Phylogenetic analysis reveals an ancient gene duplication as the origin of the MdtABC efflux pump

Abstract

The efflux pumps from the Resistance-Nodulation-Division family, RND, are main contributors to intrinsic antibiotic resistance in Gram-negative bacteria. Among this family, the MdtABC pump is unusual by having two inner membrane components. The two components, MdtB and MdtC are homologs, therefore it is evident that the two components arose by gene duplication. In this paper, we describe the results obtained from a phylogenetic analysis of the MdtBC pumps in the context of other RNDs. We show that the individual inner membrane components (MdtB and MdtC) are conserved throughout the Proteobacterial species and that their existence is a result of a single gene duplication. We argue that this gene duplication was an ancient event which occurred before the split of Proteobacteria into Alpha-, Beta- and Gamma- classes. Moreover, we find that the MdtABC pumps and the MexMN pump from Pseudomonas aeruginosa share a close common ancestor, suggesting the MexMN pump arose by another gene duplication event of the original Mdt ancestor. Taken together, these results shed light on the evolution of the RND efflux pumps and demonstrate the ancient origin of the Mdt pumps and suggest that the core bacterial efflux pump repertoires have been generally stable throughout the course of evolution.

Fig 1. Analysis of >1000 RND sequences.

Left, an unrooted phylogenetic tree, with bootstrap values represented as branch colors. Leaves are labelled with colors corresponding to their clusters (black represents proteins not belonging to the colored clusters, or singletons). Right, sequence similarity network. Clusters containing more than 10 sequences are colored and given a number. P. aeruginosa and E. coli proteins are marked with thick circles, black and white, respectively. On both panels P. aeruginosa and E. coli proteins are additionally marked with their abbreviations (E. coli proteins underlined).

Fig 2. A phylogenetic tree of the Mdt-like proteins.

The bootstrap values are represented by a branch color as in Fig 1 (the branches with bootstrap support lower than 50 were not collapsed, in order to show the genomic neighborhood of these genes). The taxonomy of each organism is represented with shading of the labels. To the right of the protein and organism names the genomic context is presented. The actual protein at the leaf tip is represented with a filled symbol: a dark red star for “progenitor-like” RNDs, a red star for “true” MexN-like proteins, a blue star for MdtB-like proteins and an orange star for MdtC-like proteins). The open symbols provide the genomic context for the RNDs. For example, in the second row, Thiobacillus denitrificans, the lack of symbols under “MdtABC” means there are no proteins from this group present in this organism. Further to the right under “MexMN”, an open green square means there is an OMP present, followed by an MFP (an open purple triangle), and an RND (a closed star). Lack of symbols under “OMP” and “MFS” means there are no further proteins in this set of genes.

Fig 3. The proposed evolutionary scenario.

The evolution within the Alpha-, Beta- and Gamma- proteobacteria groups is shown, as deduced from the phylogenetic tree in Fig 2 and a timeline of evolution of Proteobacteria (Battistuzzi, Feijao and Hedges 2004). The cladograms lengths and timepoints of evolutionary events are not to scale. As an example of horizontal gene transfer, Cupriavidus pinatubonensis is also shown.