BACKGROUND
the diverse structural and functional activities described for restriction endonucleases  is reflected by their ubiquitous abundance among prokaryotic organisms  <cit> .
these enzymes are typically components of restriction/modification  systems where they primarily serve as defensive instruments against bacteriophage infection and other invading "foreign" dna.
four distinct groups of reases are known, with the "type ii" group being the most richly represented  <cit> .
members of this group, of which there are eleven subtypes, generally recognize relatively short dna sequences and specifically cut within or reasonably close to such sequences in a mg+ <dig> dependent manner.
although generally lacking significant amino acid sequence homology, structural analysis of many of these reases has revealed in some instances a conserved core, consisting of a four/five-stranded mixed β-sheet flanked by α-helices, reviewed in  <cit> .
this core spatially localizes two carboxylates  which are believed to be involved in divalent metal ion binding, and a lysine residue, possibly involved in positioning and/or activation of an attacking nucleophile  <cit> .
together it is believed that these residues comprise the rease catalytic site, the pd...d/exk motif, responsible for phosphodiester bond hydrolysis in the target dna molecule.
for double-stranded scission this hydrolysis event must occur on both strands of the target dna thereby requiring two catalytic sites  <cit> , although single-site exceptions do exist  <cit> .
consequently, the active forms of many type ii reases are homodimers , with each identical subunit providing a single catalytic site for individual strand hydrolysis  <cit> .
of course given the multifarious nature of the type ii group, it is not surprising that unusual hetero-multimeric reases, comprised of unique subunits, consisting of the associated products of different genes, have been described  <cit> .
one such characterized enzyme, bbvci, has been shown to be heterodimeric, with each individual subunit independently responsible for cleavage of separate strands of its asymmetric recognition sequence  <cit> .
similarly, the bpu10i rease requires two different subunits for its activity however, in this instance, a direct interaction between the subunits has yet to be completely established  <cit> .
previously we have described the cloning and transcriptional regulation of llaji r/m system  <cit> .
the llaji gene cluster, encoded by the lactococcal plasmid pnp <dig>  consists of two 5mc mtase-encoding genes followed by two rease-encoding genes which mediate a prodigious barrier to bacteriophage infection  <cit> .
the specificity of the system was determined to be asymmetric but complementary, comprised of 5'gacgc' <dig> in one strand and 5'gcgtc' <dig> in the other  <cit> .
perhaps the most striking feature of this r/m system is its homology to the methyl-specific rease mcrbc.
this highly unusual enzyme, which lacks an associated mtase, has been characterized extensively  <cit> .
its orphan nature, unusual specificity and cofactor requirements define mcrbc, together with its identified homologues, as a distinct rease group  <cit> .
the mcrbc locus encodes two major products, mcrb  and mcrc , which mediate the restriction phenotype  <cit> .
mcrbc recognizes two methylated/hemimethylated rmc sites  and cleaves at random positions between the two  <cit> .
the optimal stoichiometry for cleavage in vitro has been suggested as 3–5: <dig>  mcrb:mcrc , with the cleavage reaction strictly dependent upon gtp and mg+ <dig>  <cit> .
the mcrb subunit harbours the specific dna binding activity , in addition to gtp binding and hydrolysis activities  <cit> .
the mcrc subunit contains the catalytic centre for cleavage  <cit> .
following binding to its methylated target site, mcrbc facilitates a gtp-dependent translocation of the dna, akin to the mechanisms of other ntp-dependent reases of the type i and iii groups, with cleavage ensuing upon stalling of the translocating complex  <cit> .
furthermore, it has been shown that mcrb forms high molecular weight complexes – heptameric rings, in the presence of gtp .
these complexes can further multimerise to tetradecamers, structures stabilized by the binding of mcrc – which constitutes the "active" form of the endonuclease  <cit> .
in this report, we execute a detailed genetic and phenotypic assessment of the mcrbc-like llaji endonuclease which serves to portray the complexity of this unique enzyme.
