Synthesis, uptake and excretion of ectoines in the alpha-proteobacteria Hyphomonas neptunium and Novosphingobium sp. LH128.

A broad range of Bacteria either synthesizes the compatible solutes ectoine and hydroxyectoine or employ specialized transporters for their uptake to ameliorate high osmolarity stress. Through bioinformatics of all sequenced microbial genomes, more than 4000 putative ectoine producing bacteria were recently identified (Czech et al., 2018). A subset of these putative ectoine producers encodes different types of transporters in the direct gene neighborhood of the core biosynthetic genes (ectABCD) for ectoines. Using Hyphomonas neptunium and Novosphingobium sp. LH128 as model organisms, we analyzed the synthesis, uptake and excretion of ectoines as these alphaproteobacteria possess novel types of ect gene clusters that harbor a different set of transporter genes, and in the case of Novosphingobium, also a mscS-like mechanosensitive channel gene. Here we show that H. neptunium harbors a novel sodium solute superfamily importer (EctI) exhibiting a substrate spectrum for a variety of compatible solutes. Novosphingobium sp. LH128, a pure hydroxyectoine producer, employs a major facilitator superfamily importer (EctU) with substrate-specificity for ectoine and hydroxyectoine. We were able to study the putative substrate-binding site of EctI and EctU for the substrates glycine betaine, ectoine and hydroxyectoine by modeling and docking studies, and through site-directed mutagenesis. In case of Novosphingobium sp. LH128, we showed that a knock-out mutant of EctU resulted in an hydroxyectoine secreting strain, highlighting the role of this substrate-specific importer in the recycling of the compatible solute hydroxyectoine. Furthermore, we reveal here for the first time that EctE from Novosphingobium sp. LH128 probably serves as a specific exporter for ectoines. [ABSTRACT FROM AUTHOR]