Extended Data Fig. 1: MDAΦ lysogenization of Nme strain 8013.

a, MDAΦ lysogenization requires major pilin pilE, but not the minor pilin pilX or other host factors for natural transformation. To validate host factor requirements for MDAΦ infection, we tested a panel of host gene deletion strains. Only ΔpilE abolished MDAΦ transduction, confirming type IV pili’s role as phage receptor and that Kan^R colonies are indeed transductants, not transformants caused by genomic DNA contamination in phage prep. MDAΦ transduction frequencies % (transductants per CFU of total host cells) in mutants are normalized to that of WT strain. Data are mean ± s.d., n = 3. b, Natural transformation test in the same set of strains as in a. Transformants were abolished by ΔRecA, ΔcomA, ΔdprA, ΔcomP, and ΔpilE, consistent with these host factors’ known roles in competency. c, Illustration of MDAΦ life cycle in Nme. MDAΦ infects mainly as episomes, including circular ssDNA viral genome and dsDNA replication form (RF). It can also integrate into host chromosome as a prophage (green) via conserved dRS3 sites (orange). d, Nanopore sequencing of total DNA from MDAΦ transductant pool to confirm the existence of MDAΦ prophage. e, Diagnostic PCRs detecting the circular junction of MDAΦ episome and prophage-host integration junction from total DNA of MDAΦ transductant pool. WT strain without MDAΦ infection is the negative control. Green and orange arrows indicate PCR primers used with annealing sites depicted in d. f, h, Real-time PCR determination of the relative copy numbers of circular MDAΦ (f) and dRS3-MDAΦ junction (h), normalized to Neisseria host gene NMV_1851 copy set as 1. On average, 10 copies of MDAΦ episome exist per host genome as shown by panel f and about 25% of the transductants’ genome have a MDAΦ prophage as shown in panel h. g, i, PCR standard curves created on serial 10-fold dilutions of reference plasmids. CT values plotted against the log scale of known initial copy numbers. Data are mean values from 3 independent experiments.