GALA-LRR) have a specific consensus pattern with characteristic differences from the previously described consensus sequences of LRR subfamilies, especially from the known bacterial LRR subfamilies. On the other hand, among the LRR subfamilies that are closest to R. solanacearum GALA-LRRs there is the Cysteine- Containing LRR (CC-LRR) subfamily of plant, animal and fungi proteins which can also contain the F-box domains and, therefore, may have a similar function. Thus, it was not clear, whether the GALA-LRR proteins are members of the CC-LRR subfamily or they should be assigned to a new LRR subfamily. Here we clarify this ambiguous case by using sequence analysis and molecular modeling. We also focus our analysis on the origin and evolution of GALA proteins from R. solanacearum. Results and Discussion Sequence analysis of GALA LRRs Analysis of F-box containing GALA proteins from Ralstonia solanacearum shows that their 24-residue long LRRs have a specific consensus pattern that has characteristic differences (Figure 1) from the previously described LRRs [1,3]. Comparison of GALA- LRRs with the other known 24-residue LRRs such as typical LRRs, PS-LRRs shows that GALA-LRRs frequently have Ile instead of Leu in position 5, Gly or Ala instead of Leu in position 9, Ala instead of Pro in position 10, and do not have a conserved Leu in position 16. The GALA-LRR consensus motif also has some differences with the 26-residue CC-LRR motif. For example, positions 3 and 16 of the GALA-LRR motif do not have a conserved Cys and position 6 is frequently occupied by Gly instead of Thr. Using the generalized profile technique [7], a sensitive method for sequence database searches, we found the GALA-LRR type of repeats in about 40 proteins including proteins of the cucurbit crops pathogenic b-proteobacterium Acidovorax avenae subsp. Citrulli, the human pathogen, and c-proteobacterium Legionella pneumophila [8], as well as in the aquatic planctomycete bacterium Gemmata sp. Wal 1. These proteins, unlike R. solanacearum’s GALA proteins, don’t contain an F-box domain. Sometimes their entire sequence corresponds to the LRR domain (Figure 1A). Some proteins have LRRs that are similar to GALA-LRR (GALA-like or GL-LRR hereafter), however, their consensus sequence has several charac- teristic differences from GALA-LRRs such as Val instead of Ile in position 5, Leu instead of Ala in position 10, presence of conserved Leu in position 16 (Figure 1B). Remarkably, isolated examples of GALA-LRR are found in GL-LRR domains of two F-box containing proteins from plants (Figure 1A). Sequence database searches with generalized profiles revealed GL-LRRs in more than a hundred LRR proteins. Among them are plant proteins, and also proteins from bacteria (Gemmata sp. Wa1-1, Parachlamydia sp., Legionella pneumophila, Rhodopirellula baltica), protists (Entamoeba histolytica, Leishmania, Trypanosoma, Dictyostelium) and animals (Danio, Tetraodon, Drosophila, Anopheles gambiae, Xenopus, Strongylo, and Homo sapiens). Interestingly, some of the GL-LRR proteins from plant, animal and protista also contain F-box domains. Place of GALA-LRRs and GL-LRRs in the classification of LRR proteins Although, the newly identified GALA-LRRs and GL-LRRs do not perfectly fit any of the previously described consensus sequences of seven LRR subfamilies [1,3], they have some similarities in the consensus sequences with CC-LRRs (Figure 1B). In particular, a characteristic ITD-motif of CC-LRRs (positions 5 to 7) is aligned with similar Igd- and Vtd-motifs of GALA- and GL-LRRs respectively. Furthermore, conserved Evolution of GALA Proteins Figure 1. Specific consensus patterns of GALA-LRRs and GL- LRRs. (A) An arrangement of LRRs (rhombs), F-boxes (rectangles) and BTB domain (ellipse) within new representative proteins of CC-LRR subfamily. The following proteins are shown: GALA4 from R. Solanacearum, strain GMI1000 GenBank accession number CAD15502; GALA protein from Legionella pneumophila subsp.pneu- mophila str. Philadelphia 1, AAU27032; hypothetical protein from Arabidopsis thaliana, AAF82144; putative regulatory subunit from Gemmata sp. Wa1-1, AAX07517; hypothetical protein from Parachla- mydia sp. UWE25, CAF23996; hypothetical protein from Parachlamydia sp. UWE25, CAF24006. (B) Alignment of some known (indicated by *) and newly identified LRR consensus sequences. (C) A consensus sequences of an updated CC-LRR. The boxes over the alignment outline known or putative a-helical and b-structural regions. Bold uppercase and lowercase letters indicate more than 60% and 20% identity, correspondingly. ‘‘O’’ denotes an apolar residue, ‘‘x’’ denotes any residue, ‘‘-‘‘ is a position of a gap. Numbers below the GALA-LRR consensus sequence show the positions of conserved residues (see also Figure 2A). doi:10.1371/journal.pone.0001694.g001 apolar residues in positions 5, 10, 13, 19, 22 and 24 of the 24- residue-long GALA- and GL-LRRs can be aligned to the 26- residue-long CC-LRRs by deleting a residue in each of the two connecting loop regions of the CC-LRRs (Figure 1B). These loop regions are known to be the most accommodative for such length differences. Interestingly, many of the CC-LRR proteins, similarly PLoS ONE | www.plosone.org 2 February 2008 | Volume 3 | Issue 2 | e1694