Identification and Functional Analysis of Novel SNPs in Enterocin Genes of Enterococcus faecium GHB21.

Abstract

This study investigates the functional and structural impact of single nucleotide polymorphisms (SNPs) in the enterocin and associated immunity genes of Enterococcus faecium GHB21, a strain known for producing potent antimicrobial peptides. Enterocins, most of them classified as class IIa bacteriocins, exhibit strong activity against pathogens such as Listeria monocytogenes, making them promising candidates for food preservation and therapeutic interventions. Using cloning, sequencing, and bioinformatics tools, we analyzed key enterocin genes (entA, entB and entP) and their associated immunity genes (entAi and entPi). Two novel SNPs were identified that result in amino acid substitutions: G15N in pre-enterocin P (EntP), located within the leader sequence, and V36I in the EntPi immunity protein. Additionally, the V9I mutation within the conserved YGNGV motif of the mature EntP peptide and the G48S mutation in the EntAi immunity protein were analyzed. Protein Variation Effect Analyzer classified all mutations as neutral, indicating minimal disruption to protein function. DynaMut analysis revealed that V9I stabilizes EntP but slightly reduces its flexibility, potentially influencing its interaction with target bacteria. Despite these mutations, the enterocins retained critical structural features, including disulfide bonds and β-sheet arrangements, ensuring their antimicrobial efficacy. These findings underscore the structural resilience of enterocins, supporting their application in food safety and in combating multidrug-resistant pathogens.