Ea22 Proteins from Lambda and Shiga Toxin-Producing Bacteriophages Balance Structural Diversity with Functional Similarity

Abstract

Enterohemorrhagic Escherichia coli (EHEC) outbreaks are commonly associated with contaminated food sources. Unlike normal intestinal bacteria, EHEC are lysogens of lambdoid bacteriophages that also carry a gene for Shiga toxin. Oxidative attack by the immune system or other stressors on the bacterial host can activate the lytic pathway of the latent phage genome to produce phage progeny and the release of Shiga toxin into the surrounding tissues. Within the genomes of bacteriophage λ and Shiga toxin-expressing (Stx + ) phages such as φ24 B and φP27, there is a conserved set of open reading frames that is located between the exo and xis genes that influences the lysogenic−lytic decision. In this report, we have focused on the largest exo−xis region open reading frame termed ea22 that has been shown previously to have prolysogenic properties. Using a variety of biophysical and bioinformatic methods, we demonstrate that λ and φP27 Ea22 proteins are tetrameric in solution and can be considered in terms of an aminoterminal region, a central coiled-coil region, and a carboxy-terminal region. The carboxy-terminal regions of λ and φ24 B Ea22, expressed on their own, form dimers with exceptional thermostability. Limited proteolysis of φP27 Ea22 also identified a C-terminal region along the predicted boundaries. While the three Ea22 proteins all appear to have the hallmarks of a domain in their respective C-terminal regions, each sequence is remarkably dissimilar. To reconcile this difference among Ea22 proteins from λ and Stx + phages alike, we speculate that each Ea22 may achieve the same function by targeting different components of the same regulatory process in the host.

Molecular Modeling the Proteins from the exo-xis Region of Lambda and Shigatoxigenic Bacteriophages

“…The ea22 gene provides some contrast to the three exo-xis genes that precede it. Not only is it the largest protein of the exo-xis region and multidomain in composition [ 52 ], but its function also favors lysogenic development over lytic development [ 27 ]. With this function in mind, Ea22 may serve as a new focal point for understanding how Stx + phage lytic program can be delayed during EHEC infections to limit the release of Shiga toxin.…”

“…From sequence comparisons and experimental evidence derived from deletion studies, and biophysical analysis of purified proteins and protein fragments, λ Ea22 may be considered in terms of a variable N -terminal region, a central tetrameric coiled-coil region, and a dimeric C-terminal region. The C-terminal region demonstrates the hallmarks of a domain since it can be separated from the protein by limited proteolysis treatment while still retaining exceptional thermostability [ 52 ]. As shown earlier in Figure 3 , a comparison of the AlphaFold and RoseTTAFold predictions for the λ Ea22 C-terminal domain as a monomer differ only in the positioning of the second helix with an overall backbone RMSD of 1.82 Å.…”

NMR structure of the carboxy-terminal domain of the Ea22 pro-lysogenic protein from lambda bacteriophage

“…The most variability among the Ea22 proteins occurs at the C-terminal region of the protein. In a few cases where purified proteins can be studied, the C-terminal region is dimeric like λ regardless of sequence, and the full-length protein is tetrameric suggesting there is evolutionary pressure to maintain a specific oligomeric state even when the sequence and structure are drifting 5 . It is possible, therefore, that Stx phages have acquired new domains that improve fitness while maintaining a similar architecture.…”

Inhibitor tolerance and bioethanol fermentability of levoglucosan-utilizing Escherichia coli were enhanced by overexpression of stress-responsive gene ycfR: The proteomics-guided metabolic engineering

“…MurR is a putative transcription factor that could repress the murPQ operon, which is required for the cellular catabolism of the bacterial cell wall [ 55 ]. Also, Ea22, probably derived from bacteriophages, is an uncharacterized protein involved in the cell cycle control, cell division, and chromosome partitioning of E. coli [ 56 ]. Deletion-substitution mutagenesis has shown that Ea22 is responsible for the block to initiation of DNA replication [ 57 ].…”