Background
==========

Most RNAs undergo a number of post-transcriptional modifications that increase the chemical diversity of the nucleotides. tRNAs are extensively modified, but rRNA, snRNA and mRNA also contain many modified nucleotides \[[@B1]\]. However, the functions of these nucleotides are poorly understood and few of the modifications are strongly conserved between species. Among all the modifications, only N-1 methylation of adenosine-58 in tRNA~i~^Met^ was shown to be truly essential \[[@B2]\] and in general, modified nucleosides are of minor importance for cell growth and/or survival (reviewed in ref. \[[@B3]\]).

Among more than 100 naturally occurring nucleotide modifications, the simplest and most common are methylations of nucleotide bases or ribose moieties \[[@B1]\]. One example of this is the monomethylation of the exocyclic amine of guanine. The 16 S RNA of *E. coli* contains three such *N*^2^-methylguanine (m^2^G) residues, at positions 966, 1207, and 1516 \[[@B4]\]. m^2^G966 is located in the loop of a small stem-loop structure, which has been implicated in tRNA binding at the P site of the ribosome \[[@B5]\]. m^2^G1207 occurs in a region of the RNA that is involved in recognition of peptide chain termination codons \[[@B6]\], and m^2^G1516 is located in the 3\'-terminal stem loop of 16S rRNA, which contains also two *N*^6^, *N*^6^-dimethyladenosines (m^6^~2~A). In the ribosome structure, these m^2^G residues as well as other 7 modified residues come together forming a compact cage surrounding the location of the anticodon stem-loop structures of A and P site-bound tRNAs \[[@B7]\]. Nevertheless, these modifications are not essential for protein synthesis, since ribosomes constructed from totally unmodified 16S RNA are able to carry out all of the partial reactions of in vitro protein synthesis, albeit at ca. half-efficiency \[[@B8]\].

The enzymes responsible for the biosynthesis of m^2^G in 16S RNA are not completely characterized. The m^2^G MTases specific for G1207 and G966 (RsmC and RsmD, respectively, named for (r)ibosomal (s)mall subunit (m)ethyltransferase) have been purified \[[@B9],[@B10]\] and the gene encoding the RsmC protein was cloned and characterized \[[@B10]\]. Recently, sequence analysis revealed a family of related proteins in Gram-negative bacteria, encompassing subfamilies typified by RsmC, the YgjO open reading frame (ORF) believed to encode the RsmD protein, and the YbiN ORF suggested to encode the m^2^G1516 MTase \[[@B11]\]. Based on similarities between the highly conserved NPPF tetrapeptide in m^2^G MTases and the key motif of enzymes that methylate the exocyclic amino groups of adenine (m^6^A) and cytosine (m^4^C) in nucleic acids, it was proposed that these enzymes should be classified as a single family of N-MTases \[[@B11]\]. It has also been hypothesized that a long non-conserved sequence in the N-terminus of m^2^G MTases may be implicated in recognition of the target nucleic acid molecule, in analogy to other RNA and DNA MTases \[[@B11]-[@B13]\]. To learn more about the evolutionary origin and the sequence-structure-function relationships in the family of  MTases, we attempted to infer their structural organization in a phylogenetic context.

Results
=======

Sequence analysis
-----------------

The amino-acid sequences of *E. coli* RsmC and RsmD were used in PSI-BLAST database searches to identify orthologous proteins (hits reported in the 1^st^ iteration with e-values \<10^-40^ and sequence identity \> 40%; see Materials and Methods for details). The resulting multiple sequence alignment (Figure [1](#F1){ref-type="fig"}) was used to predict the secondary structure (separately for both protein families) and to precisely identify boundaries of the C-terminal catalytic domain. The Rsm sequences were divided into the N-terminal (variable) and C-terminal (conserved) domains (hereafter dubbed NTD and CTD) of approximately equal length. The sequences of NTD and CTD of RsmC and RsmD were used in additional PSI-BLAST searches and also submitted to the structure prediction MetaServer <>. The CTD exhibited similarity to a large class of genuine and predicted N-methyltransferases. Both the RsmC and RsmD CTD sequences showed highest similarity to each other (e-values in the range of 10^-20^--10^-19^ in the 1^st^ iteration), but also to a family of putative MTases from Gram-positive Bacteria and Archaea, typified by the Mj0882 protein from *M. jannaschii* (e-value 10^-19^ in the 2^nd^ iteration starting with RsmC, 10^-35^ in the 3^rd^ iteration starting with RsmD). Interestingly, the crystal structure of Mj0882 has been solved as a part of a structural genomics program (1dus; deposited in the Protein Data Bank in January 2000, cited as: LW Hung, L Huang, R Kim, SH Kim: Crystal structure and functional analysis of a hypothetical protein, Mj0882, from *Methanococcus jannaschii,* to be published). However, neither the biochemical characterization of this protein or the analysis of its structure has been published and its function remains a mystery. More distant homologs with shorter alignments reported in the CTD-initiated search included the HemK/YfcB family of protenr:*N*^5^-glutamine MTases \[[@B14],[@B15]\] and various distinct RNA and DNA MTase families (e-values \> 10^-16^ after the RsmD-initiated search converged in the 4^th^ iteration). Only Mj0882 and its close homologs could be aligned to the CTD of RsmC or RsmD over their entire length if the default PSI-BLAST parameters were used. Reciprocal database searches initiated with Mj0882 yielded its presumed orthologs from Archaea and Gram-positive Bacteria (detected in the 1^st^ PSI-BLAST iteration with e-values \< 10^-30^ and sequence identity to the query \> 40%) and CTD sequences of proteobacterial m^2^G MTases (RsmC detected in the 2^nd^ iteration, e-values ≥ 2\*10^-26^ and sequence identities ≤ 26%; RsmD detected in the 3^rd^ iteration with e values ≥ 2\*10^-27^ and sequence identities ≤ 24%). This result of reciprocal database searches strongly suggested an orthologous relationship \[[@B16]\] between the RsmC/RsmD and Mj0882 families. Further support for this prediction was obtained from the threading analysis. Among structurally characterized MTases, Mj0882 was reported as the definitely best structural template for the CTD of RsmC and RsmD (confident Pcons scores 5.78 and 6.09; see Table [1](#T1){ref-type="table"} and Table [2](#T2){ref-type="table"} for the summary of the threading analysis).

::: {#T1 .table-wrap}
::: {.caption}
######

Summary of the fold recognition analysis for RsmC-CTD.
:::

  Model number   Pcons score   Threading server reporting the model   PDB ace. \#   PDB entry description
  -------------- ------------- -------------------------------------- ------------- -----------------------
  **1**          **5.779**     **FFAS**                               **1dus**      **Mj0882**
  **2**          **5.46**      **INBGU**                              **1dus**      **Mj0882**
  **3**          **5.085**     **3DPSSM**                             **1dus**      **Mj0882**
  **4**          **4.701**     **SAM-T99**                            **1dus**      **Mj0882**
  **5**          **4.326**     **mGENTHREADER**                       **1dus**      **Mj0882**
  **6**          **3.271**     **FUGUE**                              **1dus**      **Mj0882**
  7              3.081         FFAS                                   1g6q          Arg MTase Hmt1
  8              3.002         SAM-T99                                1g6q          Arg MTase Hmt1
  9              2.994         SAM-T99                                1g6q          Arg MTase Hmt1
  10             2.99          SAM-T99                                1f31          Arg MTase Pmt3

Hits to the Mj0882 structure are shown in bold. Comprehensive structure prediction results are available at the URL: <>
:::

::: {#T2 .table-wrap}
::: {.caption}
######

Summary of the fold recognition analysis for RsmD-CTD.
:::

  Model number   Pcons score   Threading server reporting the model   PDB ace. \#   PDB entry description
  -------------- ------------- -------------------------------------- ------------- -----------------------
  **1**          **6.092**     **FFAS**                               **1dus**      **Mj0882**
  **2**          **5.856**     **INBGU**                              **1dus**      **Mj0882**
  **3**          **5.43**      **3DPSSM**                             **1dus**      **Mj0882**
  **4**          **5.132**     **SAM-T99**                            **1dus**      **Mj0882**
  **5**          **4.893**     **mGENTHREADER**                       **1dus**      **Mj0882**
  **6**          **4.171**     **PDB-Blast**                          **1dus**      **Mj0882**
  **7**          **3.544**     **FUGUE**                              **1dus**      **Mj0882**
  8              2.907         FFAS                                   2adm           MTase
  9              2.906         SAM-T99                                1f31          Arg MTase Prmt3
  10             2.874         SAM-T99                                1g6q          Arg MTase Hmt1

Hits to the Mj0882 structure are shown in bold. Comprehensive structure prediction results are available at the URL: <>
:::

::: {#T3 .table-wrap}
::: {.caption}
######

Summary of the fold recognition analysis for RsmC-NTD.
:::

