IGEM:IMPERIAL/2009/Encapsulation/Phase2/Sporulation

Sporulation and E.coli
E.coli is non-sporulating. Therefore the idea to clone the genes for sporulation from Bacillus subtilis into E.coli was investigated. A paper (below) used the amino-acid sequence deduced from the nucleotide sequence of the spolIAC gene of Bacillus subtilis which has been shown to be homologous to that of the sigma subunit of the Escherichia coli RNA polymerase. Results show that this gene can be cloned in E. coli only under conditions in which it is not expressed.

E.coli and sporulation.

The sigma-like products of the sporulation gene spolIA C of Bacillus subtilis is toxic to Escherichia coli. The main chassis we would therefore use is B.subtilis as it is the easiest to manipulate and well characterised.

It is now agreed that spore formation in bacteria is a form of differentiation in which there is an ordered, temporal sequence of events and a degree of commitment.

Bacteria will produce a single endospore internally. The spore is sometimes surrounded by a thin covering known as the exosporium, which overlies the spore coat. The spore coat, which acts like a [sieve] that excludes large toxic molecules like lysozyme, is resistant to many toxic molecules and may also contain enzymes that are involved in germination. The cortex lies beneath the spore coat and consists of peptidoglycan. The core wall lies beneath the cortex and surrounds the protoplast or core of the endospore. The core contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs, that protect the spore DNA from UV radiation and heat. The core also contains normal cell structures, such as ribosomes and other enzymes, but is not metabolically active.

The Sporulation Process & Genes Involved At Each Stage
Sporulation in Bacillus subtilis is a fertile system for studying development because of the detailed genetic understanding of this process. A summary from the paper is shown, identifying the key steps in the sporulation process.

1. Entry into sporulation
 * Spo0A, which in its phosphorylated form stimulates expression of early sporulation genes and inhibits the expression of genes whose function is to prevent sporulation.
 * SinR is required for the development of competence and motility, but inhibitory for sporulation and the production of exoproteases.
 * SinI binds tightly to SinR, thus preventing SinR from binding to promoter DNA.

2. Final commitment
 * SpoIIAB can bind to sigmaF and prevent sigmaF from combining with core RNA polymerase and transcribing from sigmaF directed promoters.

3. Spore coat synthesis and assembly
 * GerE is the latest acting of four regulatory factors (sigmaE-SpoIIID-sigmaK-GerE) which ensure the correct temporal appearance of gene products in the mother cell.
 * GerE is a 74 residue DNA-binding protein that acts both as a repressor and as an activator of expression of a number of coat proteins. Its function is to control the synthesis and assembly of the spore at the level of transcription.
 * SpoIVA induced by sigma E and controls the assembly of a ring of CotE proteins.
 * Coat protein genes (eg CotG) induced under the control of sigma K and the DNA-binding protein GerE.

Control, Genes Involved and Potential Solution?
There are a number of transcription factors that regulate each stage of the sporulation process. Each has a number of operons that it influences.

At the sporulation initiation stage, Spo0A controls 62 operons (found using DBTBS). The exact function of each gene is not known. However, there have been various studies done on isolating and mutating a specific gene to find the effect it has on the coat formation produced. Therefore, the mechanism known is more of a 'linked' pathway and the role of all individual genes is not known.

Therefore, instead of finding the minimum number of genes required for each stage, an idea we came up with was to keep as many genes as possible and add in an inducible promoter for production of the initial stage of sporulation. Therefore, we can control Spo0A without subjecting it to nutrient starvation processes. This can be done in a plasmid in vitro and then inserting this into the cell.

Since the forespore will be the part producing the spore (as the mother cell lyses), it is important for our protein produced and contained within the forespore. As a result we would try to target the sigma factors that are only expressed in the forespore to produce the protein we require to transport. An example is sigma G. For this a promoter needs to be found that acts on sigma G, and the protein gene placed under the control of this promoter. The next steps in the sporulation process will be the same.

However, the process produces a number of extracellular and intracellular proteases that may degrade the protein we are trying to deliver. For this purpose, the genes involved in the production of protein degrading enzymes has been investigated (see paper below).


 * So if we want to control the initial step we would need to modify the chromosome, which in B.subtilis is more feasible than in other chassis such as E.coli. Tom and Chris have recently ordered a B.subtilis sporulation deficient strain, might be interesting to read how this was done.
 * Id be interested to hear more of a comparison about the physical properites of the spore vs the other encapsulation methods you have looked at. Tianyi has outlined some key features that need to be met, e.g. resistance to pH as low as 2, stable at body temperature

The spore offers unique resistance properties and can survive under extreme conditions, such as excessive temperature, desiccation, and exposure to solvents and other noxious chemicals. These features would make the spore an ideal vehicle for delivery of heterologous antigens to extreme environments such as the gastrointestinal tract.

Oral administration of a Bacillus subtilis spore-based vaccine

Useful Links
Link to a patent describing the surface display of enzymes on spore proteins

Structural studies of Key Regulators of Bacterial Sporulation