HME, NFE, and HAE-1 efflux pumps in Gram-negative bacteria: a comprehensive phylogenetic and ecological approach

Abstract

The three primary resistance-nodulation-cell division (RND) efflux pump families (heavy metal efflux [HME], nodulation factor exporter [NFE], and hydrophobe/amphiphile efflux-1 [HAE-1]) are almost exclusively found in Gram-negative bacteria and play a major role in resistance against metals and bacterial biocides, including antibiotics. Despite their significant societal interest, their evolutionary history and environmental functions are poorly understood. Here, we conducted a comprehensive phylogenetic and ecological study of the RND permease, the subunit responsible for the substrate specificity of these efflux pumps. From 920 representative genomes of Gram-negative bacteria, we identified 6205 genes encoding RND permeases with an average of 6.7 genes per genome. The HME family, which is involved in metal resistance, corresponds to a single clade (21.8% of all RND pumps), but the HAE-1 and NFE families had overlapping distributions among clades. We propose to restrict the HAE-1 family to two phylogenetic sister clades, representing 41.8% of all RND pumps and grouping most of the RND pumps involved in multidrug resistance. Metadata associated with genomes, analyses of previously published metagenomes, and quantitative Polymerase Chain Reaction (qPCR) analyses confirmed a significant increase in genes encoding HME permeases in metal-contaminated environments. Interestingly, and possibly related to their role in root colonization, genes encoding HAE-1 permeases were particularly abundant in the rhizosphere. In addition, we found that the genes encoding these HAE-1 permeases are significantly less abundant in marine environments, whereas permeases of a new proposed HAE-4 family are predominant in the genomes of marine strains. These findings emphasize the critical role of the RND pumps in bacterial resistance and adaptation to diverse ecological niches.

Keywords: RND; antibiotic resistance; efflux pumps; metal resistance; phylogeny; plant.

Figure 1

Phylogenetic tree from 83 reference RND permeases; sequences of the three families HAE-1, NFE, and HME were obtained from the Transport Classification Database (http://www.tcdb.org/; in January 2021); the color of the sequence names refers to the RND family indicated in TCDB (red for the HME-1 family, blue for HAE-1, and green for NFE); known substrates supported are indicated: a few antibiotics (AB), MDR, heavy metals (HM), organic components (OC), or not determined (ND); substrates written in bold type are outliers with respect to their position in the clades; numbers on tree branches report bootstrap results (1000 replicates); because Clade C slightly disturbs the tree by decreasing some bootstrap values, this clade was excluded for the phylogenetic reconstruction and then placed it as an indication on the tree.

Figure 2

Phylogenetic tree from 6205 RND permeases; sequences of the three families HAE-1, NFE, and HME were obtained from 920 reference proteomes of Gram-negative bacteria (UniProt, release 2015_09); colored dots indicate positions of the 83 reference RND permeases described in Fig. 1; the color of the dots refers to the RND family indicated in TCDB (red for the HME-1 family, blue for HAE-1, and green for NFE); a single ancestral duplication probably occurred in Clade B (node in orange).

Figure 3

RND gene diversity in function of strain origin; heat map showing the proportion of the RND clades in function of strain habitat from our genomic database; the UC group corresponds to sequences UnClassified among the various clades described.

Figure 4

Proportion of genes encoding RND of HAE-1 (Clades A and B) and HAE-4 (Clades G and H) as a function of salinity; the dotted line indicates the number of RND permease genes per Gram-negative genome; results are indicated as mean value ± standard deviation (n = 3); asterisk indicates a significant difference (P < .05; Wilcoxon test); metagenome data from Tee et al. [30].