RESULTS
sequence analysis of llaji
r <dig> llaji is predicted to be a  <dig>  kda protein which exhibits sequence homology to a number of partially characterized and putative reases in the database .
these homologies primarily correspond to a region proposed to represent a domain responsible for gtp binding and hydrolysis .
this domain consists of three signature motifs, namely  the phosphate binding loop ),  the switch ii region , and  the guanine recognition loop   <cit> .
this domain has been investigated biochemically in the case of the r <dig> llaji homologue, the mcrb subunit of the mcrbc endonuclease – and has revealed interesting variations from the archetypal gtpase family   <cit> .
additional conserved modules  can be discerned within r <dig> llaji and its homologues; however, at this point no functional significance can be assigned to any of these regions .
it was suggested for mcrb, that some of these conserved modules, at the time identified using completely different protein sets, may contribute in some manner to the diverged functionality of the gtp-binding domain  <cit> .
hence, it is likely that, as for mcrb, r <dig> llaji represents yet another member of a distinct subclass of the classical gtpase family.
for most of the identified r <dig> llaji homologues, significant alignment was only achieved with portions of the sequences corresponding to the c-terminal half of each, with the n-terminal portions apparently representing non-conserved sequence.
the two exceptions to this, namely llaib and bsumib , which are smaller  than r <dig> llaji , aligned completely along the full length of their respective sequences.
interestingly, both appear to be encoded by "restriction cassettes" comprised of three orf's as opposed to the two observed for the remaining homologues .
database searches with r <dig> llaji  revealed a set of similar proteins that are encoded by genes located adjacent to those that had been identified as encoding homologues of r <dig> llaji .
1a multiple alignment of r <dig> llaji with its homologues allowed the identification of a putative catalytic centre for dna cleavage; pd...d/exk.
this assignment is supported by inclusion of the mcrc subunit of mcrbc in the alignment, whose catalytic centre has essentially been confirmed  <cit> .
as for r <dig> llaji above, r <dig> llaji shares significant, but scattered modules of identity with other proteins, but again the functional significance of this similarity remains purely speculative.
these data cumulatively suggest that llaji, and indeed other restriction systems exhibit structural and therefore likely mechanistic similarities with that of the mcrbc methyl-specific endonuclease .
phenotypic and genetic analysis of llaji
previously, we have described the bacteriophage resistance phenotype conferred by the llaji operon  <cit> .
to fully explore the contribution of the individual llaji restriction "subunits" to this phage resistance activity, we adopted a genetic subcloning approach.
to this end, the llajir <dig> and llajir <dig> genes were amplified individually, and in tandem, and placed under the control of the strong nisin-inducible promoter of plasmid pnz <dig> to facilitate their over-expression.
the resulting constructs, pnz-r <dig>  pnz-r <dig>  and pnz-r1r <dig> were subsequently introduced into the expression host nz <dig>  containing the plasmid pjo-m1m <dig> , and analyzed for their ability to confer phage resistance.
as can be seen from table  <dig>  it was apparent that both r <dig> llaji and r <dig> llaji were required for the complete llaji phenotype .
furthermore, at 30°c the degree of phage resistance exhibited by the host over-expressing both llaji subunits  was 100-fold greater than that exhibited by the "wild type" llaji operon present on the low copy number plasmid pjo-j.
notably, the significant decrease in eop conferred by the host containing pjo-j, which accompanied growth at 19°c, the previously established optimum for llaji-mediated resistance, was not preserved in the pnz-r1r <dig> containing host.
in fact, at 30°c the degree of llaji-mediated resistance conferred by pnz-r1r <dig> was comparable to that obtained at 19°c with the pjo-j containing host, which would suggest that the upper threshold for llaji activity had been reached.
the fact that this optimal level of resistance may be achieved at 30°c by mere over-expression of r <dig> llaji and r <dig> llaji suggests that the temperature sensitivity of the llaji system previously reported, may be attributable to the intracellular concentration of active llaji at the respective temperatures , rather than to the temperature "activity" spectrum of assembled functional llaji, as had previously been speculated  <cit> .
as stated, the results obtained with the hosts expressing r <dig> llaji and r <dig> llaji individually  indicated that neither subunit alone was capable of restricting the growth of phage.
however, despite the lack of significant decrease in eop at either sampling temperature used, a delicate but visibly obvious variable plaque phenotype was observed for the host expressing r <dig> llaji alone .
perhaps most intriguing was the complete absence of any detectable endonuclease activity in crude extracts of lactococcal strains containing either pjo-j or the pnz-r1r <dig> over-expression plasmid .
purification of the individual llaji subunits
as it was established that both llaji subunits were required for "complete" activity, it was assumed that expression of the individual subunits separately for purification purposes could be achieved in the absence of the llaji mtases.
to this end, both llajir <dig> and llajir <dig> were cloned in plasmid pqe <dig> to produce derivatives pqe-r <dig> and pqe-r <dig>  respectively which were subsequently established in e.coli .
over-expression with pqe-r <dig> allowed purification of r <dig> llaji as a c-terminal 6xhis tagged derivative.
mass spectrometry and n-terminal sequencing confirmed the predicted molecular mass  and identity of purified r <dig> llaji.
purification of r <dig> llaji by this approach failed, possibly due to instability of the resulting recombinant protein.
