Page 122 - Read Online

Varming et al. Microbiome Res Rep 2024;3:15 https://dx.doi.org/10.20517/mrr.2023.50 Page 11 of 16

Figure 3. Purification of φ13 Mor and characterization of interaction with φ13 CI. (A) Analysis of Mor purification progress on 15%
SDS-PAGE, showing a single band for Mor in SEC elution fractions. Each type of fraction loaded is indicated above the lane. Fractions
named FT contain the flowthrough from HisTrap affinity chromatography, while numbers listed below gel filtration step indicate the
elution volume of each fraction; (B) SEC chromatograms of φ13 Mor. The absorbance at 280 and 260 nm are shown in blue and red,
respectively, and the estimated oligomeric state is marked by an arrow; (C) IEF electrophoresis of CI and Mor alone and in complex. The
pH standard values are listed on the left and the protein sample composition for each lane is above. CI: Phage repressor; SEC: selected
gel filtration; IEF: isoelectric focusing.

no template yielded a highly favorable clash score (1.83) and resolved the interface problems, leading to the
creation of a credible model for the CI-NTD:Mor interface in φ13, which is presented here for further
discussion and is available as supplementary material.

Analysis of the φ13 CI-NTD:Mor interface
A close-up of the interface region is shown in Figure 4. Several residues at the interface (Gln55, Tyr59,
Met73, and Glu69) are conserved in CI from the two species [Figure 4A]. Residue 75 is Phe in TP901-1 and
Tyr in φ13. In TP901-1 , the substitution of Met73 to Ala had a great impact in vitro and in vivo, while the
[24]
substitution of Phe75 to Ala had less impact, and the substitution with Tyr only a mild impact, which is
compatible with the residue being a Tyr in the φ13 system. Substitution of Gln55 had a large effect in vivo,
whereas substitutions of Tyr59 and the previously discussed Glu69 have not been investigated, but they
undoubtedly warrant further investigation. Tyr3, Tyr5, Trp43, and Phe67 are conserved interface aromatic
residues on Mor, forming a pocket in which Gln55 from CI can fit [Figures 2A and 4B] in both phages.