  Model number   Pcons score   Threading server reporting the model   PDB ace. \#   PDB entry description
  -------------- ------------- -------------------------------------- ------------- ------------------------
  **1**          **3.615**     **INBGU**                              **1DUS**      **Mj0882**
  **2**          **3.576**     **FUGUE**                              **1DUS**      **Mj0882**
  **3**          **3.266**     **FFAS**                               **1DUS**      **Mj0882**
  **4**          **3.237**     **SAM-T99**                            **1DUS**      **Mj0882**
  **5**          **3.225**     **3DPSSM**                             **1DUS**      **Mj0882**
  **6**          **2.624**     **mGENTHREADER**                       **1DUS**      **Mj0882**
  7              2.091         3DPSSM                                 1EIZ           O-MTase
  8              1.812         SAM-T99                                1BHJ          glycine N-MTase
  9              1.802         mGENTHREADER                           1DL5          protein isoAsp-O-MTase
  10             1.673         SAM-T99                                1G6Q          Arg MTase Hmt1

Hits to the Mj0882 structure are shown in bold. Comprehensive structure prediction results are available at the URL: <>
:::

::: {#T4 .table-wrap}
::: {.caption}
######

Summary of the fold recognition analysis for RsmD-NTD.
:::

  Model number   Pcons score   Threading server reporting the model   PDB ace. \#   PDB entry description
  -------------- ------------- -------------------------------------- ------------- -----------------------
  **1**          **3.805**     **FUGUE**                              **1DUS**      **Mj0882**
  **2**          **3.573**     **INBGU**                              **1DUS**      **Mj0882**
  **3**          **3.379**     **FFAS**                               **1DUS**      **Mj0882**
  **4**          **3.242**     **3DPSSM**                             **1DUS**      **Mj0882**
  **5**          **2.687**     **mGENTHREADER**                       **1DUS**      **Mj0882**
  **6**          **2.262**     **SAM-T99**                            **1DUS**      **Mj0882**
  7              1.864         SAM-T99 1                              1F3L          Arg MTase Pmt3
  8              1.587         FFAS                                   1G6Q          Arg MTase Hmt1
  9              1.51          INBGU                                  1VID          catechol O-MTase
  10             1.426         FFAS                                   1D2H          glycine N-MTase

Hits to the Mj0882 structure are shown in bold. Comprehensive structure prediction results are available at the URL: <>
:::

::: {#T5 .table-wrap}
::: {.caption}
######

Summary of the fold recognition analysis for YbiN.
:::

  Model number   Pcons score   Threading server reporting the model   PDB ace. \#   PDB entry description
  -------------- ------------- -------------------------------------- ------------- ------------------------
  **1**          **3.406**     **FFAS**                               **1DUS**      **Mj0882**
  **2**          **3.267**     **3DPSSM**                             **1DUS**      **Mj0882**
  **3**          **3.083**     **SAM-T99**                            **1DUS**      **Mj0882**
  **4**          **2.618**     **mGENTHREADER**                       **1DUS**      **Mj0882**
  **5**          **2.552**     **PDB-Blast**                          **1G6Q**      **Arg MTase Hmt1**
  **6**          **2.547**     **INBGU**                              **1DUS**      **Mj0882**
  7              2.481         PDB-Blast                              1DL5          protein isoAsp-O-MTase
  8              2.467         PDB-Blast                              1F3L          Arg MTase Pmt3
  9              2.462         FFAS                                   1G6Q          Arg MTase Hmt1
  10             2.4           PDB-Blast                              1G6Q          Arg MTase Hmt1

Hits to the Mj0882 structure are shown in bold. Comprehensive structure prediction results are available at the URL: <>
:::

![Multiple alignment of the Mj0882, RsmC, RsmD, and YbiN families. Sequences are denoted by the species\' name and the NCBI gene identification number; sequences lacking the number were obtained from the unfinished genome data. Numbers on the left side indicate the index of the first N-terminal residue shown, numbers in parentheses indicate how many residues in an insertion were omitted for clarity. Conserved motifs are labeled according to the nomenclature described for the AdoMet-dependent MTase superfamily \[[@B12]\]. Conserved residues are highlighted in black; the residues with invariant physicochemical character (hydrophobic, small etc.) are highlighted in gray. Conserved carboxylates predicted to bind AdoMet are shown in black on red background; the pair of residues proposed to make van der Waals contacts to guanine is shown in green on a yellow background; the Asn residue (His in Archaeal YbiN orthologs) proposed to catalyze the methyltransfer is shown in yellow on red background; the carboxylate proposed to bind the ribose of the target nucleoside is shown in yellow on magenta background. Secondary structure elements of Mj0882, extracted from the 1dus coordinates and virtually identical for the secondary structure independently predicted for other families (see Methods), are shown above the alignment.](1471-2105-3-10-1){#F1}

In contrast, querying PSI-BLAST with the NTD sequences did not reveal any significant similarities. The NTDs could only be aligned to each other, displaying a number of conserved residues, if the full-length RsmC or RsmD sequences were used in the PSI-BLAST search. This suggested that the NTD could represent a novel type of a variable domain fused to the common catalytic domain or a strongly diverged version of some known nucleic acid binding domain. Quite unexpectedly, all threading algorithms (see Methods) reported that the sequences of the NTD of RsmC and RsmD are compatible with the MTase fold. Moreover, the majority of servers reported the Mj0882 structure as the best template with significant scores (Table [3](#T3){ref-type="table"} and Table [4](#T4){ref-type="table"}). In the rankings produced by the consensus server Pcons, the top 6 hits corresponded to the Mj0882 structure with the best score of 3.615 (RsmC NTD) and 3.805 (RsmD NTD). These results strongly suggested that the NTD and the CTD in bacterial m^2^G MTases are related to Mj0882 and to each other. Nevertheless, we found that the NTD lacked many residues that may be important for the enzymatic activity of Mj0882 and the CTD (see below). Hence, the NTDs could be catalytically inactive versions of the MTase domain. Therefore, the sequences of NTD were not included in subsequent studies of the m^2^G MTase AdoMet-binding site and the active site based on analysis of conserved residues.

The YbiN family of predicted N-MTases has also been subjected to sequence comparisons and threading analyses. These results confirmed the previously observed similarity to sequences of RsmC and RsmD CTD \[[@B11]\] as well as to the Mj0882 family. PSI-BLAST reported similarity of YbiN to RsmC CTD in the 3^rd^ iteration with the e-value of 8 \* 10^-5^, whereas Pcons ranking of the top 4 hits corresponded to the Mj0882 structure with the best score of 3.41 (Table [5](#T5){ref-type="table"}). In the PSI-BLAST searches and in sequence profile comparisons using the FFAS algorithm \[[@B17]\], the YbiN family showed similarity to members of the HemK/YfcB family with scores comparable to that of the RsmC/RsmD/Mj0882 family (data not shown). It has been recently shown that the HemK family comprises  MTases, suggesting that the (D/N/S)-P-P-(F/Y/W/H) motif is characteristic for a subset of MTases that methylate -NH~2~ groups of various substrates, rather than only bases in nucleic acids \[[@B14],[@B15]\].

Although the predicted key catalytic motif of the YbiN family (NPPFH) resembles more that of *bona fide* guanine-N2 MTases RsmC and RsmD than of any other N-MTase family, it cannot be excluded that these proteins methylate other substrates than guanine in RNA. We hope that this analysis will facilitate the choice of representative targets for experimental characterization, which would allow more precise inference of biological function (target specificity) and relationships between various families of N-MTases.

Another piece of information that should shed light onto the broad picture of the N-MTase family is the previously unnoticed high similarity of the YbiN family to uncharacterized proteins from Eukaryota and Archaea (BLAST e-values 10^-27^, and 4\*^-04^, respectively). Their close similarity (confirmed in reciprocal searches with similar e-values) indicates an orthologous relationship to bacterial YbiN proteins. This was confirmed in reciprocal searches initiated with the eukaryotic and archaeal members of the family. Interestingly, while the predicted eukaryotic YbiN orthologs possess all sequence features typical for the bacterial MTases, their archaeal counterparts lack the key Asn residue in motif IV, suggesting that they may be inactive, unless the catalytic side chain \"migrated\" to another position in the primary sequence (see below). Regardless of the possible loss of functional residues, the presence of common motifs in the YbiN and Mj0882 families as well as in the NTD and CTD sequences of proteobacterial m^2^G MTases was confirmed using the Gibbs sampling procedure \[[@B18]\]. Motifs I, VI and VIII were detected with probability of occurring by chance \< 10^-13^. In addition, there is a good correlation of secondary structure elements independently predicted for all protein groups (Figure [1](#F1){ref-type="fig"}).