to overcome this presumed instability, llajir <dig> was inserted downstream of the male gene of plasmid pmal-c2x under the control of the ptac promoter  which, upon induction resulted in the expression of an r2-mbp fusion of the predicted molecular mass .
this fusion was purified by affinity chromatography using amylose resin and the r <dig> llaji subunit was subsequently released from the r2-mbp preparation by cleavage with factor xa protease.
rebinding of mbp to amylose resin resulted in purification of r <dig> llaji to ~ <dig> % homogeneity.
assignment of the specificity determining llaji subunit
given the domain architecture shared between r <dig> llaji and mcrb, and the established dna binding activity of the latter, we investigated the possibility of r <dig> llaji constituting the dna recognition element of llaji.
mobility shift assays were therefore performed with r <dig> llaji using probes containing both single and multiple llaji sites, in addition to a non-specific probe .
as can be seen from figure 2b, an obvious and specific dna binding activity was evident using probes j <dig> and j <dig>  each containing a single llaji recognition site.
for both probes, binding did not result in a defined shifted band , but rather appeared as a "strewn" binding pattern, suggestive of a poorly defined protein:dna stoichiometry of the retarded complex.
at high r <dig> llaji concentrations, binding to probe j  resulted in the formation of more apparent yet still "diffuse" and extremely slow migrating complexes , reminiscent of the high molecular weight complexes formed by mcrb with its specific probe  <cit> .
incubation of probe j <dig>  with excess r <dig> llaji did not reveal any binding activity , and this finding, combined with competition experiments with unlabelled "specific" probes confirmed the specificity of the observed interactions .
inclusion of mg+ <dig> in the binding buffer had no discernable effect on r <dig> llaji specific binding, nor did its addition post protein:dna complex formation.
interestingly however, and in contrast to mcrb, inclusion of gtp did not stimulate the r <dig> llaji dna interaction .
in fact it appeared that gtp, and to a lesser extent atp, had a very modest inhibitory effect on the observed binding activity.
a similar analysis using partially purified r <dig> llaji did not reveal any dna binding to the llaji site containing probes.
in addition, r <dig> llaji did not appear to have any discernable effect on the r <dig> llaji binding activity.
investigation of the biological activity of the purified llaji subunits
as mentioned above, it was not possible to detect specific endonuclease activity in crude cell extracts of lactococcal strains expressing the llaji system .
subsequent attempts to reconstitute active llaji by combination of the partially purified subunits in various ratios, and with an assortment of cofactors  failed to reveal any specific nuclease activity.
furthermore, neither subunit was found to exhibit a dna nicking activity.
possible explanations for these observations are discussed below.
the presence of the putative gtpase domain in r <dig> llaji prompted us to investigate whether or not this subunit displayed such an activity.
analysis of gtp hydrolysis, conducted by colorimetric assay  suggested that r <dig> llaji did not exhibit an intrinsic gtpase activity.
surprisingly, r <dig> llaji dependent gtp hydrolysis could not be activated by the inclusion of "specifc" dna and/or partially purified r <dig> llaji in the reaction.
similar results were obtained with alternative nucleotides.
phenotypic analysis of heterologous llaji expression
in an attempt to explain the lack of endonuclease activity in the protein preparations, a phenotypic investigation of the efficacy of llaji in the heterologous e. coli host was performed.
as can be seen from table  <dig>  no significant resistance to bacteriophage proliferation was observed with a host containing plasmid pjo-j, encoding the complete llaji system.
a similar phenotype was associated with the host over-expressing r <dig> llaji or r <dig> llaji alone .
when both subunits were over-expressed in the same host  however, and in agreement with results for the lactococcal over-expression host above, a phage resistance phenotype was apparent .
the degree of this resistance was significantly lower than expected given the number of llaji sites on the λ genome and the obvious relationship between eop and llaji site frequency for the lactococcal phage used .
the lack of phage resistance associated with the pjo-j containing host was therefore likely attributable to the lower copy number of this replicon, coupled to the lower efficiency of in vivo llaji restriction in this host.
investigation of the temperature induced efficacy of llaji in e.coli further revealed that, although the observed resistance  was not evident at 37°c, it was not significantly enhanced with growth at low temperatures .
again, as with the lactococcal hosts, crude extracts of e.coli over-expressing r <dig> llaji and r <dig> llaji  did not exhibit detectable endonuclease activity.
discussion
the molecular mechanism by which the lactococcal plasmid-encoded llaji r/m system achieves dna restriction has remained largely elusive.
previous studies have characterized the phage resistance capabilities of this four-gene cluster which is accompanied by an intricate transcriptional regulatory mechanism  <cit> .
