EOHA ASM 2026

Abstract

Background:

Bacteriophages are critical mediators of bacterial population dynamics in natural, agricultural, and food-associated ecosystems, impacting human and animal health. In Listeria monocytogenes, both virulent and temperate phages shape ecological balance, drive evolution, and contribute to the pathogen’s persistence. Although phage lifestyles are well-characterized, genome-wide differences—particularly in gene content and synonymous codon usage affecting translation efficiency—remain poorly understood.
Objectives: This study aimed to characterize genome-wide differences in gene content and synonymous codon usage between virulent and temperate L. monocytogenes phages, providing insights into lifestyle-specific adaptations in translational efficiency and codon optimization.

Methods:

We analyzed 1,516 genes from 10 virulent and 686 genes from 10 temperate phages. Metrics including gene length, codon adaptation index (CAI), effective number of codons (Nc), total GC content, and positional GC content (%GC1, %GC2, %GC3) were computed. Differences were evaluated using Welch’s t-test and the Mann–Whitney U test. Principal component analysis and Pearson correlation analysis examined codon usage patterns and associations.

Results:

Temperate phages had smaller genomes (41,023 ± 3,225 kb) and shorter genes (555 ± 647 nt) than virulent phages (101,089 ± 44,030 kb; 629 ± 584 nt). CAI was higher in temperate phages (0.711 ± 0.045), indicating stronger host codon adaptation, while virulent phages showed higher GC content (35.84 ± 2.99%) and greater codon usage bias (Nc = 44.80 ± 5.86), particularly at the third codon position. CAI and %GC3 were strongly negatively correlated (r = −0.77 to −0.97).

Conclusions:

Differences in genome composition and codon usage reflect phage lifestyle, with temperate phages favoring host-adapted codons and virulent phages favoring biased codons most likely for rapid protein synthesis. These findings provide a framework for codon usage–based biomarkers to rapidly distinguish phage lifestyles and inform synthetic biology strategies to enhance phage-based therapeutics, diagnostics, and biocontrol.