and CC-LRR have an energetically favorable ‘‘knobs-into-holes’’ interface while the superposition of LRRs from the other analyzed subfamilies with the CC-LRR results in ‘‘knobs-into-knobs’’ packing with steric tensions and voids (Figure 3). For example, the RI-LRR and CC-LRR, PS-LRR and CC-LRR, and SDS22- LRR and CC-LRR interfaces have distances between Ca and (or) Cb atoms of 2.1-2.7 A˚ that are 0.5–1.1 A˚ closer than normally allowed limits for such distances [11]. This steric tension could be alleviated by a deformation of the LRR b-structure, but the distortion of the b-structural H-bonds would eventually also lead to the loss in the structure stability. The superposition of the typical LRR and CC-LRR domains does not lead to such close contacts, however, it results in an energetically unfavorable ‘‘knobs-into-knobs’’ packing with voids (Figure 3). Thus, our analysis suggests that some LRRs with different sequence motifs have an energetically favorable (‘‘permissive’’) packing, while simultaneous occurrence of the other ones in the same structure results in unfavorable (‘‘mutually exclusive’’) packing. The permissive packing of repeats with different consensus sequences may serve as a criterion for their membership in the same subfamily, at the same time as the mutually exclusive packing defines the boundaries between the LRR subfamilies. It is worth mentioning that GALA-LRRs are erroneously assigned to the RI-LRR subfamily in the annotation of protein databases on the NCBI Web site (http://www.ncbi.nlm.nih.gov/ entrez). In order to dissipate any doubt, Figure 2B displays the apparent difference of GALA-LRR and RI-LRR through the backbone superposition. Inferring origin of R. solanacearum GALA proteins GALA F-box domains are functionally related to plant F-box domains [4]. Although some bacteria have a proteasome-like Figure 3. Side-by-side packing of LRRs having different consensus sequence motifs. ‘‘Knobs-into-holes’’ packing of GALA- LRRs against CC LRRs (contoured by a thin line box) and ‘‘knobs-into- knobs’’ packing of PS-LRRs, RI-LRRs, SDS22-LRRs, typical LRRs, bacterial LRRs against CC-LRRs. The CC-LRR-domain is in blue color. The side- chains that are involved in the inter-domain packing are shown by ball- and-stick representation. Arrows point at the clash sites where Ca and (or) Cb atoms have distances between of 2.1–2.7 A˚ . doi:10.1371/journal.pone.0001694.g003 Evolution of GALA Proteins compartmentalized protease system [12], they do not have an ubiquitin-dependent protein degradation system like in eukaryotes. Still several bacteria have in their genome typical eukaryotic E3 ubiquitin ligase-like proteins among which F-box proteins, like the GALA proteins from R. solanacearum [13]. These bacterial F-box proteins also often contain eukaryote-like protein-protein interac- tion domains like LRR, ankyrin and WD40. We systematically searched all the sequenced eubacterial genomes available (353 genomes available through TIGR Comprehensive Microbial Resource, release 24.0) for the presence of the F-box domain (automatic search with Pfam Hiden Markov Model for F-box (PF00646) service available at TIGR CMR). We only found F-box domains present in one chlamydiae species out of 11 complete sequence available (Candidatus Protochlamydia amoebophila strain UWE25) and in 9 proteobacteria out of 184 complete sequences available (alphaproteobacteria: Mesorhizobium loti, Agrobacterium tumefaciens; betaproteobacterium: Ralstonia solana- cearum, gammaproteobacteria: Pseudomonas syringae, Sodalis glossini- dius, Coxiella burnetii, legionella pneumophila, Xanthomonas campestris and X. axonopodis). All these positive hits correspond indeed to the presence of a canonical F-box domain. The evidence for functional F-box domains is available for both A. tumefaciens and R. solanacearum F-box containing proteins [13,14]. A few low scoring hits (in proteins from Borrelia burgdorferi, B. garinii, Chlamydophila caviae, Rhizobium etli, Salmonella tiphimurium and Streptococcus pneumoniae) were inspected and clearly ruled out as being F-box domains (by constraints in primary sequence, see e.g. [15]). Within the proteobacteria phylum, 9 out of 184 completely sequenced bacteria clearly contain at least one F-box-containing predicted protein. Among the 175 negatively scoring bacteria, we believe we can rule out the presence of ‘‘remnants’’ of F-box domain, which could have been indicative of gene loss. Considering such sporadic presence of this F-box domain, the scenario of systematic gene loss appears very unlikely The F-box domain has its only described function in eukaryotic cells and is overrepresented in this kingdom (interpro F-box domain (IPR001810) hits: 735 in A. thaliana, 428 in Caenorhabditis elegans, 120 in humans, and only 46 hits among all bacteria sequence available, mostly in proteobacteria, see above). It is interesting to mention that all the bacteria containing F-box domains in their genome intimately interact with eukaryotes. For example, P. amoebophila, S. glossinidius and M. loti are symbionts of amoeba, insects and plants; A. tumefaciens, R. Solanacearum, P. syringae, X. campestris and X. axonopodis are plant pathogens and C. burnetii and L. pneumophila are human pathogens. Finally, for several of these F-box-containing bacterial proteins injection into their host cells (via specialised bacterial secretion systems) has been proven (M. loti, A. tumefaciens, R. solanacearum) [16–18] or predicted (L. pneumophilae) [8]. Among the seven GALA genes from the R. solanacearum genome (strain GMI1000, (http://bioinfo.genopole-toulouse.prd.fr/ annotation/iANT/bacteria/ralsto/), GALA1(RSp0914) is located in an alternative codon usage region, GALA2(RSp0672) is flanked by a region duplicated elsewhere in the genome and GA- LA3(RSp0028) is flanked at either side by an alternative codon usage region. These genomic characteristic have been previously identified as potential signatures of LGT [19,20]. Furthermore, considering the capacity of R. solanacearum to uptake DNA [21], it is natural to suggest a lateral gene transfer (LGT) from host plant DNA that gave rise to the F-box domain (and possibly the LRRs) of the GALA proteins. One way of testing such a hypothesis is through phylogenetic analysis of the protein origins to identify putative donor for a PLoS ONE | www.plosone.org 4 February 2008 | Volume 3 | Issue 2 | e1694