in the present study we offer additional phenotypic and genetic data which alludes to an unusual mode of action for llaji.
analysis of the deduced amino acid sequence for r <dig> llaji revealed a segmented architecture, with a c-terminally located putative gtp binding and hydrolysis domain, and a largely unique n-terminal portion.
this organization was similar to that observed for the mcrb subunit of the mcrbc methyl-specific endonuclease  <cit> .
a mutational analysis of the three motifs which comprise the gtpase domain of mcrb confirmed the assignment for motif i only, which is the most highly conserved of the three with respect to r <dig> llaji and its homologues .
conservative substitutions in motifs ii and iii failed to unequivocally confirm the proposed assignments on mcrb  <cit> .
it was suggested that the proposed motif iii, although essential for gtp binding and hydrolysis , rather than define nucleotide specificity , was more likely to be involved in mcrc interaction   <cit> .
the apparent replacement of the conserved lysine of motif iii with a threonine  further substantiated the likelihood of this motif contributing an atypical function.
in addition, the relative spacing between motif's ii and iii on mcrb , appeared too short to be structurally analogous to the classical gtpases  <cit> .
however, in the case of r <dig> llaji, the spacing between these proposed motifs is, although relatively large, more canonical , and in fact the alignment reveals an alternative candidate for the mcrb motif ii .
it has been shown that it is the n-terminal  <dig> amino acids of mcrb which, like r <dig> llaji did not exhibit significant homology to the database, specified the dna binding activity of mcrbc  <cit> .
this, in addition to the placement of the gtpase domain suggested that an equivalent activity could be attributable to the unique n-terminal portion of r <dig> llaji.
we examined these predicted properties for r <dig> llaji and indeed established a specific dna binding activity for this subunit to llaji-site containing dna fragments.
interestingly, and in contrast to mcrb, r <dig> llaji did not exhibit an intrinsic gtpase activity, which could neither be stimulated by specific dna nor partially purified r <dig> llaji.
this latter finding was again in contrast to mcrb, whose intrinsic gtpase activity was stimulated 30-fold by mcrc  <cit> .
a putative catalytic centre was identified within the amino acid sequence for r <dig> llaji, which suggested a role for this subunit in substrate strand hydrolysis; however, neither r <dig> llaji nor r <dig> llaji  exhibited cleavage or nicking activity.
the absolute requirement for both r <dig> llaji and r <dig> llaji for complete in vivo activity was clearly demonstrated .
a mild phenotype was associated with over-expression of r <dig> llaji alone , although this was most likely due to dna binding only, which may have served to partially hinder the phage replication machinery.
although less dramatic, heterologous expression of r <dig> llaji and r <dig> llaji in e.coli provided resistance to phage infection where, again an absolute requirement for both r <dig> llaji and r <dig> llaji was observed .
the relative poor efficacy of the resistance provided in this host background was attributed to instability of the r <dig> llaji subunit.
this subunit, in contrast to r <dig> llaji, could not be observed in over-expressing cell extracts when analyzed by sds-page.
purification of r <dig> llaji was only achieved when stabilized by fusion to a carrier protein.
it is possible that in this fusion correct folding of the r <dig> llaji subunit had been inhibited, and was not subsequently corrected following the proteolytic removal of the carrier.
this could account for our inability to reconstitute an active endonucleolytic complex with r <dig> llaji despite the clearly evident dna binding activity of the latter.
furthermore, it is possible that the predicted gtp hydrolyzing activity  of r <dig> llaji would only become apparent following assembly of the "active" heterooligomer on its target dna.
it must be noted however, that an additional lactococcal derived cofactor, proteinacious or otherwise, can not be entirely excluded at this point, despite the absence of endonuclease activity in llaji-expressing lactococcal crude extracts.
in summary, llaji is a heterooligomeric endonuclease wherein the r <dig> llaji subunit is responsible for determining specificity.
it is suggested that r <dig> llaji harbours the catalytic centre for dna cleavage, whose site, although undetermined, is likely to be defined by the relative "placement" of r <dig> llaji on the target dna .
CONCLUSIONS
llaji was shown to retain behavioral characteristics of a conventional type iia system, in that unmethylated dna is targeted in vivo however; unlike any characterized enzyme of its kind it exhibits striking genetic similarities with the type iv enzyme mcrbc, which is specific for methylated dna.
the presence of an mcrbc-like enzyme as a component of an r/m system suggests two mutually exclusive modes of action for these enzyme types whereby they are either in isolation, cleaving methylated dna , or in combination with mtases cleaving unmethylated dna .
whilst unusual, this is not altogether surprising as such contrasting activities have been described for some conventional endonucleases   <cit> .
similarly organized r/m systems were found in the database and together with llaji, are likely to represent a new subgroup of the type ii family, combining features of both type ii and type iv systems.
further biochemical characterization of llaji, and indeed its identified and relatively unknown homologues  will hopefully disclose further revelations regarding this novel and remarkable endonuclease family.

