Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2

Abstract

The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10-2 to 10-3 and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10-4 and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90%o of Prf4 cells infected with phage p2 die. Thus, phage infections in PrI cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp.cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by TnS mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. actis subsp. cis ME2) and abi416 (Abi+, from L. lad subsp. lactis fL416). Unlike abU4, the action ofabiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.

The Lactococcal Abortive Phage Infection System AbiP Prevents both Phage DNA Replication and Temporal Transcription Switch

“…Orf1 (194 aa) shares no significant homology with proteins in the databases and presents five putative transmembrane helices (positions 7 to 29, 55 to 77, 82 to 102, 112 to 134, and 155 to 177) (27,40). Orf2 (245 aa) shares 22% identity with the lactococcal abortive phage infection protein AbiC (19). Alignment of the two proteins reveals three regions of homology separated by an insertion (on AbiC; 344 aa) or by deletion (on Orf2) of two stretches of 34 and 46 amino acids.…”

“…AbiP shares homology only with AbiC, another lactococcal Abi (19). However, the two proteins have different modes of action on phage development: AbiC does not affect phage DNA replication, whereas AbiP blocks DNA production 10 min after infection.…”

Isolation, cloning and characterisation of the abiI gene from Lactococcus lactis subsp. lactis M138 encoding abortive phage infection

“…This mechanism is considered the most powerful mechanism of phage resistance identified thus far in lactococci (Klaenhammer and Fitzgerald, 1994). A number of lactococcal genes encoding abortive phage infection have been cloned and sequenced and these include abiA (Hill et al, 1990;Coffey et al, 1991), abiB (Cluzel et al, 1991), abiC (Durmaz et al, 1992), abiD (McLandsborough et al, 1995), abiD1 (Anba et al, 1995), abiE and abiF (Garvey et al, 1995), abiG (O'Connor et al, 1996) and abiH (Prevots et al, 1996). In this paper we report a new plasmid encoded abortive infection gene abiI…”

Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1

“…In every case, phage infection induced deleterious effects for highly conserved cellular components involved either in the translation apparatus or in membrane potential. This results in cell death and phage growth arrest, which blocks the spread of phages to other cells.Four lactococcal genes coding for an Abi phenotype have been cloned and sequenced (2,6,7,9,15). Except for AbiA (16, 23), which has been proposed to interfere with phage DNA replication, the molecular mechanisms of lactococcal phage growth impairment in Abi cells are unknown.…”

“…Four lactococcal genes coding for an Abi phenotype have been cloned and sequenced (2,6,7,9,15). Except for AbiA (16, 23), which has been proposed to interfere with phage DNA replication, the molecular mechanisms of lactococcal phage growth impairment in Abi cells are unknown.…”