The orthologous/paralogous relationships inferred from the results of PSI-BLAST searches were confirmed by phylogenetic analysis using the neighbor-joining method of Saitou and Nei \[[@B19]\]. The bacterial YbiN sequences and their archaeal and eukaryotic homologs grouped together with 100% bootstrap support and the RsmC/RsmD NTD, RsmC/RsmD CTD and Mj0882 families formed groups with bootstrap support \> 50% (data not shown). Taken together, these findings strongly suggest that the two domains of the proteobacterial m^2^G MTases RsmC and RsmD, and the predicted MTases YbiN and Mj0882 evolved from the common ancestor. An alternative scenario can be envisaged, in which the pseudodimeric structure of RsmC and RsmD results not from duplication but a fusion of the ancestral CTD with another distantly related MTase that has now degenerated. Nevertheless, fold-recognition results suggest that among various MTases of known structure, the NTD clearly shows highest similarity to Mj0882 (the archaeal counterpart of the CTD) (Table [3](#T3){ref-type="table"} and Table [4](#T4){ref-type="table"}). In addition, comparison of the sequence profile of the NTD sequences with profiles of other MTase families (unpublished data) using the FFAS algorithm \[[@B17]\] confirmed that the Mj0882/CTD family is the best match (data not shown), as reported in the course of fold recognition searches against the profiles comprising proteins from PDB. Moreover, the scenario involving intragenic duplication of the catalytic domain in the ancestor of the RsmC/RsmD lineage, which generated a functional pseudodimer, is supported by the dimeric structure of Mj0882 in the crystal (see below). Determination of the crystal structure of a member of the RsmC/RsmD lineage will provide insight into the relationship of the inactivated NTD to known MTase structures and the mutual orientation of the two domains.

Structure-based interpretation of sequence alignment and prediction of the active site
--------------------------------------------------------------------------------------

The Mj0882 structure was solved in the absence of any ligands. Prompted by the identification of close relationships between the family of m^2^G MTases and Mj0882, we sought to identify the cofactor- and guanine-binding sites in the 1.8 A crystal structure of the latter protein based on comparison with structures of known N-MTases. Searching the Protein Data Bank \[[@B20]\] using VAST \[[@B21]\]<> and DALI \[[@B22]\]<> revealed that the Mj0882 structure is more similar to various MTases alkylating hydroxyl groups, than to other nucleic acid N-MTases. This is not surprising, since previous evolutionary analysis of MTase structures and sequences demonstrated that some nucleic acid N-MTase families do not group together in the superfamily tree. The indication is that the common motif (N/D/S)-(I/P)-P-(F/Y/W/H) evolved independently several times on the common framework \[[@B23]\]. It will be interesting to determine, if m^2^G MTases share a relatively recent common ancestor with any of the m^6^A or m^4^C MTase families and if the similarities in their active site are synapomorphic or homoplastic.

The issue of divergent or convergent evolution notwithstanding, the \"catalytic loop\", corresponding to motif IV, assumes a strikingly similar conformation in all N-MTase structures solved to date (Figure [2](#F2){ref-type="fig"}). This suggests that the interactions between the target amino group and the side chain of the first residue and the carbonyl oxygen of the second residue of the tetrapeptide are highly similar. The same conformation is retained in the Mj0882 structure, consistent with the prediction that this protein belongs to the N-MTase family. Nevertheless, in the superimposed structures there is a substantial variation in the conformation of the cofactor AdoMet or its analogs, and in their relative orientation with respect to the catalytic site (Figure [2](#F2){ref-type="fig"}). This variation was noted previously in the course of crystallographic analyses, and was attributed to subtle structural differences between cofactor-binding pockets of various MTases that impose distinct conformation of the same ligand \[[@B12],[@B24]\] and to the fact that different ligands (AdoMet, AdoHcy, and their analogs) make different interactions and do not necessarily retain the common conformation when bound to the same MTase \[[@B24],[@B25]\].

![Comparison of the conformations of the \"catalytic loop\" (motif IV) of N-MTases in stereoview.  MTases: *M.Taq*I (1g38 in PDB) \[[@B29]\] in yellow, *M.Rsr*I (leg2) \[[@B55]\] in red, *M.Dpn*M (2dpm) \[[@B56]\] in green;  MTase ErmC\' (1qao) \[[@B24]\] in blue;  MTase *M.Pvu*II (1boo) \[[@B57]\] in cyan; Mj0882 (1dus) in magenta. The ligand for each protein is shown: AdoMet for *M.Dpn*M and ErmC\', S-adenosylhomocysteine for *M.Pvu*II, 5\'-methylthioadenosine for *M.Rsr*I, and 5\'-\[2-(amino)ethylthio\]-5\'-deoxyadenosine (cofactor analog) and the target deoxyadenosine for *M.Taq*I. In case of *M.Rsr*I, ErmC\' and *M.Pvu*II, whose structures were solved in the absence of the target, the Tyr or Phe sidechain can be easily rotated to the M.TaqI-like orientation to make a face-to-face π-stacking interaction with the target base.](1471-2105-3-10-2){#F2}

In order to dock the AdoMet molecule to the 1dus coordinates, we sought to identify those MTase structures, which display greatest similarity to the Mj0882 structure in the cofactor-binding region. VAST and DALI searches confirmed that the region spanning residues 55 to 117 in the Mj0882 sequence is the most similar to MTases that modify hydroxyl groups (data not shown), as well as the entire protein structure (see above). The catechol *O*-MTase structure (COMT) \[[@B26]\](1vid) was reported as the best hit by VAST (Score 7.1, RMSD 1.3). It was used as a structural template, because it was the only well-scored structure of a MTase complexed with AdoMet and not with its non-reactive analogs or the reaction product AdoHcy. Accordingly, the AdoMet moiety was copied from COMT to the Mj0882 structure based on superposition of the 1vid and 1dus coordinates. The obtained Mj0882-AdoMet complex showed no severe atomic overlaps and the cofactor seemed to fit the groove on the protein surface very well. According to AutoDock \[[@B27]\] the energy of the interactions between the 1dus structure and AdoMet in the docked complex is very favorable (-15.03 kcal/mol) even though it is lower from that calculated for the template COMT-AdoMet complex (-22.04 kcal/mol) in the 1vid structure \[[@B26]\]. From the docked model, in striking analogy to most MTase structures (reviewed in ref. \[[@B12]\]), the following three crucial contacts can be predicted: i) D^61^ from motif I coordinates the methionine amino group of AdoMet *via* an ordered water molecule. Even though the corresponding acidic residue is conserved in nearly all MTases analyzed to date, this contact has been identified only recently in the high-resolution structure of the RrmJ MTase \[[@B28]\]. ii) D^84^ from motif II coordinates the ribose hydroxyl groups, iii) D^113^ from motif III coordinates the amino group of the adenine moiety. Non-polar interactions between the side-chains of I^85^ and L^114^ and the adenine ring further contribute to the binding (Figure [3](#F3){ref-type="fig"}).

![The docking model of AdoMet and guanosine to the Mj0882 structure. Residues predicted to participate in cofactor and target binding are shown in wireframe representation and are labeled in cyan and in green, respectively.](1471-2105-3-10-3){#F3}

Docking of the target guanine to the Mj0882 structure (Figure [4](#F4){ref-type="fig"}) was guided by superposition of the \"motif IV loop\" of Mj0882 and *M.Taq*I, the only N-MTase co-crystallized with the nucleic acid substrate \[[@B29]\]. Under the assumption that both enzymes bind their targets in the same plane, the N2 group of guanine could be aligned with the N6 group of the adenine amino only if the purine ring was rotated by 120 degrees (*i.e.* with the atoms C2 and N3 of guanine superimposed onto atoms C6 and N1 of adenine). According to AutoDock \[[@B27]\] the energy of the interactions between the Mj0882-AdoMet complex and the docked guanine is quite favorable and comparable with that calculated for the target adenine in the *M.Taq*I structure (1g38) (-6.14 kcal/mol and -7.59 kcal/mol, respectively). These values are much lower than the energy of interactions between the protein and the cofactor (see above), because the AdoMet-binding groove is very deep and hydrophobic, while the base-binding site is relatively shallow. An alternative orientation of the target guanine in complex with 1dus could be obtained if its atoms C2 and N1 were superimposed onto atoms C6 and N1 of adenine. However, the latter model resulted in steric clashes between the ribose moiety and I^132^ (data not shown). Even if the flexible ligand docking option of AutoDock was used, which alleviated steric clashes, energies for the guanine in the \"alternative\" position (a cluster of similar conformations) were significantly worse (data not shown). In the first docked model the guanine moiety fits the groove on the protein\'s surface surprisingly well; a residue was identified, whose side chain might coordinate the ribose hydroxyl groups, namely D^41^ (Figure [3](#F3){ref-type="fig"}). Analysis of the multiple sequence alignment (Figure [1](#F1){ref-type="fig"}) revealed that this residue is highly conserved in the Mj0882, RsmD, RsmC, and YbiN families, supporting its functional importance. In the \"alternative\" docked model, D^41^ is too remote from the target base to make direct contacts to the target nucleoside (data not shown). However, a substantial conformational change upon substrate binding cannot be excluded, and direct participation of D^41^ in catalysis as well as other modes of guanine binding can be envisaged. Identification of this conserved carboxylate as a potentially important residue will hopefully prompt site-directed mutagenesis experiments and experimental determination of its role in binding and/or catalysis.

