Bioinformatics analysis of bacterial annexins--putative ancestral relatives of eukaryotic annexins

Abstract

Annexins are Ca(2+)-binding, membrane-interacting proteins, widespread among eukaryotes, consisting usually of four structurally similar repeated domains. It is accepted that vertebrate annexins derive from a double genome duplication event. It has been postulated that a single domain annexin, if found, might represent a molecule related to the hypothetical ancestral annexin. The recent discovery of a single-domain annexin in a bacterium, Cytophaga hutchinsonii, apparently confirmed this hypothesis. Here, we present a more complex picture. Using remote sequence similarity detection tools, a survey of bacterial genomes was performed in search of annexin-like proteins. In total, we identified about thirty annexin homologues, including single-domain and multi-domain annexins, in seventeen bacterial species. The thorough search yielded, besides the known annexin homologue from C. hutchinsonii, homologues from the Bacteroidetes/Chlorobi phylum, from Gemmatimonadetes, from beta- and delta-Proteobacteria, and from Actinobacteria. The sequences of bacterial annexins exhibited remote but statistically significant similarity to sequence profiles built of the eukaryotic ones. Some bacterial annexins are equipped with additional, different domains, for example those characteristic for toxins. The variation in bacterial annexin sequences, much wider than that observed in eukaryotes, and different domain architectures suggest that annexins found in bacteria may actually descend from an ancestral bacterial annexin, from which eukaryotic annexins also originate. The hypothesis of an ancient origin of bacterial annexins has to be reconciled with the fact that remarkably few bacterial strains possess annexin genes compared to the thousands of known bacterial genomes and with the patchy, anomalous phylogenetic distribution of bacterial annexins. Thus, a massive annexin gene loss in several bacterial lineages or very divergent evolution would appear a likely explanation. Alternative evolutionary scenarios, involving horizontal gene transfer between bacteria and protozoan eukaryotes, in either direction, appear much less likely. Altogether, current evidence does not allow unequivocal judgement as to the origin of bacterial annexins.

Figure 1. Sequence logos (weblogo.berkeley.edu) showing amino-acid residue conservation in eukaryotic annexins (upper logo), and bacterial annexins (lower logo).

For the bacterial annexins, alignment from Fig. 3 used.

Figure 2. Domain composition of bacterial annexins.

Domain architectures for bacterial annexins. HMMER3 and HHpred assignments of Pfam domains shown, as well as transmembrane region (TM) predictions. Broader rectangles with gold edges indicate weak similarities to full annexin tetrads (see also Fig. S3). Proteins identified by NCBI gi identifiers, preceded by species acronyms (see Fig. 3 caption).

Figure 3. Multiple sequence alignment (Promals3D) of selected bacterial annexin domains.

Alignment is manually edited in the GxGTDE region, sequence redundancy at 70% identity removed. Alignment columns containing mostly gaps hidden (as marked by blue markers above). Secondary structure prediction shown (Jnet algorithm), red bars represent alpha-helices. The JnetConf histogram represents the confidence of secondary structure prediction at each position. Proteins identified by NCBI gi identifiers preceded by species acronyms: Aa, Aquimarina agarilytica ZC1, Bb, Burkholderiales bacterium HQ_001, Cc, Corallococcus coralloides DSM 2259, Cg, Capnocytophaga gingivalis ATCC 33624, Ch, Cytophaga hutchinsonii ATCC 33406, Fb, Flavobacteriaceae bacterium HQM9, Fi, Fulvivirga imtechensis AK7, Ga, Gemmatimonas aurantiaca T-27, Ho, Haliangium ochraceum DSM 14365, Ko, Kordia algicida OT-1, Mm, Microscilla marina ATCC 23134, Ms, Marinilabilia salmonicolor JCM 21150, Mst, Myxococcus stipitatus DSM 14675, Nm, Nakamurella multipartita DSM 44233, Ri, Rhodococcus imtechensis RKJ300, Sl, Spirosoma linguale DSM 74.

Figure 4. CLANS graph – sequence similarity-based clustering of bacterial annexins and known eukaryotic annexins.

A) very relaxed sequence similarity threshold, B) relaxed sequence similarity threshold, C) strict sequence similarity threshold. Symbols colouring by taxonomy: red – Bacteria, blue - Metazoa, orange - Fungi, cyan - other Opisthokonts, green - plants, magenta - Stramenopiles, brown - Excavata, black - Amoebozoa. 774 representative sequences included The P-value sequence similarity thresholds used for graph building: A) 0.1, B) 1e-3, C) 1e-7.

Figure 5. Phylogenetic tree of 774 representative annexin domain sequences.

Multiple sequence alignment built using the MAFFT program. Phylogeny built using the PhyML algorithm. Branch colouring by taxonomy, as in Fig. 4. Approximate bootstrap values obtained using the aLRT test. Branches with bootstrap values below 0.75 collapsed, dots on branches indicate bootstrap values above 0.9.