Functional and practical insights into three lactococcal antiphage systems

Abstract

The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities.

Keywords: Abi; Lactococcus; bacterial immunity; lactic acid bacteria; phage defense.

Fig 1

(A) One-step phage growth curve of phage sk1 and (B) ECOI and burst size of phage sk1 on L. cremoris NZ9000::pNZ44 and its phage-resistant derivatives harboring Rhea, Aristaios, and Kamadhenu antiphage systems. Experiments were performed in biological triplicate, and data are presented as means ± SD.

Fig 2

Quantification of phage sk1 DNA on different time intervals by qPCR in L. cremoris NZ9000::pNZ44 and its phage-resistant derivatives harboring Rhea, Aristaios, Kamadhenu, and AbiP antiphage systems. Experiments were performed in biological triplicate, and data are presented as means ± SD. Asterisks mark statistically significant differences between the phage-resistant derivatives and the reference strain at respective time points (unpaired t-test, P value < 0.01).

Fig 3

Quantification of phage sk1 cDNA on different time intervals by qPCR in L. cremoris NZ9000::pNZ44 and its phage-resistant derivatives harboring Rhea, Aristaios, and Kamadhenu antiphage systems. Experiments were performed in biological triplicate, and data are presented as means ± SD. Asterisks mark statistically significant differences between the phage-resistant derivatives and the reference strain at respective time points (unpaired t-test, P value < 0.01).

Fig 4

Detection of sk1 phage capsid (A), tail (B), and RBP (C) in L. cremoris NZ9000::pNZ44 and its phage-resistant derivatives harboring Rhea, Aristaios, and Kamadhenu antiphage systems with western blot in different time points.

Fig 5

Milk acidification curves of L. cremoris NZ9000::pNZ44 and its phage-resistant derivatives harboring Rhea, Aristaios, and Kamadhenu antiphage systems. Experiments were performed in biological triplicate, and data are presented as means ± SD.

Fig 6

Plasmid map of the pMRC01-like conjugative plasmid pUCCL624B carrying Kamadhenu antiphage system among other antiphage systems.