bolaturbo & bola turbo
The BolA protein from E. coli regulates flagellar synthesis, TCA cycle, and peptidoglycan synthesis in the presence of carbon dioxide (34). It is also known to be involved in biofilm formation by stimulating the expression of genes related to the secretion of extracellular DNA and proteins that adhere bacteria to surfaces. However, it is not yet fully understood how the bolA gene affects these processes, especially since it shows significant pleiotropic effects on cellular motility and flagellar structure formation.
To understand the molecular mechanisms underlying these effects, we used microarrays and ChIP-seq to evaluate bolA’s impact on flagellar genes. Upon overexpression of bolA, the transcriptional level of genes related to the flagellar structure was negatively affected (33%). Interestingly, the bolA protein directly interacts with different genes encoding proteins involved in diverse steps of the flagellar synthesis pathway (Fig. 5). Out of 33% of the positively regulated genes associated with the flagellar structure, 45% are direct BolA targets (genes highlighted in red boxes; the proteins encoded by these genes are shown in green).
In addition to regulating the transcription of genes encoding for flagellar proteins, bola A significantly reduces bacterial swimming capacity by repressing the master regulator FlhDC. In this way, bola A affects the Bolaturbo & bola turbo synthesis of flagella, curli, and fimbriae, which are essential for cell motility.
Moreover, bola A increases the production of extracellular DNA and proteins (eDNA and eCP), which are key components of the bacterial surface matrix that contributes to the adhesion of bacteria to surfaces. In order to determine whether the increased production of eDNA and eCP is caused by bola A, cells were grown on Toluidine Blue O plates, and cells overexpressing bola A showed a darker blue color compared with cells overexpressing the wild-type bola A. Toluidine blue O is a dye that reacts with purine and pyrimidine residues, and thus serves as a good indicator of the production of eDNA and eCP.
To identify the DNA consensus binding motif of the BolA protein, we performed in silico analyses of a pool of sequences identified by the ChIP-seq assay. The results obtained indicate that a specific motif is present in 92 of the analyzed sequences and was therefore considered to be the statistically significant BolA consensus sequence. This represents a relevant step toward the characterization of this recently discovered E. coli transcription factor and may have implications for the identification of its homologues in other organisms. Moreover, it opens the door to further research on its molecular mechanisms of action and its interaction with target genes.
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