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

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.