![Comparison of positions of the ligands in the active site of *M.Taq*I and the docked model of Mj0882. *M.Taq*I is shown in yellow, Mj0882 is shown in magenta.](1471-2105-3-10-4){#F4}

Although the presented docking model of Mj0882 complexed with AdoMet and guanine should be regarded as preliminary, it provides a useful platform for correlating the structure with sequence conservation in the Mj0882/RsmD/RsmC/YbiN family. The predicted AdoMet- and ribose-binding Asp residues (see above), as well as other invariant and highly conserved surface-exposed residues, map in the vicinity of the docked ligands, strongly supporting the model (Figure [5](#F5){ref-type="fig"}). The N2 group of the docked guanine is hydrogen bonded to the side chain of N^129^ and the carbonyl oxygen of P^130^. This is similar to the contacts made by the N6 group of the target adenine in the co-crystal structure of *M.Taq*I \[[@B29]\], which served as a docking template. As proposed for *M.Taq*I, these interactions could increase electron density of N2, change its hybridization from sp^2^ towards sp^3^, where the lone pair of N6 is no longer conjugated with the aromatic system, and thus contribute to its activation for nucleophilic attack on the activated methyl group of AdoMet. From the Mj0882 structure it is not obvious which residue may be responsible for eliminating the proton from the methylated N2 amine. The only conserved acidic residue in the vicinity of the active site corresponds to D^41^, and its localization is incompatible with the localization of the protons on N2 in the above-mentioned mechanism. If no massive conformational change of the m^2^G MTase occurs during RNA binding and catalysis, we propose that the proton is transferred directly to a bulk solvent molecule. In *M.Taq*I, the target base forms a face-to-face π-stacking interaction with Y^108^ from the NPPY tetrapeptide and a hydrophobic interaction with V^21^ from motif X. In Mj0882 these contacts are reversed (*i.e.* in motif IV Y^108^ is substituted by non-aromatic I and V^21^ is replaced by aromatic F^35^). Interestingly, all members of families RsmC, RsmD, and YbiN possess aromatic residues at both positions (Figure [1](#F1){ref-type="fig"}). It has been hypothesized that the methyltransfer reaction in N-MTases proceeds *via* a positively charged intermediate, which could be stabilized by cation-Ti interactions \[[@B30]\]. In m^2^G MTases, the electron-rich aromatic rings on both sides of the target could contribute to such stabilization.

![Molecular surface of Mj0882 colored by the conservation score. The score have been calculated using SWISS-PDB VIEWER \[[@B54]\] based on superposition of 1dus coordinates with homology models of YbiN and the CTD of RsmC and RsmD (all proteins from *E. coli*). The color scale varies from blue to yellow, with invariant residues in blue and most variable in yellow. The docked AdoMet and guanine moieties are shown in wireframe representation.](1471-2105-3-10-5){#F5}

The predicted guanine-binding mode implies that the target nucleotide is \"flipped out\" of the rRNA into the catalytic site, similarly to the mechanism discovered for DNA: m^5^C MTases \[[@B31]\] and extended to DNA N-MTases \[[@B29],[@B32]\]. In bacterial 16S rRNA G966 and G1516 occur in extrahelical regions, which should facilitate their binding in the catalytic pocket of the enzyme, but G1207 is base-paired in the helix 34 stem \[[@B7]\]. The m^2^G1207 MTase RsmC reacts with the 30S ribosomal particles but barely at all with 16S RNA, while the m^2^G966 MTase RsmD requires only the S7 and S19 proteins to methylate its target in the 16S RNA \[[@B10]\]. It has been inferred that both these nucleotides are modified after the association of several ribosomal proteins with the 16S rRNA, which is likely to assume the native secondary structure, but before assembly of the 30S subunit is complete \[[@B10]\]. The positively charged surface of Mj0882 in the area next to the docked guanine suggests charge complementarity to the negatively charged phosphate groups in rRNA (Figure [6](#F6){ref-type="fig"}). It will be interesting to study the base-flipping capabilities of RsmC and RsmD, since they may shed the light on the evolution of presumed mechanistic differences between the nucleic acid MTases that flip their target out of the double helix and those modifying extrahelical bases.

![Molecular surface of Mj0882 colored by the electrostatic potential. The values of surface potential are expressed as a spectrum ranging from -5 kT/e (deep red) to +5 kT/e (deep blue). The docked AdoMet and guanine moieties are shown in wireframe representation.](1471-2105-3-10-6){#F6}

Analysis of the apparently \"degenerated\" members of the family
----------------------------------------------------------------

Analysis of the multiple sequence alignment (Figure [1](#F1){ref-type="fig"}) suggested that the NTD of RsmC and RsmD lack several conserved residues. However, only in connection with the docking model of Mj0882 it can be concluded, which of these residues may be involved in binding or catalysis. Specifically, all NTD sequences lack the guanine N2-coordinating Asn and the aromatic or aliphatic residue in motif IV. The NTD of all RsmC, with the exception of *T. thermophilus,* lacks the N-terminal extension, which includes the aromatic residue predicted to make the face-to-face π-stacking interactions with the target guanine (F^35^ in Mj0882). This conserved aromatic residue is also missing from the longer N-terminus of RsmD and *T. thermophilus* RsmC. The NTD of RsmD retained the predicted ribose-binding acidic residue at the position corresponding to D^41^ in Mj0882, while in the NTD of RsmC this residue is often substituted, in most cases with Ser or Thr. All NTD sequences, with the possible exception of *C. crescentus* and *T. thermophilus* RsmC, lack the AdoMet-coordinating acidic residue in motif I, corresponding to D^61^ of Mj0882. The NTD of RsmD retained the Asp residue corresponding to D^84^ in Mj0882, however in nearly all RsmC sequences this residue is substituted with various polar amino acids. A region corresponding to motif III was so diverged in the NTD of both RsmC and RsmD that it could not be aligned with confidence and the position corresponding to D^113^ in Mj0882 could not be identified unambiguously. It is noteworthy that the residue corresponding to D^113^ (motif III) is weakly conserved in the MTase superfamily, but the acidic residue corresponding to D^84^ (motif II) is conserved in nearly all MTase families studied to date \[[@B12]\](JMB, unpublished data).

In summary, the NTD of RsmC and RsmD lacks all residues, which were predicted to be a part of the active site based on the docking model and which are conserved between Mj0882 and the CTD. The NTD of RsmC also lacks most of the acidic residues predicted to coordinate AdoMet and the ribose moiety of the target nucleoside. Nevertheless, some of these residues are retained in the NTD of RsmD and in the NTD of *T. thermophilus* RsmC. Hence, it is inferred that the NTD of RsmC and RsmD lost the m^2^G MTase activity and are probably also deficient in the ability to bind the cofactor. There are known precedents of proteins with apparent severe defects in conserved MTase motifs. One example includes the yeast Kar4p protein required for expression of karyogamy-specific genes during mating \[[@B33]\]. Using fold recognition and homology modeling it was demonstrated that Kar4p is related to  MTases and the \"permuted\"  and m^4^C MTases, although it lost its active site and has the AdoMet-binding site blocked by an insertion. It is quite probable that Kar4p exerts its function due to the retained ability to bind to RNA or DNA (JM Bujnicki and coworkers, manuscript submitted). Another example is the eukaryotic heterodimeric  MTase, in which one subunit binds AdoMet and presumably carries out the catalysis, while the other is indispensable for tRNA binding \[[@B2]\]. It has been demonstrated that the tRNA-binding subunit is homologous to the \"active\" subunit, but lost the cofactor-binding and catalytic side-chains. We have also identified a single \"active\"  MTase gene in Bacteria and Archaea, suggesting that the ancestral prokaryotic form was a homodimer \[[@B34]\]. This prediction has been recently confirmed by the crystal structure of the Rv2118c protein from *Mycobacterium tuberculosis,* which forms a dimer of tight homodimers \[[@B35]\].

Interestingly, according to the EBI Macromolecular Structure Database, the suggested biologically relevant molecule for the Mj0882 structure is a homodimer <>. The Mj0882 homodimer exhibits 5 interchain salt bridges within the complex and shows a loss of 2045.5 Ang\*\*2 of solvent accessible surface area upon complex formation. However, in each subunit the active site is completely blocked by a loop from the other subunit. Nevertheless, it is tempting to speculate that the active form of Mj0882 is a homodimer that undergoes a conformational change to allow the target 16S rRNA to bind and that RsmC and RsmD MTases function as pseudodimers. If this is true, the relationship between Mj0882 and the RsmC/RsmD families would be similar to the relationships between prokaryotic and eukaryotic  MTases.

