Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

Abstract

The gene encoding the LlaCI methyltransferase (M.LlaCI) from Lactococcus lactis subsp. cremoris W15 was overexpressed in Escherichia coli. The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of M r 31 300±1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 59-AAGCTT-39. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 59 end of the specific sequence to N 6 -methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzae Rd) and EcoVIII (Escherichia coli E1585-68) restriction-modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among N 6 -adenine b-class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg 2+ for phosphodiester bond cleavage. Mg 2+ was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg 2+ may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed. INTRODUCTIONLactic acid bacteria (LAB) are probiotic micro-organisms that have been used for millennia in the processing of dairy products. It was shown recently that dairy establishments in Britain can be dated back to 5000 years BC (Copley et al., 2003). Manufacturing of food such as cheese and yogurt, but also sauerkraut and dill pickles, as well as the existence of some regional cuisine, is heavily dependent on lactic fermentation. LAB and food processed by them therefore have armies of admirers, but on the other side, these bacteria have also powerful enemies. Their enemies are not people who hate buttermilk, but virulent phages whose existence relies on propagation within bacterial cells. To combat phages, LAB have developed multiple molecular strategies.These measures can be divided into four groups: (i) inhibition of phage adsorption, (ii) blockage of phage DNA injection, (iii) abortive infection mechanisms, and (iv) restriction of phage DNA (Forde & Fitzgerald, 1999;Coffey & Ross, 2002). These defence mechanisms are often located on plasmids. That feature can facilitate their horizontal spread among LAB and when acquired affords them protection against phage invasion (Allison & Klaenhammer, 1998). Among the defence mechanisms listed above, restriction-modification (R-M) systems form the most diverse group. Based on their molecular structure and cofactor requireme...

Novel conjugative plasmids from the natural isolate Lactococcus lactis subspecies cremoris DPC3758: A repository of genes for the potential improvement of dairy starters

“…The plasmid complement of L. lactis DPC3758 harbors a significant array of defense mechanisms against phage attack, including 1 type I R/M and 2 type II R/M systems, which confirms suggestions that the latter mechanisms are the most common in lactococci (Mruk et al, 2003). Type I R/M systems are typically made up of 3 genes (hsdRMS), transcribed in the same direction, and coding for 3 subunits that are responsible for DNA restriction (HsdR), modification (HsdM), and specificity (HsdS), respectively (Murray, 2000).…”

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

“…Zn 2ϩ acted as a strong inhibitor of M.PabI-catalyzed methylation, in contrast to Mg 2ϩ , Ca 2ϩ , and Mn 2ϩ , all of which showed no significant inhibition. It has been reported that the methylation activities of M.HindIII, M.LlaCl, and M.EcoVIII were inhibited by Zn 2ϩ (35). An inhibitory effect of Mg 2ϩ was also observed with these enzymes.…”

Kinetics of Methylation by EcoP1I DNA Methyltransferase

“…The presence of metal ions is not an absolute requirement for M.BcgI [29], M.AloI [30], M.Eco57I [31], and M.MmeI [32] but the methylation activity of these enzymes was enhanced in the presence of Mg 2+ . On the other hand, M.HindIII, M.LlaCI, M.EcoVIII [33], M.PabI [18], M1.MboI, M2.MboII, M1.NcuI, M.NcuII [34, 35], and M.FokI [36] showed inhibition of methylation activity in the presence of various metal ions. It is clear that the activities of DNA MTases characterized so far respond differently in the presence of metal ions.…”