In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage

Abstract

The conjugative plasmid pTR2030 confers bacteriophage resistance to lactococci by two independent mechanisms, an abortive infection mechanism (Hsp+) and a restriction and modification system (R+/M+). pTR2030 transconjugants of lactococcal strains are used in the dairy industry to prolong the usefulness of mesophilic starter cultures. One bacteriophage which has emerged against a pTR2030 transconjugant is not susceptible to either of the two defense systems encoded by the plasmid. Phage nck2O2.50 (450) is completely resistant to restriction by pTR2030. A region of homology between pTR2030 and 450 was subcloned, physically mapped, and sequenced. A region of 1,273 bp was identical in both plasmid and phage, suggesting that the fragment had recently been transferred between the two genomes. Sequence analysis confirmed that the transferred region encoded >55% of the amino domain of the structural gene for a type II methylase designated LlaI. The LlaI gene is 1,869 bp in length and shows organizational similarities to the type II A methylase FokI. In addition to the amino domain, upstream sequences, possibly containing the expression signals, were present on the phage genome. The phage 450 fragment containing the methylase amino domain, designated LlaPI, when cloned onto the shuttle vector pSA3 was capable of modifying another phage genome in trans. This is the first report of the genetic exchange between a bacterium and a phage which confers a selective advantage on the phage. Definition of the LlaI system on pTR2030 provides the first evidence that type II systems contribute to restriction and modification phenotypes during host-dependent replication of phages in lactococci.

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…For example, phages of Bacillus subtilis, SPR, 3T, ␦l1, and SP␤, have evolved protection mechanisms against host restriction enzymes by self-methylating their DNA at various sequences (53). The methylation of phage genomes is caused by the acquisition of host-encoded MTases by the phages (54).…”

Movers and shakers

“…This strategy has enabled phages to overcome resistance mechanisms, a phenomenon that has been observed for dairy starter cultures in a factory setting. [114][115][116] For example, lactococcal phage 50 became completely resistant to the type II R/M system encoded on the lactococcal plasmid pTR2030 after it acquired a copy of the plasmid-encoded methylase gene through an in vivo genetic exchange event. 114 Four phage mutants resistant to the abortive infection mechanisms AbiK, AbiT or both emerged after extensive genome re-shuffling with resident prophage sequences.…”

“…[114][115][116] For example, lactococcal phage 50 became completely resistant to the type II R/M system encoded on the lactococcal plasmid pTR2030 after it acquired a copy of the plasmid-encoded methylase gene through an in vivo genetic exchange event. 114 Four phage mutants resistant to the abortive infection mechanisms AbiK, AbiT or both emerged after extensive genome re-shuffling with resident prophage sequences. 116 Thus, potential phage resistance mechanisms in the intestine may serve to amplify the phage population.…”

Control of expression of LlaI restriction in Lactococcus lactis

“…lactis ME2 encodes the LlaI restriction modification (R/M) system (Klaenhammer and Sanozky, 1985). The LlaI methylase gene (llaIM ) encodes a methylase with two functional domains, each bearing consensus regions of type IIS methylases (Hill et al, 1991). The full LlaI R/M system is encoded on a 6.9 kb polycistronic operon .…”