In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030

Abstract

The LlaI restriction and modification (R/M) system is encoded on pTR2030, a 46.2-kb conjugative plasmid from Lactococcus lactis. The llaI methylase gene, sequenced previously, encodes a functional type IIS methylase and is located ϳ5 kb upstream from the abiA gene, encoding abortive phage resistance. In this study, the sequence of the region between llaIM and abiA was determined and revealed four consecutive open reading frames (ORFs). Northern (RNA) analysis showed that the four ORFs were part of a 7-kb operon with llaIM and the downstream abiA gene on a separate transcriptional unit. The deduced protein sequence of ORF2 revealed a P-loop consensus motif for ATP/GTP-binding sites and a three-part consensus motif for GTP-binding proteins. Data bank searches with the deduced protein sequences for all four ORFs revealed no homology except for ORF2 with McrB, in three regions that coincided with the GTP-binding motifs in both proteins. To phenotypically analyze the llaI operon, a 9.0-kb fragment was cloned into a high-copy-number lactococcal shuttle vector, pTRKH2. The resulting construct, pTRK370, exhibited a significantly higher level of in vivo restriction and modification in L. lactis NCK203 than the low-copy-number parental plasmid, pTR2030. A combination of deletion constructions and frameshift mutations indicated that the first three ORFs were involved in LlaI restriction, and they were therefore designated llaI.1, llaI.2, and llaI.3. Mutating llaI.1 completely abolished restriction, while disrupting llaI.2 or llaI.3 allowed an inefficient restriction of phage DNA to occur, manifested primarily by a variable plaque phenotype. ORF4 had no discernible effect on in vivo restriction. A frameshift mutation in llaIM proved lethal to L. lactis NCK203, implying that the restriction component was active without the modification subunit. These results suggested that the LlaI R/M system is unlike any other R/M system studied to date and has diverged from the type IIS class of restriction enzymes by acquiring some characteristics reminiscent of type I enzymes.The biological role of restriction and modification (R/M) systems is to protect cells from invasion by foreign DNA, such as promiscuous plasmids or infecting bacteriophages. The restriction enzyme can discriminate between foreign and host DNA by the presence of methylation on certain nucleotides (reviewed in reference 4). Traditionally, these enzymes have been classified into three types. Type I enzymes consist of three subunits which are responsible for modification, restriction, and specificity. A complex of the three subunits is required to restrict DNA in the presence of Mg 2ϩ , ATP, and S-adenosylmethionine. The site of cleavage is generally a large distance (often Ͼ1 kb) from the recognition site. Type II enzymes are the most common and the simplest, with over 1,000 different varieties identified (45). They consist of one subunit, require Mg 2ϩ as a cofactor, and restrict at or near their recognition sites. Those that restrict outside their recognition sites ...

Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain The GenBank accession numbers for the sequences reported in this paper are AF153410–AF153414.

“…These have been subdivided as type IIs systems. A lactococcal example of a type IIs R\M system is the LlaI system from the conjugative plasmid pTR2030 (O'Sullivan et al, 1995). This system also differs from other type II R\M systems because the restriction activity is encoded by three genes rather than one.…”

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

“…In a previous study, a 13.6-kb BglII fragment of pTR2030 was cloned into shuttle vector pSA3 to produce plasmid pTK6 (30). This fragment encodes the phage resistance determinants for the LlaI restriction/modification operon and also carries the abortive gene abiA (26,39). However, phage resistance from pTK6 (EOP of 10 Ϫ4 with phage 31) is expressed at a lower level than that from pTR2030 (EOP of Ͻ10 Ϫ10 with phage 31) (32).…”

“…Plasmid pTR2030 has previously been partially subcloned and sequenced to reveal an R/M system, LlaI, and the abortive gene abiA (originally designated hsp) (26,39). However, testing of AbiA and R/M subclones of pTR2030 suggested that a third mechanism could be encoded on this plasmid (27,32,39,42). Additional subcloning and sequencing of pTR2030 revealed a novel abortive resistance mechanism downstream of abiA.…”

“…Two self-transmissible plasmids, pTR2030 and pTN20, each encode at least one R/M system and one Abi system. Plasmid pTR2030 has previously been partially subcloned and sequenced to reveal an R/M system, LlaI, and the abortive gene abiA (originally designated hsp) (26,39). However, testing of AbiA and R/M subclones of pTR2030 suggested that a third mechanism could be encoded on this plasmid (27,32,39,42).…”

Molecular characterization of the restriction endonuclease gene (scrFIR) associated with the ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503

“…R/M systems are the most frequently encountered resistance mechanism in Lactococcus, where they are typically plasmid-encoded (Hill, 1993 ;Klaenhammer & Fitzgerald, 1994;Garvey et al, 1995). The nucleotide sequences of three complete systems have been pub-0002-1 480 0 1997 SGM lished: LlaI (Hill et al, 1991;O'Sullivan et al, 1995), LlaDCHI (previously referred to as LlaII; Moineau et al, 1995) and LlaBI (Nyengaard et al, 1996). In addition, the sequences of two chromosomally encoded methyltransferases (MTases) associated with the ScrFI R / M system from L. lactis subsp.…”