In bacterial chemotaxis, the signaling effects of ligand binding are counterbalanced by reversible methylation of specific glutamate residues within the cytoplasmic domains of chemoreceptors, resetting the receptor signaling output back to the prestimulus level. While signaling and adaptation have been most extensively characterized in E. coli
and S. enterica
, T. maritima
has emerged as a valuable organism for structural analysis of chemotaxis proteins (2
). In this study we have taken initial steps towards characterizing in vitro methylation of T. maritima
receptors and have identified specific sites within the cytoplasmic domains that can be methylated by methyltransferase CheR in the absence of stimuli.
Including the previously determined deamidation sites (6
) and the methylation and deamidation sites identified here, a total of 19 sites have been identified. Interestingly, the identified methylation and deamidation sites clustered to four regions within the receptors and in many cases were found to be arranged in tandem pairs spaced six to seven residues apart. Sequence alignments of the receptor cytoplasmic domains demonstrated that the first tandem pair (methylation region 2) mapped to a similar location for three of the four receptors (Fig. ). However, a second tandem pair mapped to two different locations. Sites in TM0429 and TM1428 aligned with one another (methylation region 3), but the sites in TM1143 did not (methylation region 4). Mapping the methylation regions onto the crystal structure of the TM1143 cytoplasmic domain (20
) demonstrated that methylation regions 2 and 3 lie spatially near one another, while methylation region 4 of TM1143 is positioned eight helical turns away in the C-terminal direction compared to methylation region 3 (Fig. ).
There were, however, several instances where identified sites were not found in tandem, with the majority of those cases limited to TM1146. In this receptor, all four identified methylation sites (Glu255, Glu284, Glu479, and Glu515) did not have a corresponding site pair. However, for methylation sites Glu479 and Glu515, this finding was similar to what was observed for methylation site Glu281 in TM1143 and Glu506 in TM1428. In those instances, only after alteration of specific glutamines to glutamates (Q274E in TM1143c and Q499E in TM1428c) were the paired deamidation/methylation sites identified (Table ). Analysis of the TM1146 sequence shows two glutamine residues, Gln472 and Gln508, which align perfectly with previously determined deamidation sites from the other T. maritima
receptors (Fig. ), making both Gln472 and Gln508 possible deamidation sites which could potentially form tandem pairs with identified methylation sites Glu479 and Glu515. Chao et al. (6
) identified consecutive deamidation sites (Gln282 and Gln283) in TM1428 that mapped upstream of methylation region 2 (Fig. ). Similarly, methylation site Glu255 in TM1146 was also found to map to this region (methylation region 1). Despite the presence of glutamate pairs (Glu275, 276 and Glu289, 290 in TM1428 and Glu248, 249 in TM1146) flanking these methylation and deamidation sites, none were identified as methylation sites. It should be noted, however, that similar to methylation regions 2 and 3, mapping methylation regions 1 and 4 onto the TM1143c structure showed methylation region 1 to lie spatially near methylation region 4 on the opposite strand (Fig. ). Clustering of the identified sites located within these four methylation regions to two distinct areas of the receptors likely facilitates the accessibility and efficiency with which the modifying proteins (CheB, CheD, and CheR) alter these sites.
Previous studies directly identifying sites of methylation in E. coli
) and S. enterica
) receptors yielded the methylation consensus sequence Glx-Glx
-X-X-Ala-Ser/Thr (modified residue is in bold). Since then, this consensus sequence has been used to predict putative methylation sites in chemoreceptors from other organisms (16
) and as the basis for identification of methylation sites through indirect methods (10
). The lack of direct chemical analyses has precluded assessment of the universality of this consensus sequence.
