Histone-like nucleoid structuring (H-NS) protein silences the beta-glucoside (bgl) utilization operon in Escherichia coli by forming a DNA loop.

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.

Effects of Global and Specific DNA-Binding Proteins on Transcriptional Regulation of the E. coli bgl Operon.

Using reporter gene (lacZ) transcriptional fusions, we examined the transcriptional dependencies of the bgl promoter (Pbgl) and the entire operon regulatory region (Pbgl-bglG) on eight transcription factors as well as the inducer, salicin, and an IS5 insertion upstream of Pbgl. Crp-cAMP is the primary activator of both Pbgl and the bgl operon, while H-NS is a strong dominant operon repressor but only a weak repressor of Pbgl. H-NS may exert its repressive effect by looping the DNA at two binding sites. StpA is a relatively weak repressor in the absence of H-NS, while Fis also has a weak repressive effect. Salicin has no effect on Pbgl activity but causes a 30-fold induction of bgl operon expression. Induction depends on the activity of the BglF transporter/kinase. IS5 insertion has only a moderate effect on Pbgl but causes a much greater activation of the bgl operon expression by preventing the full repressive effects of H-NS and StpA. While several other transcription factors (BglJ, RcsB, and LeuO) have been reported to influence bgl operon transcription when overexpressed, they had little or no effect when present at wild type levels. These results indicate the important transcriptional regulatory mechanisms operative on the bgl operon in E. coli.

Comparative population genomic analyses of transporters within the Asgard archaeal superphylum.

Upon discovery of the first archaeal species in the 1970s, life has been subdivided into three domains: Eukarya, Archaea, and Bacteria. However, the organization of the three-domain tree of life has been challenged following the discovery of archaeal lineages such as the TACK and Asgard superphyla. The Asgard Superphylum has emerged as the closest archaeal ancestor to eukaryotes, potentially improving our understanding of the evolution of life forms. We characterized the transportomes and their substrates within four metagenome-assembled genomes (MAGs), that is, Odin-, Thor-, Heimdall- and Loki-archaeota as well as the fully sequenced genome of Candidatus Prometheoarchaeum syntrophicum strain MK-D1 that belongs to the Loki phylum. Using the Transporter Classification Database (TCDB) as reference, candidate transporters encoded within the proteomes were identified based on sequence similarity, alignment coverage, compatibility of hydropathy profiles, TMS topologies and shared domains. Identified transport systems were compared within the Asgard superphylum as well as within dissimilar eukaryotic, archaeal and bacterial organisms. From these analyses, we infer that Asgard organisms rely mostly on the transport of substrates driven by the proton motive force (pmf), the proton electrochemical gradient which then can be used for ATP production and to drive the activities of secondary carriers. The results indicate that Asgard archaea depend heavily on the uptake of organic molecules such as lipid precursors, amino acids and their derivatives, and sugars and their derivatives. Overall, the majority of the transporters identified are more similar to prokaryotic transporters than eukaryotic systems although several instances of the reverse were documented. Taken together, the results support the previous suggestions that the Asgard superphylum includes organisms that are largely mixotrophic and anaerobic but more clearly define their metabolic potential while providing evidence regarding their relatedness to eukaryotes.

The Transporter Classification Database (TCDB): 2021 update.

The Transporter Classification Database (TCDB; tcdb.org) is a freely accessible reference resource, which provides functional, structural, mechanistic, medical and biotechnological information about transporters from organisms of all types. TCDB is the only transport protein classification database adopted by the International Union of Biochemistry and Molecular Biology (IUBMB) and now (October 1, 2020) consists of 20 653 proteins classified in 15 528 non-redundant transport systems with 1567 tabulated 3D structures, 18 336 reference citations describing 1536 transporter families, of which 26% are members of 82 recognized superfamilies. Overall, this is an increase of over 50% since the last published update of the database in 2016. This comprehensive update of the database contents and features include (i) adoption of a chemical ontology for substrates of transporters, (ii) inclusion of new superfamilies, (iii) a domain-based characterization of transporter families for the identification of new members as well as functional and evolutionary relationships between families, (iv) development of novel software to facilitate curation and use of the database, (v) addition of new subclasses of transport systems including 11 novel types of channels and 3 types of group translocators and (vi) the inclusion of many man-made (artificial) transmembrane pores/channels and carriers.

Expansion of the Major Facilitator Superfamily (MFS) to include novel transporters as well as transmembrane-acting enzymes.

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.