EF0176 and EF0177 from Enterococcus faecalis V583 are substrate-binding lipoproteins involved in ABC transporter mediated ribonucleoside uptake

Abstract

One of the ABC transporter systems in Enterococcus faecalis V583 is encoded by the ef0176-ef0180 gene cluster, which differs from orthologous operons in related bacteria in that it contains two genes putatively encoding substrate-binding proteins (SBPs). These SBPs, EF0176 and EF0177, have previously been identified on the surface of E. faecalis. By phenotypic studies of single and double knockout mutants, we show here that EF0176 and EF0177 are specific for ribonucleosides and, by inference, that the EF0176-EF0180 ABC transporter plays a role in nucleoside uptake. The specificity of the SBPs was mapped using growth experiments on a medium, RPMI 1640, that only supports growth of E. faecalis when supplemented with purine nucleosides or their corresponding bases. This analysis was complemented by studies with toxic fluorinated pyrimidine ribonucleoside analogues and competition experiments. The data show that EF0176 and EF0177 have broad and overlapping, but not identical, substrate specificities and that they, together, are likely to bind and facilitate the transport of all common ribonucleosides. Comparative sequence analysis and inspection of an available crystal structure of an orthologue, PnrA from Treponema pallidum, showed that the strongest binding interactions between the protein and the ligand involve the ribose moiety and that sequence variation in the binding site primarily affects interactions with the base. This explains both the broad substrate specificity of these binding proteins and the observed variations therein. The presence of two SBPs in this nucleoside ABC transporter system in E. faecalis may improve the bacterium's ability to scavenge nucleosides.

Omadacycline Efficacy against Enterococcus faecalis Isolated in China: In Vitro Activity, Heteroresistance, and Resistance Mechanisms

“…Phylogenetic and homology analyses indicated that the efflux protein encoded by OG1RF_RS00630 is an ATPbinding cassette family protein expressed mainly in the cellular membrane. A previous study showed that the ortholog of OG1RF_RS00630 (EF0177) from E. faecalis V583 is involved in ABC transporter-mediated ribonucleoside uptake (30). Our data demonstrate for the first time that OG1RF_RS00630 participates in the development of OMC heteroresistance in E. faecalis.…”

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA

“…The D39∆pnrA strain did not show significant changes in growth behavior compared to the wild-type in THY (data not shown) or in chemically defined medium RPMImodi. Likewise, no changes in growth behavior of homologous proteins in other bacteria were observed after the deletion of RnsB of S. mutans [17] and EF_0176 and EF_0177 of Enterococcus faecalis [32].…”

“…These are e.g. PnrA (TmpC) of T. pallidum [15], RnsB of S. mutans [17], EF_0176 and EF_0177 of Enterococcus faecalis [32], NupN of B. subtilis [5,33] and the substrate binding protein BpmA of L. lactis [16,34]. The transporters or their respective substrate binding proteins exhibit different substrate specificities.…”