The bacteriophage defense mechanism AbiR from L. lactis
KR2 is encoded by three genes, abiRa
, and abiRc
, organized in an operon that is involved in the expression of the AbiR phenotype (38
). However, the presence of the abiR
operon alone failed to exhibit an AbiR phenotype in L. lactis
, implying that extra DNA was necessary to complete the expression of the AbiR phenotype. The findings of this study surprisingly demonstrate that the extra DNA required for expression of AbiR is the methylase gene from the LlaKR2I R/M system, which is located downstream of the AbiR operon. To date, almost 500 R/M systems have been characterized at the gene level (http://rebase.neb.com/cgi-bin/statlist
), and the role of the methylase in each case is to protect the bacterial DNA from digestion by the cognate restriction enzyme. The LlaKR2I methylase gene was previously shown to be an active component of the LlaKR2I R/M system (37
), and in this study, it was shown to protect L. lactis
from the toxic effects of AbiR (Fig. and ). This latter protective role for the LlaKR2I methylase differs significantly from its protective role in the LlaKR2I R/M system. The sole role of a methylase in an R/M system is to protect the host genome from restriction by the cognate restriction enzyme, as methylated DNA would successfully escape the restriction system. Unlike an R/M system, AbiR terminates the proliferation of both unmethylated and methylated bacteriophage DNA. In addition, mechanistic studies have shown that AbiR acts by impeding bacteriophage DNA replication (38
). This substantiates a novel role for the methylase from the LlaKR2I R/M system in L. lactis
since besides protecting the chromosome from the LlaKR2I restriction enzyme, it also protects L. lactis
from the toxicity of the AbiR operon.
To uncover the extra DNA necessary to express the AbiR mechanism in L. lactis, numerous deletion and insertion strategies were attempted. Digestion with single-cut restriction enzymes allowed targeted deletions in all ORFs on the plasmid whose function was not known. Of these, ORF5 and -6 contained a G+C content of 36.4% and 34.5%, respectively, similar to the L. lactis typical range, while ORF4 contained a significantly lower G+C content of 30.5%, consistent with atypical abi genes. Surprisingly, deletion of ORF4, -5, or -6 had no effect on the AbiR phenotype in L. lactis (Fig. ). The only other genes present on the plasmid were the IS982 transposase genes and the LlaKR2I restriction and modification genes. The IS982 element positioned directly upstream from the abiR operon was disrupted by in vitro transposition, and the downstream IS982 element was successfully deleted without affecting the AbiR phenotype (Fig. and ). The LlaKR2I restriction gene was also successfully deleted without altering the AbiR phenotype (Fig. ), leaving the LlaKR2I methylase gene as the only possible candidate gene on the plasmid to be investigated for involvement in the expression of the AbiR phenotype. However, numerous attempts to delete or introduce mutations specifically in llaKR2IM either failed or resulted in further unintended deletions in the abiR operon. The culmination of these data indicated that removal of the LlaKR2I methylase gene could not occur while the abiR operon was intact, suggesting that the establishment of this operon in L. lactis required this methylase gene. It is proposed that AbiR inhibits the host chromosomal DNA replication in a mechanism similar to phage DNA replication and that the presence of the LlaKR2I methylase is necessary to shield L. lactis from AbiR effects. Confirmation of the protective role for the LlaKR2I methylase gene in the AbiR phenotype was obtained by trans-complementation of these two components in L. lactis, whereby the AbiR operon alone was toxic to the cell, and the llaKR2IM gene was necessary to protect the cell from the AbiR toxicity and allow expression of the AbiR phenotype in L. lactis (Fig. and ). This observation uncovered not only an original makeup for an Abi bacteriophage defense system for L. lactis but also a novel protective role for the LlaKR2I methylase gene besides its traditional role to protect the host DNA from its cognate restriction enzyme.
There are two classes of methylases that do not have a cognate restriction enzyme and whose roles are involved in cell cycle replication and other cellular regulatory functions. These are the E. coli
Dam methylase and the cell cycle-regulated methyltransferases, termed CcrM, which have been found in a number of gram-negative bacteria (23
). Like the Dam methylase, the LlaKR2I methylase recognizes the DNA sequence 5′-GATC-3′. However, Dam methylates at the adenine residue while LlaKR2IM methylates at the cytosine residue (37
). Dam methylase plays an important role in regulation of several cellular functions, such as directing mismatch repair, coordinating the timing of DNA replication, and segregating chromosomal DNA. However, this global regulator is not essential to the survival of E. coli
cells as Dam-negative mutants of E. coli
can readily be obtained. In contrast, Dam was shown to be essential for the viability of Yersinia pseudotuberculosis
and Vibrio cholerae
). In fact, disruption of the dam
genes in these two bacteria resulted in no growth unless a copy of the wild-type dam
gene was present in trans
. The importance of Dam methylation in Y. pseudotuberculosis
and V. cholerae
is analogous to the essential role of CcrM for viability of other gram-negative bacteria.
