the indicated forms of development depend on 0A and on the phosphorelay that governs the phosphorylation of this transcription factor. This figure is intended to summarize many of the major interactions mentioned in the text that govern the developmental processes. It is not exhaustive, although it may be exhausting. Lines ending in perpendiculars and arrows denote negative and positive effects, respectively. Arrows associated with right-angled lines denote transcription initiation. The dotted line from the cannibalism module indicates that the release of nutrients from dead cells delays sporulation. Several kinases deliver phosphoryl groups to the phosphorelay, which results in the formation of 0A~P. Under some conditions one or more kinase can dephosphorylate 0F~P, draining phosphate from 0A~P. RapA is one of several related proteins that can also dephosphorylate 0F~P. RapC acts by preventing ComA~P from interacting with its DNA target. These Rap proteins are inhibited by cognate secreted peptides (e.g., PhrA and PhrC), which are internalized by the oligopeptide permease Spo0K. ComX is a modified and secreted peptide which activates the autophosphorylation of ComP. ComP~P donates a phosphate to ComA, and ComA~P then activates the transcription of srfA. Embedded in the srfA operon is the gene for ComS. This small protein binds to the protease complex of MecA plus ComP plus ClpC, preventing the degradation of the transcription factor ComK. ComK is then free to activate its own expression by antagonizing the repressor Rok, activating a positive autoregulatory loop. When ComK accumulates, it, in turn, activates the transcription of many downstream genes, resulting in the induction of competence (the K-state). A low level of 0A~P is also essential for competence due to its direct interaction with the comK promoter and its repression of abrB. A low to intermediate concentration of 0A~P also activates the sinI promoter. SinI antagonizes SinR, lifting the repression of several transcription units that are essential for biofilm formation, as well as the repression of slrR. SlrR binds to SinR, further derepressing the biofilm operons. The SinR-SlrR heterocomplex represses the genes for motility as well as those that encode the autolysins that separate daughter cells following division. This results in the formation of chains of sessile cells. Low concentrations of 0A~P also activate genes that encode toxins. Toxin-producing cells (cannibals) benefit by killing other cells, thus deriving nutrients. Finally, high concentrations of 0A~P activate the sporulation genes.

Diagram of the uptick mechanism ( 34 ). The top portion shows a graphical representation of the Rok and 0A~P concentrations, as well as the availability of RNApol and the rate of comK basal transcription (solid black line) during the transition to stationary phase. (RNApol availability is used as a plausible stand-in for the cause of the global increase in transcription that was observed.) The peak rate of transcription coincides with T0, the time of departure from exponential growth. When the concentrations of available RNApol and of 0A~P are low ( 1 ), Rok is dominant and the rate of comK transcription is also low. As the concentration of 0A∼P increases further, Rok is antagonized at sites A1, A2, and A3 and at the same time RNApol becomes more available. As a result, the rate of comK transcription increases ( 2 ). Finally, the 0A∼P concentration reaches a level that is able to repress at R1 and R2 and comK transcription slows ( 3 ). In reality, of course, three demarcated periods of time do not exist. Note that the concentration of Rok remains constant throughout and both RNApol and 0A~P work to counteract its effects. Rok works at an unidentified site in addition to A1 to A3, shown here between A3 and R1. For simplicity, the availability of RNApol is shown as constant after T0, although the data would suggest that it varies somewhat ( 34 ).

Two proposed mechanisms controlling chaining and motility (A) The epigenetic switch ( 43 ). (Adapted with permission from the authors and the publisher from Fig. 1 in reference 43 .) (a) SinI sequesters SinR, relieving repression of slrR. SlrR then binds to SinR, and the resulting complex represses the autolysin and motility genes and prevents repression of the matrix genes by SinR. (b) This circuitry allows for two metastable states. In one, when SlrR is low, the autolysin and motility genes are ON and the resulting cell is motile. In the other, when the SlrR concentration is high, these genes are OFF and the cells form chains and do not swim. The central feature of the circuitry that permits this bistable switch is the double-feedback mechanism involving repression of slrR by SinR and the inactivation of SinR for matrix gene repression by binding to SlrR. If SlrR is high, repression locks the cell in the motility OFF state, and vice versa. The transition between states can be stochastic, due to fluctuations in protein concentration (noise), or deterministic, in the sense that it is a programmed developmental switch. (B) Diagram of the gene position mechanism ( 50 , 51 ). SigD is the penultimate gene in the 27-kb fla-che operon. For unknown reasons, the probability that promoter-distal genes are included in the operon transcript falls off with distance. Thus, if the mean number of transcripts per cell is low, some cells will have more SigD than others, and these cells will be motile. The distance-dependent fall-off in transcript abundance is reported to be due to the action of the SinR-SlrR heterocomplex ( 51 ).

Cell type determination in biofilms ( 60 , 98 , 104 , 107 ). Pre-ComX is processed and ComX is secreted with the aid of ComQ. ComX interacts with ComP at the cell surface, resulting in the phosphorylation of ComA and the transcriptional activation of srfA. The surface-active SrfA molecule induces potassium flux in a susceptible cell, activating KinC and the formation of small amounts of 0A~P. For unknown reasons, the surfactin-producing cell itself becomes refractory to activation by surfactin. In the susceptible cell, 0A~P activates the transcription of sinI, which interacts with SinR, relieving repression of the matrix genes. For unknown reasons, matrix producers are not activated to produce surfactin. The presence of matrix downregulates the phosphatase activity of KinD, permitting the 0A~P concentration to rise further, inducing sporulation. Matrix producers also become cannibals, because their intermediate 0A~P concentration triggers toxin production. These toxins kill nonproducers, which release nutrients, delaying sporulation. As a result, matrix producers proliferate, increasing the population of eventual sporulating cells.