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Chassis

Introduction

The main aim of our project is to sequester cadmium in the environment into the spores of our engineered B. subtilis, but what happens after the cadmium has been sequestered?

Do we attempt to retrieve the sequestered cadmium? Or, do we simply leave the sequestered cadmium in the spores of our engineered B. subtilis?

For our project, we have chosen the latter. We will not be attempting to retrieve the sequestered cadmium. However, then comes the question of, would there not be chances of the cadmium entering the environment again?

Our solution to this question would be to disable germination of the spores, thus retrieval of the sequestered cadmium becomes unnecessary, as the spores can persist intact for thousands of years.

In order to disable germination of the spores, we would require non-germinating spores, and we were fortunate enough that Prof. Anne Moir from Sheffield University kindly sent us two non-germination spores, with inactivated genes, namely cwlD, and sleB and cwlJ.

While we would like to disable germination for the spores that contain sequestered cadmium, not all the cells would have sequestered cadmium, and it is also essential that we still have some cells germinating, so that our population of bacteria can continue to live and grow, reaching a balance, and not simply deplete totally.

Therefore, a mechanism is needed to allow us to choose to turn on germination, when the cell is not a "metal container".

Using the treatment protocol for the non-germination spores from Prof. Anne Moir, we performed lab experiments for the two non-germination spores, and concluded that the double-knockout mutant, sleB and cwlJ would be more ideal for our project as it had more colonies growing after treatment, and less colonies growing without treatment, as compared to the single knock-out mutant, cwlD.

We propose that we could use IPTG as a switch for germination.

Bacillus subtilis cwlD mutant

Together with the non-germination spores and protocol for recovery which Prof. Anne Moir sent to us, information on the cwlD mutant was given as well, quoted in the following paragraph.

"The cwlD (Cm^r) mutation was transformed in our lab strain of Bacillus subtilis (1620) to create a mutant lacking muramic lactam in the cortex peptidoglycan. As this is a target for the activated cortex lytic enzymes, there is no cortex breakdown, limited rehydration, no gross protein changes in the spore coat, and hence the strain is germination defective."

From the reference recommended by Prof. Anne Moir, we found that, the mutation in the cwlD gene of Bacillus subtilis is predicted to encode a muramoyl L-alanine amidase, which results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/outgrowth process to produce viable cells.^[1]

Germination in the presence of lysozyme allows the cwlD spores to produce viable cells with normal heat resistance properties.^[1]

Bacillus subtilis sleB and cwlJ double-knockout mutant

The sleB and cwlJ proteins are cortex-lytic enzymes, partially redundant in function, and are required together for effective cortex hydrolysis during Bacillus subtilis spore germination. Enzymic hydrolysis of spore-cortex peptidoglycan is essential for spores to complete rehydration during germination and to commence outgrowth,^[2] thus the SleB and CwlJ proteins are important enzymes in normal spore germination of Bacillus subtilis.

References:

[1] Popham, D., Helin, J., Costello, C & Setlow, P. (1996). Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore rehydration or heat resistance. Proc. Natl. Acad. Sci. 93; 15403-15410

[2] Chirakkal, H., O'Rourke, M., Atrih, A., Foster, S. J., Moir, A. (2002.) Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination. Microbiology 148; 2383-2392