The broad distribution, functional versatility, and modular assembly of the energy-coupling module-dependent transport systems are summarized in Fig. and Fig. S2 in the supplemental material. The ECF transporters form a novel class of membrane transporters that can be classified into two groups: group I includes transporters from diverse microbial lineages (170 species) that have a dedicated AT module encoded in the same gene cluster as an S component, and transporters of group II (a total of 459 transporters in 91 species) employ a universal energy-coupling module (EcfAA′T) that is encoded by a separate gene cassette and shared by many different unlinked S components. Group II is ubiquitous in the phyla Firmicutes and Thermotogales and also occurs in some members of the Archaea.
The S components identified in this work could be classified into at least 20 protein families that correspond to different substrate specificities (Table ). Most of them are integral membrane proteins of comparable sizes (155 to 230 residues) that have six predicted transmembrane domains. The NikM and CbiM proteins, which form a single family in the Pfam database, are larger (210 to 250 residues) and are predicted to have seven transmembrane domains. Apart from the CbiM/NikM family, only five other S-component families (BioY, MtsT, HtsT, QueT, and ThiT) are present in the Pfam database, and all of them are annotated as hypothetical membrane proteins. Sequence comparisons of representative S components from 18 families revealed very little overall pairwise identity between the proteins from different families (see Table S3 in the supplemental material). A detailed phylogenetic comparison will be needed to establish whether different S components are related.
The ECF transporters identified in this study are mechanistically unique. Their substrate specificity is mediated by integral membrane proteins (S components), which form active transporters in the presence of the energy-coupling AT module. The stoichiometry of the transporter components is unknown, but domain fusions in various ECF transport systems give some clues. First, the nik, cbi, and bio gene cassettes encode a single A component (ATPase), but as noted above, dual A components are more common. Second, in some cases, the two A components are fused (Fig. and ). Third, rare fusions of transporter components include two “SAA” fusions, four “TAA” fusions, and one “ST” fusion in the Archaea, the Chloroflexi, and the Actinobacteria (Fig. and see Table S1 in the supplemental material). On this basis, and because shared EcfAA′T components and specific S components formed quadripartite complexes (Fig. ), we propose a quadripartite model in which the S component binds and translocates the substrate across the membrane. The translocation process is coupled to ATP hydrolysis mediated by an AT module that contains two ATPase domains and one transmembrane T component.
How do ECF transporters relate to the ABC transporter superfamily? The latter transporters couple ATP hydrolysis to substrate uptake or efflux (3
). ABC importers and exporters share a four-component architecture comprised of two transmembrane and two ATP-hydrolyzing domains. Prokaryotic ABC importers have additional extracytoplasmic soluble proteins that mediate substrate binding and delivery to the respective transmembrane components. Fundamental differences between ECF transporters and classical ABC importers include (i) the absence of extracytoplasmic substrate binding proteins and their replacement by integral membrane proteins and (ii) the shared use of energy-coupling AT modules by many highly diverse S components. Such sharing is occasionally seen in classical ABC transporters, but it always involves very similar substrates and substrate binding proteins (17
). A less fundamental but nonetheless marked characteristic of ECF transporters is a predilection for vitamins.
Finally, as noted at the outset, numerous human pathogens such as Mycoplasma
, and Streptococcus
strains rely totally upon transporters to obtain vitamins and other essential metabolites due to the absence of the corresponding de novo biosynthetic pathways. Many of these microorganisms use ECF transporters, and indeed, certain ecf
genes have been found to be essential for the growth and survival of Streptococcus pneumoniae
and Mycoplasma genitalium
). All components of ECF transporters, especially the unique S and T proteins, are thus potential targets for antibiotic development. In fact, the centrality of the T component to the uptake of multiple compounds makes it a classic Achilles' heel.