The bacteria B.subtilis is unusual in being able to live as a normal free swimmi

Question

The bacteria B.subtilis is unusual in being able to live as a normal free swimming “vegetative” bug when nutrient conditions are good (rich) but when starved for carbon (glucose) it can go into a dormant state called a spore. This ability to sporulate requires the expression of “sporulation genes” that are regulated by specialized sigma factors.

a) After purifying the RNA Pol holoenzyme from vegetative and sporulating B.subtilis -- you compare the subunit composition of these 2 enzymes by SDS Gel electrophoresis. What would you expect to see (i.e. Which subunits of RNA Pol would you expect to be the same and which ones different?) Explain the reason for your answer. Show pattern on bands with relative migration.

b) Let’s assume that the B.subtilis genome encodes a total of 7300 genes and 5125 are expressed in the vegetative state while 3532 are expressed during sporulation. What modern technique would you use to quickly identify the 5125 vegetative and 3532 sporulation genes? Briefly describe the steps needed to determine pattern of gene expression. Notice that these 2 numbers don’t add up to 7300 – how might you explain this? (Assume the entire B.subtilis genome has been sequenced and all gene coding sequences determined.)

Explanation / Answer

a) RNA polymerase has many subunits. In vegetative cell of B. subtilis, principle subunit is CA. This subunit maintains the vegetative cell character. In sporulating bacteria, the principle subunit is CA is replaced by CS. Two other subunits which aid the replacement process for maintaining the sporulation character are CE and CK.

So, during gel electrophoresis, one band will be shown in vegetative cell; CA subunit. In sporulating cell, two bands of CE and CK will be seen.

b) For finding out genes, the best method is 'Ab initio'. This is the simplest and most modern method to find out genes. It is a computer aided program in which computer looks for 'start' sites in the DNA. The total number of 'start' sites is the total number of genes.

There are certain genes which aid both in sporulation as well as vegetative character. These genes include cell division genes, structural genes like those of cell wall and cell membrane etc. Therefore, the total number does not add up to 7300.

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