|dc.description.abstract||In bacteria, genetic transformation is a physiological process of taking up exogenous DNA from the extracellular environment. In S. pneumoniae, this process is a transient state and subject to tight regulation. To initiate competence, two sets of gene expressions need to be induced by a signal peptide, CSP. The early gene expression produces ComX and ComW. ComX and ComW work in concert to turn on late gene expression, which eventually make cell competent.
Among late genes, there are many uncharacterized ones whose functions remain unknown. To find out if those unknown late genes are required for competence development, classic genetic analysis is necessary. In order to have a gene disruption tool without leaving the selective marker in the genome, I sought to construct a lox/cre/lox cassette for creating efficient marker-less gene deletion. In this new cassette, two mutant lox sites, lox66 and lox71, flank a erythromycin-resistance marker, ermAM, that can be used as a temporary marker for selection of desired recombinants, and a cre gene, which is under the control of a native regulated pneumococcal promoter. The cassette may subsequently be removed by the recombination of the two lox sites induced by the controllable induction of the cre gene, with retention of 34 bp from the cassette as an inert residual double-mutant lox72 site. We named this cassette “Cheshire”. Compared to other gene deletion strategies used in streptococcus, the Cheshire cassette has shown to be more efficient and reliable.
As soon as competence is turned on, it is shut off immediately. Proteolysis of ComX and ComW has been proposed to be one factor that shuts off competence in time. However, it seems that there is another factor from the late gene products that might also play a role in competence termination. I decided to screen on the ComX-induced late genes, especially the transformation-essential late genes. Among the 20 genes tested, ΔdprA displayed a prolonged late gene expression pattern, whereas mutants lacking cbpD, cibABC, cglEFG, coiA, ssbB, celAB, cclA, cglABCD, cflAB, or radA, exhibited a wild-type temporal expression pattern. Combined with previous lab work showing that DprA limits the amounts of ComX and another early gene product ComW, I concluded that DprA controls their amounts by inhibiting early gene expression rather than by promoting the degradation of ComX and ComW. To ask what target DprA might work on in turning off early gene expression, yeast two-hybrid assay was employed to investigate protein interactions between DprA and ComD, DprA and ComE. My results suggest that DprA could interact with ComE, but not with ComD, ComX, and ComW. Therefore, I further hypothesize that DprA shuts off early gene expression and competence by a direct interaction with ComE, which makes ComE unable to be phosphorylated.
On the other hand, ComW has been found to be required for competence development along with ComX. However, how ComW participates in this process is not known. To ask if ComX and ComW interact directly, I took advantage of yeast two-hybrid system by fusing ComX with the activation domain and ComW with the DNA-binding domain of the GAL4 transcription factor. My results revealed a direct interaction between ComX and ComW. Taken together with previous results, my data indicate that the role of ComW in turning on competence might be via a direct interaction with ComX. Considering that ComW is a small protein S. pneumoniae, I hypothesize that ComW might work as an adaptor for the activation of ComX.||en_US