 the diverse structural and functional activities described for restriction endonucleases is reflected by their ubiquitous abundance among prokaryotic organisms. these enzymes are typically components of restriction/modification systems where they primarily serve as defensive instruments against bacteriophage infection and other invading "foreign" dna. four distinct groups of reases are known, with the "type ii" group being the most richly represented. members of this group, of which there are eleven subtypes, generally recognize relatively short dna sequences and specifically cut within or reasonably close to such sequences in a mg+ dependent manner. although generally lacking significant amino acid sequence homology, structural analysis of many of these reases has revealed in some instances a conserved core, consisting of a four/five-stranded mixed β-sheet flanked by α-helices, reviewed in. this core spatially localizes two carboxylates which are believed to be involved in divalent metal ion binding, and a lysine residue, possibly involved in positioning and/or activation of an attacking nucleophile. together it is believed that these residues comprise the rease catalytic site, the pd...d/exk motif, responsible for phosphodiester bond hydrolysis in the target dna molecule. for double-stranded scission this hydrolysis event must occur on both strands of the target dna thereby requiring two catalytic sites, although single-site exceptions do exist. consequently, the active forms of many type ii reases are homodimers, with each identical subunit providing a single catalytic site for individual strand hydrolysis. of course given the multifarious nature of the type ii group, it is not surprising that unusual hetero-multimeric reases, comprised of unique subunits, consisting of the associated products of different genes, have been described. one such characterized enzyme, bbvci, has been shown to be heterodimeric, with each individual subunit independently responsible for cleavage of separate strands of its asymmetric recognition sequence. similarly, the bpu10i rease requires two different subunits for its activity however, in this instance, a direct interaction between the subunits has yet to be completely established. previously we have described the cloning and transcriptional regulation of llaji r/m system. the llaji gene cluster, encoded by the lactococcal plasmid pnp consists of two 5mc mtase-encoding genes followed by two rease-encoding genes which mediate a prodigious barrier to bacteriophage infection. the specificity of the system was determined to be asymmetric but complementary, comprised of 5'gacgc' in one strand and 5'gcgtc' in the other. perhaps the most striking feature of this r/m system is its homology to the methyl-specific rease mcrbc. this highly unusual enzyme, which lacks an associated mtase, has been characterized extensively. its orphan nature, unusual specificity and cofactor requirements define mcrbc, together with its identified homologues, as a distinct rease group. the mcrbc locus encodes two major products, mcrb and mcrc, which mediate the restriction phenotype. mcrbc recognizes two methylated/hemimethylated rmc sites and cleaves at random positions between the two. the optimal stoichiometry for cleavage in vitro has been suggested as 3–5: mcrb:mcrc, with the cleavage reaction strictly dependent upon gtp and mg+. the mcrb subunit harbours the specific dna binding activity, in addition to gtp binding and hydrolysis activities. the mcrc subunit contains the catalytic centre for cleavage. following binding to its methylated target site, mcrbc facilitates a gtp-dependent translocation of the dna, akin to the mechanisms of other ntp-dependent reases of the type i and iii groups, with cleavage ensuing upon stalling of the translocating complex. furthermore, it has been shown that mcrb forms high molecular weight complexes – heptameric rings, in the presence of gtp. these complexes can further multimerise to tetradecamers, structures stabilized by the binding of mcrc – which constitutes the "active" form of the endonuclease. in this report, we execute a detailed genetic and phenotypic assessment of the mcrbc-like llaji endonuclease which serves to portray the complexity of this unique enzyme.  sequence analysis of llaji r llaji is predicted to be a kda protein which exhibits sequence homology to a number of partially characterized and putative reases in the database. these homologies primarily correspond to a region proposed to represent a domain responsible for gtp binding and hydrolysis. this domain consists of three signature motifs, namely the phosphate binding loop ), the switch ii region, and the guanine recognition loop. this domain has been investigated biochemically in the case of the r llaji homologue, the mcrb subunit of the mcrbc endonuclease – and has revealed interesting variations from the archetypal gtpase family. additional conserved modules can be discerned within r llaji and its homologues; however, at this point no functional significance can be assigned to any of these regions. it was suggested for mcrb, that some of these conserved modules, at the time identified using completely different protein sets, may contribute in some manner to the diverged functionality of the gtp-binding domain. hence, it is likely that, as for mcrb, r llaji represents yet another member of a distinct subclass of the classical gtpase family. for most of the identified r llaji homologues, significant alignment was only achieved with portions of the sequences corresponding to the c-terminal half of each, with the n-terminal portions apparently representing non-conserved sequence. the two exceptions to this, namely llaib and bsumib, which are smaller than r llaji, aligned completely along the full length of their respective sequences. interestingly, both appear to be encoded by "restriction cassettes" comprised of three orf's as opposed to the two observed for the remaining homologues. database searches with r llaji revealed a set of similar proteins that are encoded by genes located adjacent to those that had been identified as encoding homologues of r llaji. 