We also found that the archaeal members of the YbiN family lack the counterpart of the conserved N^129^ in Mj0882. However, analysis of the multiple sequence alignment revealed that they retained all other predicted binding residues, with the possible exception of the poorly conserved D^113^ (Figure [1](#F1){ref-type="fig"}). This prompted us to build a provisional homology model of the PH0266 protein, which revealed that the \"missing\" Asp sidechain in motif IV is spatially replaced by a His residue from motif X (data not shown). The alignment (Figure [1](#F1){ref-type="fig"}) reveals that the His residue is conserved at this position in all archaeal YbiN homologs, whereas bacterial and eukaryotic YbiN proteins have Pro or Ala and the Mj0882/RsmC/RsmD lineage have Gly or Ala (G^43^ in Mj0882). \"Migration\" of catalytic and cofactor binding residues have been documented in many cases (for a review see ref. \[[@B36]\]), including RNA MTases that generate m^5^C in rRNA \[[@B37]\] and m^7^G in mRNA \[[@B38]\]. Nonetheless, no active nucleic acid MTase have been reported bearing the His substitution for the catalytic residue in motif IV. We hope that this analysis will stimulate experimental characterization of archaeal YbiN homologs, which may eventually lead to validation of a novel version of the methyltransfer active site.

Conclusions
===========

Based on a comprehensive bioinformatic analysis of m^2^G MTases it was inferred that the prokaryotic RsmC and RsmD MTases are pseudodimers. The C-terminal catalytic domain is closely related to the structurally characterized Mj0882 protein, while the N-terminal domain lacks the cofactor-binding and catalytic side-chains. Based on analysis of sequence similarities within and between subfamilies, it is suggested that bacterial m^2^G MTases evolved from a duplicated Mj0882-like enzyme, although a fusion with another distantly related MTase cannot be definitely excluded. The supposed intragenic duplication or gene fusion followed by degeneration of the N-terminal domain was ancient and predated the gene duplication leading to radiation of RsmC and RsmD lineages. However, we were not able to determine the precise timing of the divergence, presumably due to the high noise-to-signal ratio in the multiple sequence alignment. This evolutionary scenario, along with the analysis of the crystal structure of Mj0882, suggests that m^2^G MTases function as dimers, with the one subunit carrying out catalysis and the other probably participating in recognition and binding of the target rRNA. An analogous case has been described for archaeal tRNA splicing enzymes, which exist in two forms. The *M. jannaschii* EndA nuclease is a homotetramer, in which two pairs of identical subunits have non-equivalent roles in tRNA binding and catalysis \[[@B39]\], whereas its *A. fulgidus* homolog is a homodimer of pseudodimers, comprising diverged domains of which only one retained the active site \[[@B40]\]. Analysis of the Mj0882 docking model in the light of the sequence conservation in the m^2^G MTase family allowed prediction of cofactor-binding and catalytic residues. In the YbiN family of predicted N-MTases (suggested to be  MTases, but this specificity is uncertain) new eukaryotic and archaeal members have been identified. The latter exhibited an intriguing correlated loss of a conserved Asn residue in motif IV with acquisition of an atypical His residue in motif X. Both side chains are likely to occupy a similar spatial position, suggesting a novel version of the MTase active site. All these predictions can be verified experimentally. We hope that this analysis will significantly improve the insight into the molecular mechanism of m^2^G methylation, facilitate further structural and functional studies on m^2^G MTases and contribute to better understanding of relationships between them and other nucleic acid MTases.

Methods
=======

Sequence database searches
--------------------------

The position-specific, iterative (PSI-) version of BLAST \[[@B41],[@B42]\] was used to search the non-redundant (nr) version of current sequence database and the publicly available complete and incomplete genome sequences available via the websites of the National Center for Biotechnology Information (Bethesda, USA; <> and the Bioinformatics Laboratory of International Institute of Molecular and Cell Biology (Warsaw, Poland; <>). All searches were initiated with stringent profile inclusion expectation (e) values (\<10^-20^) to avoid \"explosion\" of hits to multiple paralogous MTase families and the cutoff was relaxed in subsequent iterations as the profile become balanced (i.e. included more orthologs of the query, which reduced the probability of assigning a significant score to a non-ortholog that exhibited a fortuitous similarity to the query, but not to the orthologs of the query). The stringent criteria for provisional assignment of orthologous relationship in PSI-BLAST searches were: the alignment spanning more than 80% of both the query and the reported hit, e-value in the 1^st^ iteration \< 10^-30^, sequence identity \> 40%. In all searches initiated with RsmC, RsmD, and YbiN sequences, hits to the catalytic domain of HemK family members were reported before relaxation of the cutoff with e-values ranging from 10^-5^ to 10^-18^ (i.e. subsequently to the presumed members of the above-mentioned families). The choice of the relaxed cutoff was dictated by the assumption that the HemK family of protein MTases should be regarded as an outgroup and its members shall not be included in the profile. The multiple sequence alignment was retrieved from the PSI-BLAST, degapped, and used as a profile, to which all the full-length sequences were realigned using CLUSTALX \[[@B43]\].

Structure prediction
--------------------

Protein structure predictions were carried via the MetaServer interface \[[@B44]\]<> using publicly available online services for fold recognition: FFAS \[[@B17]\], 3DPSSM \[[@B45]\], BIOINBGU \[[@B46]\], GenThreader \[[@B47]\], SAM-T99 \[[@B48]\], and FUGUE \[[@B49]\], and secondary structure prediction: JPRED2 \[[@B50]\] and PSI-PRED \[[@B51]\]. The results were processed by the Pcons consensus server \[[@B52]\], which produced a ranking of potentially best target-template alignments and evaluated the likelihood of the models to be correct.

Protein structure analysis
--------------------------

Comparison of atomic coordinates with the protein structures from the Protein Data Bank \[[@B20]\] were carried out using VAST \[[@B21]\] and DALI \[[@B53]\]. SWISS-PDB VIEWER \[[@B54]\] was used for all protein structure manipulation, calculation of conservation scores and electrostatic potential distribution, and to generate the figures. Provisional homology modeling was carried out using the SWISS-MODEL/PROMOD II server <>\[[@B54]\].

Authors\' contributions
=======================

J.M.B. carried out the sequence and structure analysis and drafted the manuscript. L.R. provided the laboratory space and the access to the MetaServer, BIB-VIEW, local version of BLAST and sequence databases.

Acknowledgments
===============

We would like to thank all the genome sequencing groups that make their preliminary data publicly available, without which this work could not be done. Especially, the use of data generated at the Department of Energy Joint Genomic Institute, the Institute for Genomic Research, and the Stanford Genome Technology Center is gratefully acknowledged. We are indebted to Krzysztof Ginalski for help with AutoDock and to Richard Leach for critical reading of the manuscript. This work was supported by the Polish State Committee for Scientific Research (grants 8T11F01019 and 6P04 B00519)


Background
==========

Enterohaemorrhagic *Escherichia coli* (EHEC) is considered to be common causes of haemorrhagic colitis (HC), thrombotic thrombocytopenic purpura, and haemolytic-uraemic syndrome (HUS) in humans \[[@B1],[@B2]\]. In a previous paper, we have demonstrated that EHEC are commonly found in the intestines of livestock, specially young cattle; and infections in humans are, in part, a consequence of consumption of undercooked meat or raw milk \[[@B3]-[@B6]\]. Several outbreaks have occurred mainly in USA, Canada, United Kingdom and Japan during the last decade \[[@B7]-[@B10]\]. Argentina has one of the highest records of HUS (300--400 cases/year; 22/100.000 children under 4 years of age). This rate can be compared with the anual incidence of 2.6/100.000 in Oregon, 3.0 in King County-Washington, 3 to 5 in Canada, United Kingdom, Chile and Uruguay \[[@B11]\]. In spite of EHEC strain O157:H7 has been reported as a cause of HUS in this country \[[@B12]\], this strain does not seem to be so common in this country as in the USA \[[@B11]\]. We have recently published an extensive list of non-O157:H7 EHEC serotypes isolated from cattle and foods in Argentina \[[@B13]\], several of them involved as cause of HC and HUS in somewhere.

In Europe, eighty percent of the VTEC isolated from patients with diarrhoea corresponded to non-O157 serogroups such as O26, O91, O103, O111, O113, O128, O145 \[[@B14]\]. In 1984, Kudoh et al. \[[@B15]\] described the first outbreak of disease due to *E. coli* O145:H- in Japan, affecting to 100 children. Beutin et al. \[[@B16]\] have considered this serotype as a new emergen. Afterthere, several investigators notified the isolation of this serotype from cattle and humans in USA, Canada and Belgium \[[@B17]-[@B22]\]. Another serotype, , was found associated to HUS cases in U.K. \[[@B19]\], meanwhile O145:H8 and  were isolated from healthy cattle in Canada \[[@B22]\] and Germany \[[@B23]\], respectively.

Aside from known differences among the pathogenicity of VT1, VT2 and its variants, factors that may affect the virulence of EHEC are the ability to cause attaching and effacing lesions (*eae* gene) in the intestinal mucosa and possession of a 60 MDa megaplasmid (Mp) \[[@B1]\]. The aim of this work is to communicate the first isolation of O145:H- from cattle in this country and characterize the virulence cassette of several strains, providing useful information to evaluate the risk of foodborne transmission of this non-O157:H7 serotype.

