Team:Newcastle/Bistability in B.Subtilis

=Bistability in B.subtilis=

The Sin (Sporulation Inhibition) operon
The Sin (Sporulation Inhibition) operon can be used as a stochastic switch. It is a natural bistable switch.



During normal conditions, SinR is expressed constitutively by P3 and keeps its concentration at a constant level repressing promoter 1. When transcription of promoter1 is activated by phosphorylated Spo0A, both sinI and sinR are expressed from promoter 1. SinI inactivates SinR by forming a complex with sinR upregulating both proteins from promoter1. This cross repression, inhibition of SinR by SinI and  the transcriptional represion of sinI by SinR, forms the basis of the bistability. While the positive feedback in the production of SinI enhances the bistability, it also causes increase in sinR levels because of the expression from promoter 1 hence causing oscillations.[1]

The Sin operon controls the early stages of sporulation and has a key role to control the sporulation without disturbing its regulation. The threshold of this switch to progress into sporulation can be controlled by varying some parameters, hence providing population heterogeneity. Tight binding of SinR to the first promoter region with fewer sinR molecules increases this heterogeneity. By mutating the first promoter, the binding affinity of sinR the promoter can be increased. With variations on SinI:SinR interaction strength, transcription rate of sinR from the third promoter and the expression rate of SinR, dynamics of the system can be altered.[1] SinR and SinI can be used to regulate the heavy metal sequestration. Only in a sub population of the bacteria, sinI will be expressed at sufficient levels to trigger our system. Whereas sinR will be expressed in all cells. So with this approach we can say that only a small population of the cells will get the heavy metals. If we want the other way we can switch the roles of SinI and sinR.With the normal conditions only 2% of the population express enough sinI.[2]

Sin operon details
Sin operon in GenBank

sinR: Positive regulation of comK; negative regulation of aprE, kinB, sigD, spo0A, spoIIA, spoIIE, spoIIG

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_000964.2&from=2551885&to=2552220&dopt=gb

sinI:Antagonist of SinR

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_000964.2&from=2551678&to=2551851&dopt=gb


 * 1) http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1449569
 * 2) http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2430929&blobtype=pdf
 * 3) http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.micro.62.081307.163002

The comK system in B. subtilis
The positive feedback in comK regulation provides the bistability in B. subtilis. we can either knock out the competence system in B. subtilis or use a homolog system from another bacteria.

ClustalW aligment of comK genes (Figure 2)

There is no evidence of bistability in in pneumococcal competence so we can not use the genes from S.pneumoniae (http://dx.doi.org/10.1038/nrmicro1613 )
 * Streptococcus pneumoniae:

Again, nearly all the cells become competent. (http://dx.doi.org/10.1016/0168-9525(96)10014-7)
 * H. influenzae

It has two homologues of B. subtilis comK gene. comK1(BC1134, 62%), comK2(BC5250, 48%). But the regulation and function of these two comK genes are unknown. B. cereus showed competence when comK gene from B. subtilis is placed into B. cereus (http://gbb.eldoc.ub.rug.nl/root/2008/MicrobBiotnMironczuk/)
 * B. cereus

Mathematical Modelling of comK
-An excitable gene regulatory circuit induces transient cellular differentiation

-And its supplementary information with math equations and parameters

ClustalW aligment of comK genes
 * Related Links:
 * 1) Bistability using positive autoregulation of ComK
 * 2) Regulatory inputs for the synthesis of ComK
 * 3) Functional analysis of the competence transcription factor ComK of Bacillus subtilis by characterization of truncation variants

Sigma D
It was porposed that perhaps sigma D could be used to make a stochastic switch however perhaps not enough is known about how it is regulated?

The factors that are responsible for the stationary-phase elevation in sigD levels are unknown. sigD-dependent transcription declines after the onset of sporulation, and inactivation of sigD itself causes no overt defect in sporulation, therefore sigD’s contribution to sporulation, if any, is modest. Present evidence demonstrates that sigD is primarily involved in the expression of flagellar, motility, and autolysin genes and their regulators.

The Sigma Factors of Bacillus subtilis, WILLIAM G. HALDENWANG MICROBIOLOGICAL REVIEWS, Mar. 1995, p. 1–30

Synthetic Switches
Synthetic bistable toggle switches constructed from LacI and Cl repressors can also be used. Theese repressors repress each other creating a bistable switch with two negative feedback loops. Bistable switch with two positive feedback can also be considered.

Some useful links for bistability

 * Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability
 * Bistability in Bacteria
 * Initiation of sporulation
 * Stochasticity and Cell Fate