The role of the LlaKR2I methylase in AbiR-containing strains of L. lactis
is also analogous, as it also cannot be disrupted. As the AbiR phenotype was shown to result in the inhibition of bacteriophage DNA replication (38
), it is likely that it may be toxic to the host because of interference with DNA replication, and the role of the LlaKR2I methylase may be to prevent this in a manner similar to that of the CcrM methylases in gram-negative bacteria. Interestingly, as with the LlaKR2I methylase, CcrM also methylates at the cytosine residue in its recognition sequence, 5′-GANTC-3′ (36
). In addition, it was demonstrated that CcrM methylation was involved in regulating the cell cycle and controlling gene expression and DNA replication of some 20 α-proteobacteria. The striking observation, in all instances, was that CcrM methylation was essential for cell viability (33
). Although Dam is not essential for cell viability, except in a few cases, it has functional analogies with CcrM, suggesting that they may have evolved from an existing 5′-GATC-3′ R/M system into the global regulators they are now.
Interestingly, the LlaKR2I methylase protects L. lactis
from AbiR while it does not protect bacteriophage DNA replication. Indeed, replication of unmethylated and methylated bacteriophage DNA is affected identically by AbiR (38
). Although how the LlaKR2I methylase works in protecting the host from AbiR is unclear, it is possible that the LlaKR2I methylase prevents AbiR from impeding the host DNA replication by methylating 5′-GATC-3′ sites in the origin of replication region. Interestingly, sequence analysis of the bacteriophage c2 genome shows only two 5′-GATC-3′ sequences (9,543 bp and 20,487 bp) that are neither contained in the bacteriophage ori
region nor located close to it. Although the precise location of the ori
of L. lactis
IL1403 is unknown, it was mapped approximately in a region containing conserved elements (3
). Sequence analysis shows 10 5′-GATC-3′ sequences localized within a 4.1-kb DNA stretch (coordinate 2,363,401; 2,100 bp) that includes the L. lactis ori
. Alignment of these 5′-GATC-3′ sequences, extended by 10 bp on either side, with the corresponding 5′-GATC-3′ sites on bacteriophage c2 did not reveal any other sequence identities (Table ). Similarly, sequence alignment of a 2-kb DNA region containing the bacteriophage ori
with the corresponding region from the L. lactis
IL1403 genome did not reveal any significant sequence identities or relevant motifs (data not shown). However, ori
regions have elaborate secondary structures, and it is feasible that AbiR may bind DNA via specific secondary structures rather than sequence motifs. As AbiR impedes bacteriophage replication, and possibly the host DNA replication in the absence of the LlaKR2I methylase, it is likely that the LlaKR2I methylase protects L. lactis
DNA either by physically interfering with the binding of the AbiR complex to the ori
or by hindering its action due to methylation.
DNA sequence alignment of 5′-GATC-3′ sequences from the bacteriophage c2 genome with 5′-GATC-3′ sequences from the L. lactis IL1403 ori
The genetic organization of the LlaKR2I R/M system is unique for a type II R/M system, as it is encoded by an endonuclease gene (llaKR2IR) and a methylase gene (llaKR2IM) that are divergently transcribed and separated by a copy of the insertion element IS982, which is located within the intergenic region. The presence of the IS982 element between the two genes is ambiguous as the LlaKR2I R/M system can operate without that element and with more efficiency. It is interesting to observe that the presence of the two IS982 elements, flanking the AbiR genetic determinants, generates a likely transposable AbiR composite. This striking observation not only substantiates the novel function of the LlaKR2I methylase in AbiR expression but also illustrates the evolution of the LlaKR2I methylase from its role in the LlaKR2I R/M system toward a new and separate cellular function. The unique organization of the LlaKR2I R/M system may have contributed to the evolution of the LlaKR2I methylase toward a separate role comparable to that of Dam and CcrM methylases.
In this study, the three-gene AbiR operon was shown to require the LlaKR2I methylase for expression in L. lactis. This is the first example in which a methylase gene that is associated with a cognate endonuclease gene has another role within the cell besides its traditional role of protecting the host DNA from its cognate restriction enzyme. It is now apparent that the LlaKR2I methylase has evolved a novel cellular role independent from the R/M system that resembles the function of Dam and CcrM methylases. This may represent a snapshot in the evolution of the cell cycle-regulated methylases from an existing R/M system.