Results and discussion
======================

Nine Shiga toxin-producing *Escherichia coli* serogroups and eleven non-typable strains were identified from a feedlot in Argentina (data no shown). One of them (O145) corresponded to a serogroup never described before in this country. All O145 strains were characterized as H-. This non-O157:H7 serotype (O145:H-) had been previously involved in haemolytic uraemic syndrome outbreaks in Japan \[[@B15]\]. The genotype of those strains were characterized, corresponding to several virulence cassettes: VT2^+^*eaeA*^+^ Mp^+^ (n = 5), VT2^+^*eaeA*^+^ (n = 1), VT1^+^*eaeA*^+^ Mp^+^ (n = 2), and VT1^+^*eaeA*^+^ (n = 1). All these strains were able to ferment sorbitol within 18 h of incubation at 37°C. Strains isolated from the same animal were considered only when showed a different virulence pattern.

O145:H- strains were distributed in two RAPD profiles, which corresponded to the presence of either, VT1^+^ or VT2^+^ genotype (RAPD profiles 2 and 1, respectively). No difference was detected by RAPD analysis between Mp^+^ or Mp^-^ strains (Fig. [1](#F1){ref-type="fig"}). This fact suggests that would be necessary to carry on RAPD studies by using new primers or to apply pulse field electrophoresis technique in the study of clonal relationships.

![**RAPD profiles generated using primer M13**. Lane MW contains DNA molecular size marker (100 bp ladder, Promega, Madison, WI).](1471-2180-2-6-1){#F1}

The presence of the virulence genes codifying for verotoxin (1 or 2), the ability to develop attaching and effacing phenomenon on enterocyte surface (*eaeA* gene) and the expression of an EHEC haemolysin (Hly~EHEC~), afford the potential of O145:H- as foodborne emergent human pathogen. This EHEC non-O157:H7 serotype, isolated from cattle in feedlot, was never found by us from grazing cattle in Argentina \[[@B13]\]. Our finding, and the high incidence rate of HUS in this country, put local health service authorities on guard.

Conclusions
===========

This was the first isolation of EHEC O145:H- serotype from Argentinian cattle which enlarges the list of non-O157:H7 serotypes isolated from bovine and ground beef in this country by us. All O145:H- strains carried several virulence factors which allow to predict their potential ability to develop haemolytic uraemic syndrome in humans. The strains, which had been isolated from different animal in the same feedlot, conformed two RAPD profiles based upon their verotoxin subtype, without clonal differences.

Materials and methods
=====================

Sample collection from cattle
-----------------------------

Samples were collected from an Argentinian farm. following the method described previously \[[@B6]\]. Briefly, 59 beef cattle, belonging to a feedlot of Pampeana region, were sampled fortnightly during six months by a single rectal swab from each animal. The swabs were placed in transport medium and processed immediately at the laboratory.

Bacterial colonies were grown on MacConkey agar incubating for 24 hs at 37°C. An aliquots of confluent growth, aproximately 100 colonies, was inoculated into Luria-Bertani broth for 3 h at 37°C and processed for DNA extraction \[[@B6],[@B24]\]. One ml from each culture was frozen at -70°C with the addition of glycerol for further isolation of individual VT^+^ colonies (50 to 100 colonies/sample) on a MacConkey agar plate. Microorganisms were confirmed as *E. coli* by means of biochemical test such as citrate, indole, urease and TSI profile.

Detection of VT1, VT2, *eae* and 60 MDa plasmid by PCR
------------------------------------------------------

*Escherichia coli* growth and DNA preparation were performed as described previously \[[@B6],[@B24]\]. Primers *eae-1* and *eae-2* were designed to amplify a portion of the *eae* gene which is conserved between enteropathogenic and enterohaemorrhagic *E. coli* strains. Primer sequences for VT1, VT2 and *eae* were indicated in a previous paper \[[@B6]\]. Mp primer sequences were: MFS1F 5\' ACGATGTGGTTTATTCTGGA 3\' and MFS1R 5\' CTTCACGTCACCATACATAT 3\' (166 bp amplimer) \[[@B25]\]. T~m~ were calculated for each primer using Oligo 4.0 (Primer Analysis Software, National BioSciences). Amplification of bacterial DNA was performed in a total volume of 50 μl. A negative control (reagent blank) was included without addition of sample. Another control was designed by adding DNA from the strain lacking VT1, VT2, *eae* and Mp. Two reference strains O157:H7 were used as positive controls. The conditions for the PCR amplification of VT1, VT2, *eae* and Mp were as described previously \[[@B6],[@B24],[@B25]\]. Amplified products were analyzed by submarine gel electrophoresis and UV-transillumination (300 nm).

Fermentation of sorbitol
------------------------

Sorbitol MacConkey agar was used to test the VT^+^ strains for this condition.

Serotyping
----------

O and H antigens were determined by means of a microagglutination technique in tubes and plates described by Guinée *et al.*\[[@B26]\] and modified by Blanco *et. al.*\[[@B27]\] using all available O (O1 -- O175) plus six putative new O antigens (OX176-through OX181) \[[@B28]\] and H (H1 -- H56) antisera \[[@B29]\]. Non-specific agglutinins were removed by absorption with the corresponding cross-reacting antigens. All VTEC were processed for O serogroup determination. H serotyping was performed only on those strains which, having been isolated from the same sample, differed in either one virulence factor or the O serogroup.

Random amplified polymorphic DNA (RAPD) analysis
------------------------------------------------

Bacteria were grown overnight at 37°C in LB broth with shaking. An aliquot of the culture was diluted 1/10 in water to determine the optical density at 600 nm \[39\]. For an optical density value of 0.5, a 500 μl aliquot of the stationary-phase culture was centrifuged (2 min at 12,000 × g) and suspended in 500 μl of bidestilled water. The suspension was then boiled for 10 min, centrifuged (2 min at 12,000 × g) and the supernatant was stored at -70°C. Five microliters were used as the template for PCR amplification. RAPD was performed in a final volume of 50 μl containing 20 mM (NH~4~)~2~SO~4~, 75 mM Tris-HCl pH 9.0, 0.1% (w/v) Tween 20, 2.5 mM MgCl~2~, 200 μM each dNTP, 1 μM primer, 2.5 U *Taq* DNA polymerase. Three different primers were evaluated: 1281 \[[@B31]\], 970--11 and M13 \[[@B32]\], but primers 1281 and 970--11 were not discriminatory.

Amplification was done in a Genius thermal cycler \[Techne (Cambridge) Ltd.\] as follows: initial denaturation at 94°C for 5 min (heating rate 60°C/min), followed by 40 cycles of denaturation at 94°C for 1 min (heating rate 29°C/min), annealing at 50°C for 1 ½ min (cooling rate 26°C/min) and extension at 72°C for 1 ½ min (heating rate 29°C/min). Reaction products (10 μl) were analysed in a 1.8% agarose gel stained with ethidium bromide.

::: {#T1 .table-wrap}
::: {.caption}
######

Feno-genotypic characteristics of O145:H- strains isolated from cattle.
:::

  **Serotype**   **Animal & Sample**   **Virulence cassette**   **Virulence profile**   **Lane**
  -------------- --------------------- ------------------------ ----------------------- ---------- ---- --- ---
  O145:H-        52(1)                 \-                       \+                      \+         \+   1   1
  O145:H-        18(1)                 \-                       \+                      \+         \+   1   2
  O145:H-        27(3)                 \-                       \+                      \+         \+   1   3
  O145:H-        46(3)                 \-                       \+                      \+         \+   1   4
  O145:H-        38(4)                 \-                       \+                      \+         \+   1   5
  O145:H-        27(3)                 \-                       \+                      \+         \-   2   6
  O145:H-        1(1)                  \+                       \-                      \+         \+   3   7
  O145:H-        17(11)                \+                       \-                      \+         \+   3   8
  O145:H-        1(1)                  \+                       \-                      \+         \-   4   9
  **n**                                3                        6                       9          7

**Lane** column indicates positions of RAPD study for each strain in Fig. [1](#F1){ref-type="fig"}.
:::

Acknowledgements
================

Authors thank Nutrition and Metabolism Unit (INTA-Balcarce) for its collaboration and María R. Ortiz for her technical assistance. This work was supported by the Scientific Research Commission Prov. Buenos Aires (CIC), SECYT-UNCPBA, FONCYT and Univ. Santiago de Compostela. A.E. Parma and A. I. Etcheverría are members of CIC and G.H. Arroyo is member of CONICET.