1a multiple alignment of r llaji with its homologues allowed the identification of a putative catalytic centre for dna cleavage; pd...d/exk. this assignment is supported by inclusion of the mcrc subunit of mcrbc in the alignment, whose catalytic centre has essentially been confirmed. as for r llaji above, r llaji shares significant, but scattered modules of identity with other proteins, but again the functional significance of this similarity remains purely speculative. these data cumulatively suggest that llaji, and indeed other restriction systems exhibit structural and therefore likely mechanistic similarities with that of the mcrbc methyl-specific endonuclease. phenotypic and genetic analysis of llaji previously, we have described the bacteriophage resistance phenotype conferred by the llaji operon. to fully explore the contribution of the individual llaji restriction "subunits" to this phage resistance activity, we adopted a genetic subcloning approach. to this end, the llajir and llajir genes were amplified individually, and in tandem, and placed under the control of the strong nisin-inducible promoter of plasmid pnz to facilitate their over-expression. the resulting constructs, pnz-r pnz-r and pnz-r1r were subsequently introduced into the expression host nz containing the plasmid pjo-m1m, and analyzed for their ability to confer phage resistance. as can be seen from table it was apparent that both r llaji and r llaji were required for the complete llaji phenotype. furthermore, at 30°c the degree of phage resistance exhibited by the host over-expressing both llaji subunits was 100-fold greater than that exhibited by the "wild type" llaji operon present on the low copy number plasmid pjo-j. notably, the significant decrease in eop conferred by the host containing pjo-j, which accompanied growth at 19°c, the previously established optimum for llaji-mediated resistance, was not preserved in the pnz-r1r containing host. in fact, at 30°c the degree of llaji-mediated resistance conferred by pnz-r1r was comparable to that obtained at 19°c with the pjo-j containing host, which would suggest that the upper threshold for llaji activity had been reached. the fact that this optimal level of resistance may be achieved at 30°c by mere over-expression of r llaji and r llaji suggests that the temperature sensitivity of the llaji system previously reported, may be attributable to the intracellular concentration of active llaji at the respective temperatures, rather than to the temperature "activity" spectrum of assembled functional llaji, as had previously been speculated. as stated, the results obtained with the hosts expressing r llaji and r llaji individually indicated that neither subunit alone was capable of restricting the growth of phage. however, despite the lack of significant decrease in eop at either sampling temperature used, a delicate but visibly obvious variable plaque phenotype was observed for the host expressing r llaji alone. perhaps most intriguing was the complete absence of any detectable endonuclease activity in crude extracts of lactococcal strains containing either pjo-j or the pnz-r1r over-expression plasmid. purification of the individual llaji subunits as it was established that both llaji subunits were required for "complete" activity, it was assumed that expression of the individual subunits separately for purification purposes could be achieved in the absence of the llaji mtases. to this end, both llajir and llajir were cloned in plasmid pqe to produce derivatives pqe-r and pqe-r respectively which were subsequently established in e.coli. over-expression with pqe-r allowed purification of r llaji as a c-terminal 6xhis tagged derivative. mass spectrometry and n-terminal sequencing confirmed the predicted molecular mass and identity of purified r llaji. purification of r llaji by this approach failed, possibly due to instability of the resulting recombinant protein. to overcome this presumed instability, llajir was inserted downstream of the male gene of plasmid pmal-c2x under the control of the ptac promoter which, upon induction resulted in the expression of an r2-mbp fusion of the predicted molecular mass. this fusion was purified by affinity chromatography using amylose resin and the r llaji subunit was subsequently released from the r2-mbp preparation by cleavage with factor xa protease. rebinding of mbp to amylose resin resulted in purification of r llaji to ~ % homogeneity. assignment of the specificity determining llaji subunit given the domain architecture shared between r llaji and mcrb, and the established dna binding activity of the latter, we investigated the possibility of r llaji constituting the dna recognition element of llaji. mobility shift assays were therefore performed with r llaji using probes containing both single and multiple llaji sites, in addition to a non-specific probe. as can be seen from figure 2b, an obvious and specific dna binding activity was evident using probes j and j each containing a single llaji recognition site. for both probes, binding did not result in a defined shifted