Lambda CI Binding to Related Phage Operator Sequences Validates Alignment Algorithm and Highlights the Importance of Overlooked Bonds

Abstract

Bacteriophage λ’s CI repressor protein controls a genetic switch between the virus’s lysogenic and lytic lifecycles, in part, by selectively binding to six different DNA sequences within the phage genome—collectively referred to as operator sites. However, the minimal level of information needed for CI to recognize and specifically bind these six unique-but-related sequences is unclear. In a previous study, we introduced an algorithm that extracts the minimal direct readout information needed for λ-CI to recognize and bind its six binding sites. We further revealed direct readout information shared among three evolutionarily related lambdoid phages: λ-phage, Enterobacteria phage VT2-Sakai, and Stx2 converting phage I, suggesting that the λ-CI protein could bind to the operator sites of these other phages. In this study, we show that λ-CI can indeed bind the other two phages’ cognate binding sites as predicted using our algorithm, validating the hypotheses from that paper. We go on to demonstrate the importance of specific hydrogen bond donors and acceptors that are maintained despite changes to the nucleobase itself, and another that has an important role in recognition and binding. This in vitro validation of our algorithm supports its use as a tool to predict alternative binding sites for DNA-binding proteins.

Precise microbiome engineering using natural and synthetic bacteriophages targeting an artificial bacterial consortium

“…This step is highly host-specific and phages can recognize their hosts often at the genus and species and sometimes strain levels ( Nale et al, 2016 ; Gao et al, 2018 ; Fujiki et al, 2022 ). Virulent phages can produce their replicates inside host cells and they were released from the cells with bacterial lysis, whereas temperate phages occasionally integrate and dormant their genome into the host genome ( Sedhom and Solomon, 2023 ). Considering the characteristics and life cycles of phages, virulent phages could be deployed for subtractive and precise microbiome engineering.…”