IGEM:IMPERIAL/2009/Encapsulation/Phase2
Alginate Chitosan Alginate
Sporulation
An endospore is a dormant, tough, and non-reproductive structure produced by Gram-positive bacteria. They form through the production of an encapsulating spore coat within the spore-forming cell. Examples include Bacillus and Clostridium.
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.
Link was not working for me, mayeb try to using the biblio function, will help to organise your references
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.
Useful Links
Link to a patent describing the surface display of enzymes on spore proteins
Structural studies of Key Regulators of Bacterial Sporulation
There are four main transcription factors, one for each stage of the sporulation process. Each has a number of genes that it influences.
At the sporulation initiation stage, Spo0A controls 62 genes (found using DBTBS)