band, but rather appeared as a "strewn" binding pattern, suggestive of a poorly defined protein:dna stoichiometry of the retarded complex. at high r llaji concentrations, binding to probe j resulted in the formation of more apparent yet still "diffuse" and extremely slow migrating complexes, reminiscent of the high molecular weight complexes formed by mcrb with its specific probe. incubation of probe j with excess r llaji did not reveal any binding activity, and this finding, combined with competition experiments with unlabelled "specific" probes confirmed the specificity of the observed interactions. inclusion of mg+ in the binding buffer had no discernable effect on r llaji specific binding, nor did its addition post protein:dna complex formation. interestingly however, and in contrast to mcrb, inclusion of gtp did not stimulate the r llaji dna interaction. in fact it appeared that gtp, and to a lesser extent atp, had a very modest inhibitory effect on the observed binding activity. a similar analysis using partially purified r llaji did not reveal any dna binding to the llaji site containing probes. in addition, r llaji did not appear to have any discernable effect on the r llaji binding activity. investigation of the biological activity of the purified llaji subunits as mentioned above, it was not possible to detect specific endonuclease activity in crude cell extracts of lactococcal strains expressing the llaji system. subsequent attempts to reconstitute active llaji by combination of the partially purified subunits in various ratios, and with an assortment of cofactors failed to reveal any specific nuclease activity. furthermore, neither subunit was found to exhibit a dna nicking activity. possible explanations for these observations are discussed below. the presence of the putative gtpase domain in r llaji prompted us to investigate whether or not this subunit displayed such an activity. analysis of gtp hydrolysis, conducted by colorimetric assay suggested that r llaji did not exhibit an intrinsic gtpase activity. surprisingly, r llaji dependent gtp hydrolysis could not be activated by the inclusion of "specifc" dna and/or partially purified r llaji in the reaction. similar results were obtained with alternative nucleotides. phenotypic analysis of heterologous llaji expression in an attempt to explain the lack of endonuclease activity in the protein preparations, a phenotypic investigation of the efficacy of llaji in the heterologous e. coli host was performed. as can be seen from table no significant resistance to bacteriophage proliferation was observed with a host containing plasmid pjo-j, encoding the complete llaji system. a similar phenotype was associated with the host over-expressing r llaji or r llaji alone. when both subunits were over-expressed in the same host however, and in agreement with results for the lactococcal over-expression host above, a phage resistance phenotype was apparent. the degree of this resistance was significantly lower than expected given the number of llaji sites on the λ genome and the obvious relationship between eop and llaji site frequency for the lactococcal phage used. the lack of phage resistance associated with the pjo-j containing host was therefore likely attributable to the lower copy number of this replicon, coupled to the lower efficiency of in vivo llaji restriction in this host. investigation of the temperature induced efficacy of llaji in e.coli further revealed that, although the observed resistance was not evident at 37°c, it was not significantly enhanced with growth at low temperatures. again, as with the lactococcal hosts, crude extracts of e.coli over-expressing r llaji and r llaji did not exhibit detectable endonuclease activity. discussion the molecular mechanism by which the lactococcal plasmid-encoded llaji r/m system achieves dna restriction has remained largely elusive. previous studies have characterized the phage resistance capabilities of this four-gene cluster which is accompanied by an intricate transcriptional regulatory mechanism. in the present study we offer additional phenotypic and genetic data which alludes to an unusual mode of action for llaji. analysis of the deduced amino acid sequence for r llaji revealed a segmented architecture, with a c-terminally located putative gtp binding and hydrolysis domain, and a largely unique n-terminal portion. this organization was similar to that observed for the mcrb subunit of the mcrbc methyl-specific endonuclease. a mutational analysis of the three motifs which comprise the gtpase domain of mcrb confirmed the assignment for motif i only, which is the most highly conserved of the three with respect to r llaji and its homologues. conservative substitutions in motifs ii and iii failed to unequivocally confirm the proposed assignments on mcrb. it was suggested that the proposed motif iii, although essential for gtp binding and hydrolysis, rather than define nucleotide specificity, was more likely to be involved in mcrc interaction. the apparent replacement of the conserved lysine of motif iii with a threonine further substantiated the likelihood of this motif contributing an atypical function. in addition, the relative spacing between motif's ii and iii on mcrb, appeared too short to be structurally analogous to the classical gtpases. however, in the case of r llaji, the spacing between these proposed motifs is, although relatively large, more canonical, and in fact the alignment reveals an alternative candidate for the mcrb motif ii. it has been shown that it is the n-terminal amino acids of mcrb which, like r llaji did not exhibit significant