Bolaturbo & Bola Turbo - Targets of BolA

 

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To identify the targets of the transcriptional regulator BolA, we performed chromatin immunoprecipitation followed by DNA high-throughput sequencing (ChIP-seq). Chromatin from an E. coli strain whose bolA gene was deleted (DbolA) and an isogenic strain expressing a chromosomal fusion of the bolA protein with a 3xFlag tag (wt 3xFlag) were subjected to ChIP-seq. Statistically significant peaks corresponding to the BolA binding sequences were identified. A DNA consensus binding motif was determined based on the pool of DNA sequences corresponding to the peaks.

The results from ChIP-seq showed that BolA directly binds to the promoter regions of several genes. Among these, the genes encoding for fimbriae and other adhesion proteins were most prominently induced upon overexpression of BolA (bolA++ strain). In addition, the expression levels of the genes ydeS, yfcV, ygiL, yadC, ybgD, yraH, and yehC increased by 3 h after induction of bolA expression. These genes are known to be involved in the early steps of biofilm formation, including the formation of type I fimbriae (see Fig. S1A in the supplemental material).

Furthermore, it was found that the BolA protein regulates central carbon metabolism by causing the direct activation of transcription of genes for amino acid pathways and the TCA cycle. These cellular pathways are connected to peptidoglycan synthesis, which is an important component of the bacterial cell wall. Thus, the regulation of these metabolic pathways by BolA is a general mechanism for the establishment of biofilms.

Finally, it was found that bolA significantly Bolaturbo decreases the motility of E. coli cells, and that this effect is influenced by the level of BolA overexpression. These results suggest that BolA influences the synthesis of flagellar genes and thereby affects the overall swimming capacity of bacteria. In a similar way, the repression of the bolA-encoded flagellar genes also leads to a reduction in the motility of bacteria. This confirms that BolA is a transcription factor with a specific DNA binding motif that is involved in the control of metabolic pathways that are connected to the synthesis of peptidoglycan, a major component of the bacterial cell wall. Thus, we present a model in which the BolA protein has a crucial role for the initiation and maintenance of bacterial biofilms. The findings from this study constitute a relevant step toward the understanding of this recently discovered transcription factor and will have an impact on other pathogenic bacteria that possess homologues of BolA.