The packaging signal ofXanthomonasintegrative filamentous phages

Abstract

Unlike Ff, the packaging signal (PS) and the mechanism of integrative filamentous phage assembly remain largely unknown. Here we revived twoInoviridaeprophage sequences, φLf2 and φLf-UK, as infectious virions lysogenize black rot pathogenXanthomonas campestrispv.campestris. φLf2 and φLf-UK genomes consist of 6,363 and 6,062 nucleotides and share 85.8% and 98.7% identity with φLf, respectively. To explore their assembly, we first identified 20-26-nucleotide long PS sequences of 10Xanthomonasphages. These PS consist of a DNA hairpin with the consensus GGX(A/-)CCG(C/T)G sequence in the stem and C/T nucleotides in the loop, both of which are conserved and essential for PS activity. In contrast to Ff, the 5′ to 3′ orientation of PS sequence is not conserved or critical for their competence. This is the first report to offer insights into the structure and function of integrative phage PS, revealing the diversity of filamentous phage encapsidation.

A packaging signal-binding protein regulates the assembly checkpoint of integrative filamentous phages

“…Thus far, genomic information has shown that integrative filamentous phages of Gram-negative bacteria lack an Ff gIV and gIX homologue. We also verified that Ff gVII homologues are not present in many integrative filamentous phages (Yeh et al, 2023). The absence of these crucial structures prompted us to identify the PS of these phages, what viral (structural and non-structural) factors are involved in their assembly and egress, and how the PS DNA becomes targeted to the IM for encapsidation.…”

“…To identify protein factor(s) involved in assembly, we first modified Russel and Model’s method of suppressor screening for PS-interacting protein(s) based on phage plaque morphology (Supplemental Figure S1) (Russel and Model, 1989). We generated a ϕLf-UK phage synonymous T340A/C341G mutant that lost its PS activity but kept gII protein function to facilitate phage DNA replication (Figure 1A and 1C) (Yeh et al, 2023). The ϕLf-UK T340A/C341G plaque morphology was tiny (0.1-0.2 mm) compared to the wild-type phage (1-1.2 mm).…”

“…The host bacteria, Xcc-TcR, was maintained by tetracycline as described (Yeh et al, 2023). TrxA G93D mutation was introduced in Xcc-TcR by homologous recombination in which TrxA G93D was expressed in cis under control of the native promoter (Kaniga et al, 1991; Huguet et al, 1998).…”

“…The ϕLf-UK replicative form (RF) DNA, the karamycin-resistant pORIPS plasmid with ϕLf-UK ori and PS sequence (245-365, MH206184), as well as chloramphenicol-resistant pGII plasmid was maintained as previously (Yeh et al, 2023). A stop codon was introduced into the start codon of ϕLf-UK ORF9 gene (nt 4983-5309, MH206184) of RF DNA to generate ϕLf-UK-ΔPSB15.…”

“…They have been shown to influence the virulency of their host bacteria, including Yersinia pestis biovar orientalis and Vibrio cholerae , which respectively causes plague and cholera, and the biofilm formation of Pseudomonas aeruginosa , contributing to public health consequences (Waldor and Mekalanos, 1996; Gonzalez et al, 2002; Rice et al, 2009). To date, there are more than 30 different integrative filamentous phages identified from human, animal, and plant pathogens (Mai-Prochnow et al, 2015; Hay and Lithgow, 2019; Yeh et al, 2023). However, very little is known about the PS or encapsidation processes crucial for their replication and infection life cycles.…”