Sequence overlap between two genes is common across all genomes, with viruses having particularly high proportions of these gene overlaps. The natural biological function and effects on fitness of gene overlaps are not fully understood and their effects on gene cluster and genome-level refactoring are unknown.The model bacteriophage φX174 genome displays complex sequence architecture in which ~26% of nucleotides are involved in encoding more than one gene. In this study we use an engineered φX174 phage containing a genome with all gene overlaps removed. Here we have temporally measured the proteome of a synthetically engineered and wild-type φX174 during infection. We find that almost half of all phage proteins (5/11) have abnormal expression profiles after genome modularisation.

Peptides were reconstituted in 2% (v/v) acetonitrile (ACN) and 0.1% (v/v) formic acid (buffer A) to a final concentration of 0.1 μg/µL. 1μg (10 μL) of each sample was analysed on a Q-Exactive mass spectrometer (ThermoFisher Scientific, United States) coupled to an EASY-nLC1000 system (ThermoFisher Scientific, United States). Peptide samples were injected onto the LC system using buffer A and were bound on a 75 µM x 100 mM C18 HALO column (2.7 µM bead size, 160 Å pore size). A flow rate of 300 nL/min using an increasing linear gradient of buffer B (99.9% (v/v) acetonitrile, 0.1% (v/v) formic acid) was run from 1% to 50% for 110-minutes followed by 85% buffer B for 10-minutes. An inclusion list was used to target pre-defined precursor mass-to-charge (m/z), and the mass spectrometer was operated to perform one full-ms scan (70000 resolution (at 400 m/z) across the m/z range of 320-1800 m/z) with an automatic gains control (AGC) target of 1e6 (or a maximum fill time of 200 ms), followed by sequential PRM scans using the inclusion list (loop count 39, isolation window of 2 m/z). The selected precursor ions from PRM scans had an AGC target of 1e6 and a maximum fill time of 58 ms, after which, they were transferred from the C-trap to the higher energy collision dissociation (HCD) cell for fragmentation at a normalized collision energy of 27. MS/MS spectra were collected at a resolution of 17500 (at 400 m/z).

Sample identifiers: Time collected-condition-replicate# D = engineered variant (decompressed genome) W = wild type. For example, 15D2 = 15-minutes post-infection of decompressed infection replicate number 2