In this study, methylation sites in T. maritima
receptors were directly identified by utilizing LC-MS/MS. A sequence alignment of the identified methylation and deamidation sites from this study and those recently reported (6
) revealed a novel consensus methylation sequence, Ala/Ser-sm-X-Glx
-X-sm-Ala/Ser (modified residues are in bold; sm represents a small amino acid [Gly, Ala, Ser, or Thr]), that appears as a tandem heptad repeat centered around the Glx-Glu pair and overlaps at the Ala/Ser residue (Fig. ). A previous study analyzing receptor cytoplasmic sequences proposed that the original consensus methylation sequence be extended to include residues to the N-terminal side of the Glx-Glx pair (16
), and this extended conservation appears to be a feature of the T. maritima
consensus as well. Remarkably, only 2 (E284 and E515 in TM1146) of the 19 methylation and/or deamidation sites that were identified in T. maritima
match the E. coli
consensus sequence. Moreover, while methylation of E. coli
receptors occurs strictly at the second Glx residue within the consensus sequence, methylation of T. maritima
receptors occurs primarily at the first Glx within the consensus sequence (Fig. ). Methylation of the first Glx residue within the consensus sequence has also been observed in photoreceptor HtrI of Halobacterium salinarum
). At some T. maritima
methylation and/or deamidation sites, modification occurs at the second Glu residue, and in two cases, both residues of the Glx-Glu pair are modified. No distinguishing features within the local primary sequence of the sites appear to correlate with the choice of the first, second, or both as the site(s) of modification.
FIG. 4. A consensus methylation sequence for T. maritima receptors. A consensus sequence was derived from alignment of the 15 methylation sites and the 4 deamidation sites identified in T. maritima receptor cytoplasmic domains. Residues conserved in all but two (more ...)
In the past decade the determination and publication of sequences of entire genomes have increased exponentially. Currently, 339 genomes have been completed, and a staggering 1,502 genome projects are still in progress (http://www.genomesonline.org
). As more and more genome sequences become available, comparative genomic analyses will serve as an important tool in identifying consensus sequences and mapping putative sites of posttranslational modification in homologous proteins within and among different organisms. Our results comparing the methylation sites in T. maritima
chemoreceptors with previously identified sites in E. coli
and S. enterica
suggest that direct chemical analysis will be required for conclusive identification of sites of modification. The consensus sequence for methylation sites in T. maritima
receptors is distinct from that for E. coli
and S. enterica
receptors. Even within the T. maritima
receptors there are unexpected deviations from the well-conserved T. maritima
consensus sequence. For example, methylation site 1 in TM0429 (Fig. and ) contains a Thr residue in position 1 of the consensus sequence instead of the Glu/Gln residue that is found in all other methylation sites. Because this site does not match the consensus sequence, its identification as a site of methylation would likely have been missed by methods other than direct chemical analysis of the entire receptor domain. When targeted mutagenesis of predicted sites is used to identify sites of posttranslational modification, sites that deviate significantly from the consensus may be missed. Importantly, the subset of sites identified by this route serves to reinforce the starting sequence and perpetuates a consensus sequence that may not be fully inclusive.
We expected that glutamine residues targeted by deamidation enzymes, once deamidated, would subsequently be methylated. Based on predictions and subsequent confirmation (6
) of three sites of deamidation, receptors containing Gln-to-Glu substitutions were constructed and analyzed for methylation. Q499E of TM1428c and Q274E of TM1143c were observed to be methylated, but Q498E of TM1143c was not methylated under in vitro methylation conditions. The lack of methylation at a confirmed deamidation site emphasizes the limitation of using in vitro reactions for identification of modification sites. It is likely that specific in vivo conditions and/or environmental cues may be required for utilization of all modification sites. For example, in Bacillus subtilis
, methylation site selection is coordinated by the interaction of CheY with receptors (14
). The absence of CheY as well as the rest of the chemotaxis proteins, full-length receptors, and chemoeffectors in our assays might explain the lack of observed methylation at Q498E. It is important to note that the methylation sites identified by in vitro modification are likely to represent a minimum subset of sites available for methylation in vivo.
In summary, the methylation sites identified within T. maritima receptors have established a consensus sequence that differs from the previously identified consensus sequence for E. coli and S. enterica receptors. Knowledge of the methylation sites in T. maritima receptors provides an important foundation for structural characterization of different receptor signaling states. Additionally, these findings provide cautionary anecdotal evidence that consensus sequences for posttranslational modifications in one bacterium may not necessarily be applicable to analogous modifications in other species.