Karlskås Ingrid Lea, Saleihan Zhian, Holo Helge, Mathiesen Geir, Eijsink Vincent G H
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway.
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway
Microbiology (Reading). 2015 Apr;161(Pt 4):754-64. doi: 10.1099/mic.0.000045. Epub 2015 Jan 29.
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.
粪肠球菌V583中的一种ABC转运蛋白系统由ef0176 - ef0180基因簇编码，它与相关细菌中的直系同源操纵子不同，因为它包含两个可能编码底物结合蛋白（SBP）的基因。这些SBP，即EF0176和EF0177，先前已在粪肠球菌表面被鉴定出来。通过对单敲除和双敲除突变体的表型研究，我们在此表明EF0176和EF0177对核糖核苷具有特异性，并据此推断EF0176 - EF0180 ABC转运蛋白在核苷摄取中发挥作用。利用在RPMI 1640培养基上的生长实验来确定SBP的特异性，该培养基只有在补充嘌呤核苷或其相应碱基时才支持粪肠球菌生长。通过使用有毒的氟化嘧啶核糖核苷类似物的研究和竞争实验对这一分析进行了补充。数据表明，EF0176和EF0177具有广泛且重叠但不完全相同的底物特异性，并且它们共同可能结合并促进所有常见核糖核苷的转运。对梅毒螺旋体的直系同源物PnrA的可用晶体结构进行比较序列分析和检查表明，蛋白质与配体之间最强的结合相互作用涉及核糖部分，并且结合位点的序列变异主要影响与碱基的相互作用。这既解释了这些结合蛋白广泛的底物特异性，也解释了其中观察到的变异。粪肠球菌的这种核苷ABC转运蛋白系统中存在两个SBP可能会提高该细菌清除核苷的能力。
