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The bgl operon of Escherichia coli encodes proteins mediating the metabolism of aromatic beta-glucosides, but the operon is silent in wild type cells. Insertion of an insertion sequence (IS) element in the regulatory region upstream of the bgl promoter activates expression of the operon. The repression mechanism involves the histone-like nucleoid structuring (H-NS) protein with two DNA binding sites, one in the region upstream of the promoter, and the other within the first structural gene of the operon, bglG. The detailed mechanism of repression is not well understood. Here, we first show two terminators flanking bglG are not required for bgl operon silencing. Instead, several lines of experimental evidence clearly suggest that the silencing mechanism involves looping of the DNA between H-NS's two DNA binding sites. H-NS is known to preferentially bind to AT-rich curved DNA, and such regions are found in the vicinity of both sites. We show that strong repression is abolished by (1) preventing H-NS self-oligomerization while retaining DNA binding, (2) preventing or reducing H-NS binding to the bgl operon regulatory region, and (3) preventing or reducing H-NS binding to the binding site in the bglG gene. We also show that the phase of the DNA between these two binding sites is not important, and that large insertions of DNA in the putative loop region do not diminish repression. These results imply that H-NS depends on DNA looping to exert strong repression.

The Major Facilitator Superfamily (MFS) is currently the largest characterized superfamily of transmembrane secondary transport proteins. Its diverse members are found in essentially all organisms in the biosphere and function by uniport, symport, and/or antiport mechanisms. In 1993 we first named and described the MFS which then consisted of 5 previously known families that had not been known to be related, and by 2012 we had identified a total of 74 families, classified phylogenetically within the MFS, all of which included only transport proteins. This superfamily has since expanded to 89 families, all included under TC# 2.A.1, and a few transporter families outside of TC# 2.A.1 were identified as members of the MFS. In this study, we assign nine previously unclassified protein families in the Transporter Classification Database (TCDB; http://www.tcdb.org) to the MFS based on multiple criteria and bioinformatic methodologies. In addition, we find integral membrane domains distantly related to partial or full-length MFS permeases in Lysyl tRNA Synthases (TC# 9.B.111), Lysylphosphatidyl Glycerol Synthases (TC# 4.H.1), and cytochrome b561 transmembrane electron carriers (TC# 5.B.2). Sequence alignments, overlap of hydropathy plots, compatibility of repeat units, similarity of complexity profiles of transmembrane segments, shared protein domains and 3D structural similarities between transport proteins were analyzed to assist in inferring homology. The MFS now includes 105 families.