Background
==========

Since the initial description of four patients with coeliac disease (CD) and primary biliary cirrhosis (PBC) \[[@B1]\], there have been several subsequent reports demonstrating an association of these two diseases \[[@B2]-[@B7]\]. Recently, a number of epidemiological studies reported an increased, up to 7%, prevalence of CD in patients with PBC and an increased prevalence, up to 3%, of PBC in patients with CD \[[@B8],[@B9]\]. Screening for one disease when the other is identified has therefore been suggested. However, in other studies this association was found less pronounced or has not been confirmed \[[@B10]-[@B13]\]. Autoimmune cholangitis (AIC) was first reported as an overlap syndrome of autoimmune hepatitis and PBC, but is considered by most as a discrete entity of antimitochondrial antibody (AMA)-negative PBC with autoimmune characters \[[@B14]\]. Apart from a case report \[[@B15]\], no relation of adult CD to autoimmune cholangitis has so far been described. However, there have been no formal attempts to systematically evaluate patients with AIC for CD, presumably because AIC represents a rare form of chronic cholestatic liver disease.

Although a definite diagnosis of CD still requires histopathologic examination of the small intestinal mucosa, the screening methods for large number of individuals are based on non-invasive serological tests, namely, anti-gliadin (AGA, IgG and IgA class), anti-reticulin (ARA R1-type, IgA class) and anti-endomysial (EMA, IgA class) antibodies \[[@B16]-[@B18]\]. The combined use of serum IgG-AGA (good sensitivity) and IgA-EMA (good specificity) has resulted in a reliable screening test for diagnosis of CD \[[@B19]\]. However, anti-gliadin antibodies (AGAs) are not suitable for CD screening in patients with chronic liver diseases including PBC, as an unacceptably high percentage of false-positives was reported in several studies \[[@B11],[@B20],[@B21]\]. In this respect, a previous study revealed that the serum IgA-EMA assay is the optimum test for predicting CD in patients with chronic liver disease \[[@B11]\]. Although IgA class antibodies to guinea pig liver-derived tissue transglutaminase (IgA-tTG) have been shown to be highly specific and sensitive for the diagnosis of CD \[[@B22]-[@B26]\], it was recently reported that in patients with chronic liver disease there was a high frequency of false-positives which disappeared when human tTG was used as the antigen in the ELISA system \[[@B27]\]. In the present study we evaluated AGAs, IgA-ARA, IgA-EMA and guinea pig liver-derived IgA-tTG in 62 patients with PBC and 17 patients with AIC. Intestinal biopsies have been subsequently performed in the majority of patients tested positive for at least one antibody class.

Methods
=======

Materials
---------

Serum samples from 62 patients with PBC (53 women and nine men; mean age, 59 years; range, 32--85 years) and 17 patients with AIC (16 women and one man; mean age, 62 years; range, 52--77 years) were used in the study. The samples were collected prospectively over a 2 yr. period (1999--2000) and stored in aliquots at -20°C. Diagnosis of PBC was based on standard laboratory findings, compatible liver histology and the presence of AMA in titer \> 1:80. Class 2 AMA antibodies (M2) detected by ELISA were positive in all patients. According to Scheuer\'s classification \[[@B28]\], 34 patients (54%) had stage I-II and 28 (46%) stage III-IV disease. The mean follow-up period from the time of diagnosis was 5.2 years (range, 1--9.4 years).

The diagnosis of AIC has been made on cholestatic patients with a liver biopsy compatible with PBC, without AMA or M2 positivity. ANA were positive (titers ≥ 1:160) in all 17 patients. Anti-smooth muscle antibodies were positive (titer ≥ 1:80) in 9 patients. According to Scheuer\'s classification, 10 patients (58%) had stage I-II and 7 (42%) stage III-IV disease. The mean follow-up after diagnosis was 6.6 years (range, 1--7.8 years).

Ultrasound, computed tomography and endoscopic retrograde cholangiography ruled out the possibility of biliary obstruction, when the latter was suspected. No patient had a family history of CD, or IgA deficiency. One PBC patient was HbsAg positive and one PBC patient and two AIC patients were anti-HCV positive. One hundred Cretan adults selected at random from blood donors served as controls. Eighteen serum samples from symptomatic, biopsy-proven, AMA negative adults with CD were included in the study as methodological controls. Total IgA was measured in all CD patients and healthy controls to exclude IgA deficiency.

Methods
-------

Two operators unaware of the clinical parameters performed the immunological tests. All PBC, AIC, CD and control sera were screened for AGAs, IgA-ARA, IgA-EMA and IgA-tTG. IgA- and IgG-AGA were screened by a commercial ELISA at a dilution of 1:50 (Alphadia SA/NV, Belgium). Values greater than 50 U/mL were considered positive. Results were considered doubtful and repeated for values between 45 and 50 U/mL. For IgA-ARA R1-type, sera were tested by an indirect immunofluorescence (IIF) test at a 1:10 and 1:40 dilution, using rat liver, kidney and stomach as substrates (Sanofi Diagnostics Pasteur, France). Sera were screened for IgA-EMA by IIF, using both monkey oesophagus (Biosystems, Barcelona, Spain) and human umbilical cord (Eurospital SpA, Trieste, Italy) as substrates. Titers of ≥ 1:5 were considered positive. To avoid false-negative results, IgA-EMA were also evaluated at a 1:50 dilution. IgA-tTG were tested by a commercially available guinea-pig liver-derived tTG ELISA kit at a dilution of 1:101, according to manufacturers\' instructions (QUANTA Lite™ tTG ELISA, INOVA Diagnostics, San Diego, USA). The samples were classified as negative (\<20 Units), weakly positive (20--30 Units) and strongly positive (\>30 Units). Small intestinal biopsies were performed at endoscopy using endoscopic forceps. Four biopsy specimens from opposite sites of the distal duodenum were obtained and evaluated by one observer specialized in gastrointestinal tract pathology with regard to the villi/crypts ratio and the number of intraepithelial lymphocytes (IELs) per 100 surface epithelial cells. Small bowel biopsies from the patients with PBC and AIC were evaluated blindly with those obtained from the 18 patients with untreated CD. When biopsy specimen revealed features of CD \[[@B29]\], namely, a lowering of the villous height to crypt depth ratio (normal, 3--5:1) and/or an increase in IELs (normal, 10--30 per 100 epithelial cells) a more detailed histological classification was applied \[[@B30]\].

Test results were compared using a 2 × 2 contingency table and chi-squared statistics. When appropriate, Fisher\'s exact test was used. *P* values below 0.05 were considered significant.

Results
=======

IgA class anti-gliadin antibodies and IgA class antibodies to tissue transglutaminase were detected with a significantly increased frequency in patients with both PBC and AIC as compared to healthy blood donors (Table [1](#T1){ref-type="table"}). In contrast, none of the PBC and AIC patients was tested positive for anti-reticulin or anti-endomysial antibodies. Controls were tested negative for anti-reticulin, anti-endomysial and IgA class anti-tissue transglutaminase antibodies.

::: {#T1 .table-wrap}
::: {.caption}
######

Prevalence (%) of antibodies in patients and in controls
:::

                         **PBC** n = 62 (%)   **AIC** n = 17(%)   **BD** n = 100   **CD** n = 18
  ---------------------- -------------------- ------------------- ---------------- ---------------
  **IgA-AGA**            12(19)^\*^           6 (35)^\*^          1                18
  **IgG-AGA**            3(5)                 \-                  2                16
  **Any AGA**            13 (21)^\*^          6 (35)^\*^          3                18
  **Both AGAs**          2(3)                 \-                  1                16
  **IgA-ARA**            \-                   \-                  \-               18
  **IgA-EMA**            \-                   \-                  \-               18
  **IgA-tTG**            6(10)^\*^            3(18)^\*^           \-               18
  **IgA-tTG and AGAs**   2(3)                 2 (12)^\*^          \-               18

n = number of patients; \* = where *p* less than 0.05, PBC = primary biliary cirrhosis; AIC = autoimmune cholangitis; CD = coeliac disease; BD = blood donors; IgA-AGA = anti-gliadin antibodies, IgA class; IgG-AGA = anti-gliadin antibodies, IgG class; AGAs = anti-gliadin antibodies; IgA-ARA = IgA class anti-reticulin antibodies, RI type; IgA-EMA = anti-endomysial antibodies, IgA class; IgA-tTG = antibodies to tissue transglutaminase, IgA class
:::

PBC patients (Table [2](#T2){ref-type="table"})
-----------------------------------------------

::: {#T2 .table-wrap}
::: {.caption}
######

Antibody titers and intestinal biopsies in patients with primary biliary cirrhosis
:::

