Transducer evolution could be explained by a scenario different from the

Transducer evolution could be explained by a scenario different from

the insertion/deletion (indel)-hypothesis

In the TM region-containing Htrs, as well as in McpB, the GDL-motif proximal to the coiled coil localizes to a position which would correspond to a "theoretical heptad -26", whereas in Tar and Tsr the GDL-motif localizes to a "theoretical heptad -22" (the term theoretical is used to indicate that the described repeat pattern of hydrophobic residues is not continued up to these positions). The enterobacterial HAMP region is therefore located 4 heptads or 28 residues closer to the hairpin turn than that of Htrs or McpB. Le Moual and Koshland (1996) propose that class III transducers, like Htrs or McpB, evolved to class I transducers, like Tar or Tsr, by deletions of four 14-residue-insertion/deletion (indel)-regions within the coiled-coil domain. Two of these regions are proposed to have been deleted between the signaling region and the methylation regions, and the other two between the methylation regions and the HAMP region and the C- terminus, respectively (Fig. 2.6). The concomitant presence of the well-conserved Htr methylation site within heptad -12, and of a reasonably well-conserved site in heptad +17, raises doubts about the indel-hypothesis.

When applying this hypothesis to a hypothetical class III transducer with methylation sites within these two heptads (which is the case in some of the Htrs), a rather complicated scenario would have to be assumed for its conversion to a class I transducer of the enterobacterial type. The assumed four 14-residue deletions would produce a hypothetical class I transducer with methylation sites in heptads -10 and +15. A subsequent development into an enterobacterial transducer would require the loss of both methylation sites and the creation of new sites within neighboring heptads, one of them in heptad -12, i. e. at the same distance from the signaling region as the original site in the class III transducer. The conversion of a class III transducer to a class I transducer would therefore require (i) two pairs of concomitant deletion events within the transducer gene, with each pair producing a loss of exactly two times 14 amino acids at approx. equidistant positions N-terminally and C-terminally of the hairpin turn (which does not destroy

but only shortens the coiled coil), (ii) the loss of the displaced original methylation sites and (iii) the development of several new methylation sites, which in each case would require several mutations to produce a methylation consensus sequence. The same scenario is valid when taking McpB as the original class III transducer.

In the face of such a complicated sequence of events, one is tempted to think of a more simple mechanism, which could explain not only the generation of the different "classes", defined by Le Moual and differing primarily in the lengths of their predicted coiled-coil regions, but also that of the different groups of Htrs.

In Fig. 3.31 a simple scenario is proposed, by which the different transducer classes and the different groups of Htrs could be derived from a hypothetical ancestral transducer. The scenario

Figure 3.31 Hypothetical scenario for the generation of class I and class III transducers from a proposed common ancestor.

Transducers are depicted schematically with their different regions highlighted: green, TM region; orange, HAMP region; dark grey, predicted coiled-coil region; red, highly conserved signaling region containing the central hairpin turn; hatched, region proposed to be originating from a duplication of the N-terminal part of the coiled-coil region. New transducers are suggested to result from deletions (-) within the transducer of origin, from insertions (+), or from a replacement of their N-terminal sequences (-,+). In the generated transducers, a ∆ marks the deleted region and a dotted box indicates either the inserted or the newly added N-terminal region. A possible set of methylation sites within the proposed ancestral transducer is indicated by the corresponding heptad numbers. For the other transducers heptad numbers are depicted for all putative and identified methylation sites. Transducers listed in parentheses show a slightly different set of methylation sites when compared with the corresponding depicted transducer(s), but their overall topology is very similar, with the following exceptions: Htrs 7 and 17 contain only 3 TM regions and Htr1 lacks the extracellular region between the TM regions. All transducers are proposed to have been generated by a single primary genetic event, as described by steps A to G in the schematic, which was then followed by several additional mutations leading to the loss of previous methylation sites, the generation of new ones (blue numbers), to changes in the C-terminal transducer part (like a trimming of the coiled-coil region, or shortening or elongation of the C-terminal region), and to changes within the putative N-terminal ligand-binding region.

assumes that each of the depicted transducer groups was generated by a primary genetic event, leading to either the loss of a single transducer region, the integration of an additional stretch of amino acids at a single location within the transducer, or the replacement of the N-terminal transducer region, either with a region originally encoded by a different orf, or by a longer version of the original N-terminal Htr region. As the primary event preserves the inner region of the coiled coil and thereby at least some and possibly all of the methylation sites at their original position, the adaptation mechanisms are expected to remain functional.

The transducers produced by the primary event are expected to have been further modified in an evolutionary process of functional optimization. For each transducer, such a process presumably included one or more of the following modifications: (i) the loss of some of the original and (ii) the generation of new methylation sites, (iii) the loss of the C-terminal part of the hydrophobic heptad pattern in response to the loss of the N-terminal counterpart (which is assumed to have occurred in the course of Tsr and Tar generation), (iv) a partial loss of the C- terminal part of the transducer (as in the case of the soluble Htrs), (v) an elongation of the C- terminal part (as seen for Htr14), (vi) changes in the extracellular domain leading to different ligand-binding specificities and (vii) minor sequence modifications throughout the whole transducer.

The described scenario is not meant to define the detailed sequence of evolutionary events in the course of transducer development. It rather intends to demonstrate that transducers might have evolved by single primary genetic events, instead of the two double events proposed by the indel-hypothesis. It is proposed that the primary events affected the region N-terminal of the first methylation region, and were followed by additional minor modifications, which could, for example, have lead to the formation of a new pattern of methylation sites. The analysis of Htr methylation in the course of this study has shown, that the methylation site-containing region of the presumed coiled coil does not only span 3 to 4 heptads as in the case of McpB and Tsr, respectively, but rather up to 6 (or even 7, if the deamidation in heptad -11 of Htr4 actually leads to a methylation at this site). The presence of methylation sites at different locations within such an extended part of the transducers seems to support the assumption, that a repositioning of methylation sites can take place in the aftermath of some primary genetic events, and has occurred without the interim loss of the transducers' functions and adaptive capabilities.