homology to the database, specified the dna binding activity of mcrbc. this, in addition to the placement of the gtpase domain suggested that an equivalent activity could be attributable to the unique n-terminal portion of r llaji. we examined these predicted properties for r llaji and indeed established a specific dna binding activity for this subunit to llaji-site containing dna fragments. interestingly, and in contrast to mcrb, r llaji did not exhibit an intrinsic gtpase activity, which could neither be stimulated by specific dna nor partially purified r llaji. this latter finding was again in contrast to mcrb, whose intrinsic gtpase activity was stimulated 30-fold by mcrc. a putative catalytic centre was identified within the amino acid sequence for r llaji, which suggested a role for this subunit in substrate strand hydrolysis; however, neither r llaji nor r llaji exhibited cleavage or nicking activity. the absolute requirement for both r llaji and r llaji for complete in vivo activity was clearly demonstrated. a mild phenotype was associated with over-expression of r llaji alone, although this was most likely due to dna binding only, which may have served to partially hinder the phage replication machinery. although less dramatic, heterologous expression of r llaji and r llaji in e.coli provided resistance to phage infection where, again an absolute requirement for both r llaji and r llaji was observed. the relative poor efficacy of the resistance provided in this host background was attributed to instability of the r llaji subunit. this subunit, in contrast to r llaji, could not be observed in over-expressing cell extracts when analyzed by sds-page. purification of r llaji was only achieved when stabilized by fusion to a carrier protein. it is possible that in this fusion correct folding of the r llaji subunit had been inhibited, and was not subsequently corrected following the proteolytic removal of the carrier. this could account for our inability to reconstitute an active endonucleolytic complex with r llaji despite the clearly evident dna binding activity of the latter. furthermore, it is possible that the predicted gtp hydrolyzing activity of r llaji would only become apparent following assembly of the "active" heterooligomer on its target dna. it must be noted however, that an additional lactococcal derived cofactor, proteinacious or otherwise, can not be entirely excluded at this point, despite the absence of endonuclease activity in llaji-expressing lactococcal crude extracts. in summary, llaji is a heterooligomeric endonuclease wherein the r llaji subunit is responsible for determining specificity. it is suggested that r llaji harbours the catalytic centre for dna cleavage, whose site, although undetermined, is likely to be defined by the relative "placement" of r llaji on the target dna.  llaji was shown to retain behavioral characteristics of a conventional type iia system, in that unmethylated dna is targeted in vivo however; unlike any characterized enzyme of its kind it exhibits striking genetic similarities with the type iv enzyme mcrbc, which is specific for methylated dna. the presence of an mcrbc-like enzyme as a component of an r/m system suggests two mutually exclusive modes of action for these enzyme types whereby they are either in isolation, cleaving methylated dna, or in combination with mtases cleaving unmethylated dna. whilst unusual, this is not altogether surprising as such contrasting activities have been described for some conventional endonucleases. similarly organized r/m systems were found in the database and together with llaji, are likely to represent a new subgroup of the type ii family, combining features of both type ii and type iv systems. further biochemical characterization of llaji, and indeed its identified and relatively unknown homologues will hopefully disclose further revelations regarding this novel and remarkable endonuclease family. 
the plasmid-encoded llaji restriction/modification system from lactococcus lactis recognizes an asymmetric, complementary dna sequence, consisting of 5'gacgc' in one strand and 5'gcgtc' in the other and provides a prodigious barrier to bacteriophage infection. this domain architecture was homologous with that of the "b" subunit of the gtp-dependent, methyl-specific mcrbc endonuclease from e.coli k- r 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 r llaji. detailed bioinformatics analysis confirmed the presence of a conserved gtp binding and hydrolysis domain within the c-terminal half of the r llaji amino acid sequence whilst the n-terminal half appeared to be entirely unique. 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. 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. 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.
 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' in one strand and 5'gcgtc' in the other and provides a prodigious barrier to bacteriophage infection. llaji is comprised of four similarly oriented genes, encoding two 5mc-mtases and two subunits responsible for restriction activity. 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.  detailed bioinformatics analysis confirmed the presence of a conserved gtp binding and hydrolysis domain within the c-terminal half of the r 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- r 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 r llaji. both r llaji and r 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 r llaji as the sole specificity determining subunit, whilst r llaji is believed to mediate dna cleavage.  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. 