Abortive Phage Resistance Mechanism AbiZ Speeds the Lysis Clock To Cause Premature Lysis of Phage-Infected Lactococcus lactis

Abstract

The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10 ؊6 , without affecting phage adsorption. AbiZ causes phage 31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of 31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage 31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis.The susceptibility of starter cultures to bacteriophage infection remains a problem in the cheese industry, especially with increasing reliance on defined starters and the high turnover in factories (8). Analysis of phage resistance mechanisms in starters has led to the identification of four categories of natural bacteriophage resistance in lactococci: (i) interference with phage adsorption, (ii) interference with phage DNA injection, (iii) DNA restriction/modification (R/M), and (iv) abortive infection. Numerous plasmids encoding one or more of these systems have been isolated from commercial starter strains (2,7,8). Abortive infection defenses (Abi) cause a disruption of phage development postinfection, resulting in a decrease in the number of infective particles released and death of the infected cells (7,8). Nonsterile milk fermentations provide an evolutionary proving ground, in which lactic acid bacteria are exposed to constant challenge from bacteriophages. Phage defense mechanisms are often encoded on plasmids, many of which are conjugative, facilitating their transfer within starter culture populations.There are three established groups of phages infecting L. lactis: groups c2 and 936 are composed of lytic phages which are highly homologous within the groups, while the P335 group contains both lytic and lysogenic members and is characterized by mosaic genomes showing evidence ...

The phage abortive infection system, ToxIN, functions as a protein–RNA toxin–antitoxin pair

“…Abi systems often are highly toxic when activated; they have varied targets and can act on central cellular processes to inhibit phage DNA replication, transcription, and protein synthesis (9). Specific effects include premature cell lysis by AbiZ (13) and interference of a phage RuvC-like endonuclease by AbiD1 (14).…”

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques to Lower Phage Contamination in Dairy Fermentation

“…The most likely explanation for this phenomenon is that Abi proteins interfere with processes essential not only for phage, but also for bacterial development; therefore, death of individual bacterial cells is always observed following activation of the Abi systems [17,[58][59]. As a consequence, release of progeny particles is limited and the bacterial population survives.…”

“…Based on this observation, it was concluded that the AbiV system arrests the bacterial translation apparatus [60]. AbiE, AbiI, AbiQ and AbiZ systems affect maturation of phage particles [59,65]. The AbiZ system, identified in 2007 by Durmaz and Klaenhammer, induces premature lysis of phage-infected cells, resulting in the release of the developing phage particles before completion of the maturation process.…”

“…The most known Abis have been found in the latter species. To date, 22 lactococcal Abi mechanisms have been identified and designated into various groups distinguished by a subsequent letter of the alphabet [58][59][60]. Most of Abi systems are plasmid-encoded and only three are located on chromosomal DNA (abiH, abiN, abiV) [60].…”

“…The AbiZ system, identified in 2007 by Durmaz and Klaenhammer, induces premature lysis of phage-infected cells, resulting in the release of the developing phage particles before completion of the maturation process. The timing of phage lysis is controlled by the phage holin protein; thus, AbiZ might interact cooperatively with the phage holin or with a holin inhibitor to make it active prematurely [59].…”