  **Patient**   **Disease stage^a^**   **IgA-AGA**   **IgG-AGA**   **IgA-ARA**   **IgA-EMA**   **IgA-tTG**   **Intestinal biopsy**
  ------------- ---------------------- ------------- ------------- ------------- ------------- ------------- -----------------------
  1\.           I-II                   \-            \-            \-            \-            49            Normal
  2\.           I-II                   199           58,6          \-            \-            50            Normal
  3\.           I-II                   \-            \-            \-            \-            25            Normal
  4\.           I-II                   55            \-            \-            \-            \-            ND
  5\.           I-II                   166           \-            \-            \-            \-            Normal
  6\.           I-II                   200           200           \-            \-            \-            Normal
  7\.           I-II                   70            \-            \-            \-            \-            ND
  8\.           III-IV                 244           \-            \-            \-            \-            ND
  9\.           III-IV                 144           \-            \-            \-            \-            ND
  10\.          III-IV                 \-            67,4          \-            \-            \-            ND
  11\.          III-IV                 \-            \-            \-            \-            21            ND
  12\.          III-IV                 106           \-            \-            \-            32            Normal
  13\.          III-IV                 \-            \-            \-            \-            28            Normal
  14\.          III-IV                 97            \-            \-            \-            \-            Normal
  15\.          III-IV                 53            \-            \-            \-            \-            Normal
  16\.          III-IV                 57            \-            \-            \-            \-            Normal
  17\.          III-IV                 253           \-            \-            \-            \-            ND

^a^According to Scheuer\'s classification; ND = not done
:::

Both anti-gliadin antibodies were detected in 13 out of 62 patients with PBC (21% *vs.* 3% in controls, *p* \< 0.001). IgA-AGA were tested positive in 12 patients (19%, *p* \< 0.001), of whom 5 had stage I-II disease, and 7 had stage II-IV disease. IgA-AGA positivity was significantly more pronounced in patients with stage III-IV disease (25% vs 14%, *p* \< 0.05). IgA-tTG were detected in 6 patients (10%), 3 of whom had stage I-II disease and another 3 stage III-IV disease, while tested negative in all controls (*p* \< 0.001). Serology was negative in the two patients with a superimposed viral hepatitis infection.

AIC patients (Table [3](#T3){ref-type="table"})
-----------------------------------------------

::: {#T3 .table-wrap}
::: {.caption}
######

Antibody titers and intestinal biopsies in patients with autoimmune cholangitis
:::

  **Patient**   **Disease stage^a^**   **IgA-AGA**   **IgG-AGA**   **IgA-ARA**   **IgA-EMA**   **IgA-tTG**   **Intestinal biopsy**
  ------------- ---------------------- ------------- ------------- ------------- ------------- ------------- -----------------------
  1\.           I-II                   157           \-            \-            \-            21,9          Normal
  2\.           I-II                   \-            \-            \-            \-            26            Normal
  3\.           I-II                   153           \-            \-            \-            \-            ND
  4\.           III-IV                 55,9          \-            \-            \-            \-            ND
  5\.           III-IV                 382           \-            \-            \-            32            Normal
  6\.           III-IV                 65            \-            \-            \-            \-            Normal
  7\.           III-IV                 60            \-            \-            \-            \-            Normal

^a^According to Scheuer\'s classification; ND = not done
:::

IgA-AGA were tested positive in 6 patients with AIC (35% *vs.* 3% in controls, *p* \< 0.001), two with stage I-II disease and four with stage III-IV disease. IgA-AGA positivity was significantly more pronounced in patients with stage III-IV disease (57% vs 20%, *p* \< 0.05). IgA-tTG were detected in 3 patients with AIC (18%), two patients with stage I-II disease and one patient with stage III-IV disease, while tested negative in all controls (*p* \< 0.001). Serology was negative in two AIC patients with superimposed hepatitis C.

CD patients
-----------

All 18 patients with coeliac disease were tested positive for IgA-ARA, IgA-EMA and IgA-tTG, whereas 16 of them had also both IgA and IgG anti-gliadin antibodies.

Healthy blood donors
--------------------

None of the 100 tested sera were found to be IgA-EMA or IgA-tTG positive. However AGAs were present in 3 individuals (IgA-AGA in one and IgG-AGA in two).

Duodenal biopsies
-----------------

Overall, biopsies were performed in 10 out of 17 PBC patients (59%) and 5 out of 7 AIC patients (71%) tested IgA-AGA and/or IgG-AGA and/or IgA-tTG positive. Five patients died shortly after testing positive and four patients refused any further investigation. None of the biopsied PBC and AIC patients had features suggestive of CD. Small bowel biopsies of our 18 patients with CD revealed partial villous atrophy (Marsh IIIa) in three, subtotal villous atrophy (Marsh IIIb) in eleven and total villous atrophy (Marsh IIIc) in four.

Discussion
==========

In contrast to recent reports from Wales \[[@B8]\], Scandinavia \[[@B9]\], Northern Ireland \[[@B31]\], and Canada \[[@B32]\], the present study has not been able to demonstrate an increased risk of coeliac disease in this group of 62 Cretan patients with primary biliary cirrhosis. Our results are consistent with data from Sweden \[[@B11]\] and Italy \[[@B12],[@B13]\], suggesting that if there is no clinical suspicion of CD, screening with anti-endomysial antibodies should not be performed routinely in all patients with PBC.

A possible explanation relevant to these controversies could be the methodological differences in detecting and defining CD, like the different serological tests used for screening (AGAs and/or IgA-EMA), the degree of intestinal damage, or the spectrum of clinical presentation of CD in patients studied. Patients with malabsorption, diarrhoea, weight loss, and total villous atrophy represent perhaps only 30--40% of the entire spectrum of gluten-sensitised individuals \[[@B19]\]. However, studies supporting the association between PBC and CD have included mainly symptomatic or complicated cases. In Kingham\'s et al study \[[@B8]\], CD in patients with PBC was sought by investigating features suggestive of malabsorption, a family history of CD, the presence of AGAs, or the finding of otherwise unexplained haematinic deficiency. In Sorensen\'s et al paper \[[@B9]\], it is not clear whether all patients with CD were systematically evaluated for PBC. PBC was found in patients hospitalised for CD, which presumably excluded patients with subclinical or silent disease. Furthermore, no information was available for patients treated as outpatients.

The inconsistencies between studies may also be attributed to the ethnic variability of CD prevalence in the different populations. The prevalence of CD in Greece is currently unknown, because of the lack of official epidemiological data, but it is probably low. A crude estimation concerning of the prevalence of CD in Greece is provided from an endoscopic study from Athens, reporting one new case of unsuspected CD per 520 endoscopies \[[@B33]\]. When we retrospectively looked for, only 23 adult patients with symptomatic gluten-sensitive enteropathy were found over a 9 year period in a specialized University Unit, which is the main reference center for the total population of the island of Crete (approximately 500 000 people).

We do recognize the fact that the size of the studied samples is not large enough to permit us to refute the presence of an association between CD and PBC safely. However, our inability to confirm this association is in agreement with recent data from large Italian series \[[@B13]\], consisted of 336 patients with CD and 65 cases with PBC. In this study, which can be assumed to be representative of the Mediterranean population, only one case of PBC among patients with CD was identified and no case of CD in the group of PBC.

Patients with autoimmune cholangitis were not included in the studies aiming to estimate the risk of CD in patients with PBC. In our small series, no case of CD was found among the 17 patients with autoimmune cholangitis and thus an association could not be established.

A possible limitation of our study may be the relatively low percentage of duodenal biopsies performed in patients with primary biliary cirrhosis tested positive for at least one antibody class. However, this was also the case in studies supporting the association. Our patients were screened for IgA-EMA by indirect immunofluorescence (IIF) test, using both monkey esophagus and human umbilical cord as substrates which have been shown to be highly sensitive and specific indicators of untreated coeliac disease \[[@B16],[@B17]\]. Thus missing a case of CD in our patients with PBC seems rather unlikely. It was previously reported that the development of IgA-EMA depends on the severity of intestinal histopathology of CD and a subgroup of asymptomatic coeliacs negative for AGAs or IgA-EMA will be under-diagnosed, especially those with minor tissue damage \[[@B30]\]. However, none of the studies supporting or failing to demonstrate the association have focused on this aspect.

In our series, 21% of PBC and 35% of AIC patients were tested positive for AGAs. Furthermore, IgA-AGA positivity was significantly more pronounced in patients with Scheuer\'s stage III-IV disease. Floreani et al \[[@B20]\] reported that IgA-AGA were found in 3.4%, whereas both IgA and IgG-AGA in 6.8% of PBC patients. Likewise, Sjoberg et al \[[@B11]\] reported that the occurrence of false-positive AGAs in PBC patients was as high as 16%. We therefore believe, as previously suggested \[[@B11],[@B20],[@B21]\], that the presence of AGAs in PBC and AIC seems to be secondary to liver damage per se and represent a marker of non-specific immune reactivity. Finally, our results confirm the previously reported low specificity of guinea pig liver-derived tTG ELISA in patients with chronic liver disease \[[@B27]\]. We were unable to compare our results with those obtained by a human recombinant tTG ELISA as the latter is not currently commercially available in our country.

Conclusions
===========

We confirmed the previously reported high prevalence of false-positive anti-gliadin and guinea pig liver-derived anti-tissue transglutaminase antibodies in patients with chronic liver disease. We were unable to confirm observations suggesting an over-representation of coeliac disease in patients with primary biliary cirrhosis. Our findings support the idea that in areas of low prevalence of CD, in the absence of clinical suspicion, screening all patients with PBC for coeliac disease should not be performed routinely.

Competing interests
===================

none declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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