A genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system

Abstract

Background: Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from Lactococcus lactis recognizes an asymmetric, complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a prodigious barrier to bacteriophage infection. LlaJI is comprised of four similarly oriented genes, encoding two 5mC-MTases (M1.LlaJI and M2.LlaJI) and two subunits responsible for restriction activity (R1.LlaJI and R2.LlaJI). Here we employ a detailed genetic analysis of the LlaJI restriction determinants in an attempt to characterize mechanistic features of this unusual hetero-oligomeric endonuclease.

Results: Detailed bioinformatics analysis confirmed the presence of a conserved GTP binding and hydrolysis domain within the C-terminal half of the R1.LlaJI amino acid sequence whilst the N-terminal half appeared to be entirely unique. This domain architecture was homologous with that of the "B" subunit of the GTP-dependent, methyl-specific McrBC endonuclease from E.coli K-12. R1.LlaJI did not appear to contain a catalytic centre, whereas this conserved motif; PD....D/EXK, was clearly identified within the amino acid sequence for R2.LlaJI. Both R1.LlaJI and R2.LlaJI were found to be absolutely required for detectable LlaJI activity in vivo. The LlaJI restriction subunits were purified and examined in vitro, which allowed the assignment of R1.LlaJI as the sole specificity determining subunit, whilst R2.LlaJI is believed to mediate DNA cleavage.

Conclusion: The hetero-subunit structure of LlaJI, wherein one subunit mediates DNA binding whilst the other subunit is predicted to catalyze strand hydrolysis distinguishes LlaJI from previously characterized restriction-modification systems. Furthermore, this distinction is accentuated by the fact that whilst LlaJI behaves as a conventional Type IIA system in vivo, in that it restricts un-methylated DNA, it resembles the Type IV McrBC endonuclease, an enzyme specific for methylated DNA. A number of similar restriction determinants were identified in the database and it is likely LlaJI together with these homologous systems, comprise a new subtype of the Type II class incorporating features of Type II and Type IV systems.

Figure 1

A: LlaJI and its identified homologous restriction modification systems. Genes sharing protein sequence homology to LlaJI components are linked by shading (with the percentages representative of the respective identity to LlaJI). B: Alignment of R1.LlaJI with is identified homologues. The aligned regions correspond to a section containing the three conserved motif's (indicated above each on the alignment) which comprise the predicted GTP binding and hydrolysis domain. Motif II for McrB is underlined. Additional conserved modules in this region are indicated by dashed lines above each. C: Alignment of the section of R2.LlaJI and its identified homologues predicted to contain the catalytic centre. The conserved PD...Xn....(D/E)XK motif is indicated.

Figure 2

A: Schematic representation of the 230 bp DNA probes used for mobility shift assays. The positions of the intact LlaJI recognition sequences are indicated. B: Mobility shift assay with specific and non-specific probes (as indicated) which were incubated with increasing concentrations of purified R1.LlaJI. Final concentrations of protein are specified above each lane. C: Mobility shift assay with probe J which was incubated with increasing concentrations of purified R1.LlaJI (as indicated above each lane). The presence (+) or absence (-) of 5 mM GTP/ATP is as specified.