Control and benefits of CP4-57 prophage excision in <i>Escherichia coli</i> biofilms

<jats:title>Abstract</jats:title> <jats:p>Earlier, we discovered that the global regulator, Hha, is related to cell death in biofilms and regulates cryptic prophage genes. Here, we show that Hha induces excision of prophages, CP4-57 and DLP12, by inducing excision genes and by reducing SsrA synthesis. SsrA is a tmRNA that is important for rescuing stalled ribosomes, contains an attachment site for CP4-57 and is shown here to be required for CP4-57 excision. These prophages impact biofilm development, as the deletion of 35 genes individually of prophages, CP4-57 and DLP12, increase biofilm formation up to 17-fold, and five genes decrease biofilm formation up to sixfold. In addition, CP4-57 excises during early biofilm development but not in planktonic cells, whereas DLP12 excision was detected at all the developmental stages for both biofilm and planktonic cells. CP4-57 excision leads to a chromosome region devoid of prophage and to the formation of a phage circle (which is lost). These results were corroborated by a whole-transcriptome analysis that showed that complete loss of CP4-57 activated the expression of the flg, flh and fli motility operons and repressed expression of key enzymes in the tricarboxylic acid cycle and of enzymes for lactate utilization. Prophage excision also results in the expression of cell lysis genes that reduce cell viability (for example, alpA, intA and intD). Hence, defective prophages are involved in host physiology through Hha and in biofilm formation by generating a diversified population with specialized functions in terms of motility and nutrient metabolism.</jats:p>