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The bglGFB operon in Escherichia coli K-12 strain BW25113, encoding the proteins necessary for the uptake and metabolism of β-glucosides, is normally not expressed. Insertion of either IS1 or IS5 upstream of the bgl promoter activates expression of the operon only when the cell is starving in the presence of a β-glucoside, drastically increasing transcription and allowing the cell to survive and grow using this carbon source. Details surrounding the exact mechanism and regulation of the IS insertional event remain unclear. In this work, the role of several DNA-binding proteins in how they affect the rate of insertion upstream of bgl are examined via mutation assays and protocols measuring transcription. Both Crp and IHF exert a positive effect on insertional Bgl+ mutations when present, active, and functional in the cell. Our results characterize IHFs effect in conjunction with other mutations, show that IHFs effect on IS insertion into bgl also affects other operons, and indicate that it may exert its effect by binding to and altering the DNA conformation of IS1 and IS5 in their native locations, rather than by directly influencing transposase gene expression. In contrast, the cAMP-CRP complex acts directly upon the bgl operon by binding upstream of the promoter, presumably altering local DNA into a conformation that enhances IS insertion.

Wildtype Escherichia coli cells cannot grow on L-1,2-propanediol, as the fucAO operon within the fucose (fuc) regulon is thought to be silent in the absence of L-fucose. Little information is available concerning the transcriptional regulation of this operon. Here, we first confirm that fucAO operon expression is highly inducible by fucose and is primarily attributable to the upstream operon promoter, while the fucO promoter within the 3'-end of fucA is weak and uninducible. Using 5'RACE, we identify the actual transcriptional start site (TSS) of the main fucAO operon promoter, refuting the originally proposed TSS. Several lines of evidence are provided showing that the fucAO locus is within a transcriptionally repressed region on the chromosome. Operon activation is dependent on FucR and Crp but not SrsR. Two Crp-cAMP binding sites previously found in the regulatory region are validated, where the upstream site plays a more critical role than the downstream site in operon activation. Furthermore, two FucR binding sites are identified, where the downstream site near the first Crp site is more important than the upstream site. Operon transcription relies on Crp-cAMP to a greater degree than on FucR. Our data strongly suggest that FucR mainly functions to facilitate the binding of Crp to its upstream site, which in turn activates the fucAO promoter by efficiently recruiting RNA polymerase.

Allantoin is a good source of ammonium for many organisms, and in Escherichia coli it is utilized under anaerobic conditions. We provide evidence that allantoinase (AllB) is allosterically activated by direct binding of the allantoin catabolic enzyme, glycerate 2-kinase (GlxK) in the presence of glyoxylate. Glyoxylate is known to be an effector of the AllR repressor which regulates the allantoin utilization operons in E. coli. AllB has low affinity for allantoin, but its activation by GlxK leads to increased affinity for its substrate. We also show that the predicted allantoin transporter YbbW (re-named AllW) has allantoin specificity and the protein-protein interaction with AllB. Our results show that the AllB-dependent allantoin degradative pathway is subject to previously unrecognized regulatory mechanisms involving direct protein-protein interactions.

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 cryptic β-glucoside GFB (bglGFB) operon in Escherichia coli (E. coli) can be activated by mutations arising under starvation conditions in the presence of an aromatic β-glucoside. This may involve the insertion of an insertion sequence (IS) element into a "stress-induced DNA duplex destabilization" (SIDD) region upstream of the operon promoter, although other types of mutations can also activate the bgl operon. Here, we show that increased expression of the bglG gene, encoding a well-characterized transcriptional antiterminator, dramatically increases the frequency of both IS-mediated and IS-independent Bgl+ mutations occurring on salicin- and arbutin-containing agar plates. Both mutation rates increased with increasing levels of bglG expression but IS-mediated mutations were more prevalent at lower BglG levels. Mutations depended on the presence of both BglG and an aromatic β-glucoside, and bglG expression did not influence IS insertion in other IS-activated operons tested. The N-terminal mRNA-binding domain of BglG was essential for mutational activation, and alteration of BglG's binding site in the mRNA nearly abolished Bgl+ mutant appearances. Increased bglG expression promoted residual bgl operon expression in parallel with the increases in mutation rates. Possible mechanisms are proposed explaining how BglG enhances the frequencies of bgl operon activating mutations.

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YcaN, YbhD, YgfI, YiaU, YneJ. We show that: (1) YbdO (tentatively re-named CitR) regulation has an effect on bacterial growth at low pH with citrate supplementation. CitR is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); (2) YgfI (tentatively re-named DhfA) activates the dhaKLM operon that encodes the phosphotransferase system, DhfA is involved in formate, glycerol and dihydroxyacetone utilization; (3) YiaU (tentatively re-named LpsR) regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase and resistance to oxacillin microaerobically; (4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of L-glutamate and putrescine as nitrogen/energy sources; and (5) YbhD and YcaN regulate adjacent y-genes on the genome. We have thus established the functions for four LTFs and identified the target genes for three LTFs.

Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, the analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. The inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine, inosine, or guanosine as the nitrogen source. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively.

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.

For decades it has been known that infectious agents including pathogenic protozoans, bacteria, and viruses, adapted to a particular animal host, can mutate to gain the ability to infect another host, and the mechanisms involved have been studied in great detail. Although an infectious agent in one animal can alter its host range with relative ease, no example of a plant virus changing its host organism to an animal has been documented. One prevalent pathway for the transmission of infectious agents between hosts involves ingestion of the flesh of one organism by another. In this article we document numerous examples of viral and prion diseases transmitted by eating animals. We suggest that the occurrence of cross-species viral epidemics can be substantially reduced by shifting to a more vegetarian diet and enforcing stricter laws that ban the slaughter and trade of wild and endangered species.

The prokaryotic phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) concomitantly transports and phosphorylates its substrate sugars. In a recent publication, we provided evidence that protein-protein interactions of the fructose-specific integral membrane transporter (FruAB) with other PTS sugar group translocators regulate the activities of the latter systems in vivo and sometimes in vitro. In this communication, we examine the consequences of the overexpression of several different transport systems on the activities of selected PTS and non-PTS permeases. We report that high levels of these transport systems enhance the in vivo activities of several other systems in a fairly specific fashion. Thus, (1) overexpression of ptsG (glucose porter) selectively enhanced mannitol, N-acetylglucosamine, and 2-deoxyglucose (2DG) uptake rates; (2) overexpression of mtlA (mannitol porter) promoted methyl α-glucoside (αMG) and 2DG uptake; (3) manYZ (but not manY alone) (mannose porter) overexpression enhanced αMG uptake; (4) galP (galactose porter) overexpression enhanced mannitol and αMG uptake; and (5) ansP (asparagine porter) overexpression preferentially enhanced αMG and 2DG uptake, all presumably as a result of direct protein-protein interactions. Thus, it appears that high level production of several integral membrane permeases enhances sugar uptake rates, with the PtsG and ManXYZ systems being most consistently stimulated, but the MtlA and NagE systems being more selectively stimulated and to a lesser extent. Neither enhanced expression nor in vitro PEP-dependent phosphorylation activities of the target PTS systems were appreciably affected. The results are consistent with the suggestion that integral membrane transport proteins form an interacting network in vivo with physiological consequences, dependent on specific transporters and their concentrations in the membrane.

The SLC22 family of OATs, OCTs, and OCTNs is emerging as a central hub of endogenous physiology. Despite often being referred to as "drug" transporters, they facilitate the movement of metabolites and key signaling molecules. An in-depth reanalysis supports a reassignment of these proteins into eight functional subgroups, with four new subgroups arising from the previously defined OAT subclade: OATS1 (SLC22A6, SLC22A8, and SLC22A20), OATS2 (SLC22A7), OATS3 (SLC22A11, SLC22A12, and Slc22a22), and OATS4 (SLC22A9, SLC22A10, SLC22A24, and SLC22A25). We propose merging the OCTN (SLC22A4, SLC22A5, and Slc22a21) and OCT-related (SLC22A15 and SLC22A16) subclades into the OCTN/OCTN-related subgroup. Using data from GWAS, in vivo models, and in vitro assays, we developed an SLC22 transporter-metabolite network and similar subgroup networks, which suggest how multiple SLC22 transporters with mono-, oligo-, and multi-specific substrate specificity interact to regulate metabolites. Subgroup associations include: OATS1 with signaling molecules, uremic toxins, and odorants, OATS2 with cyclic nucleotides, OATS3 with uric acid, OATS4 with conjugated sex hormones, particularly etiocholanolone glucuronide, OCT with neurotransmitters, and OCTN/OCTN-related with ergothioneine and carnitine derivatives. Our data suggest that the SLC22 family can work among itself, as well as with other ADME genes, to optimize levels of numerous metabolites and signaling molecules, involved in organ crosstalk and inter-organismal communication, as proposed by the remote sensing and signaling theory.

The universal triple-nucleotide genetic code is often viewed as a given, randomly selected through evolution. However, as summarized in this article, many observations and deductions within structural and thermodynamic frameworks help to explain the forces that must have shaped the code during the early evolution of life on Earth.

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.

Treponema is a diverse bacterial genus, the species of which can be pathogenic, symbiotic, or free living. These treponemes can cause various diseases in humans and other animals, such as periodontal disease, bovine digital dermatitis and animal skin lesions. However, the most important and well-studied disease of treponemes that affects humans is 'syphilis'. This disease is caused by Treponema pallidum subspecie pallidum with 11-12 million new cases around the globe on an annual basis. In this study we analyze the transportome of ten Treponema species, with emphasis on the types of encoded transport proteins and their substrates. Of the ten species examined, two (T. primitia and T. azonutricium) reside as symbionts in the guts of termites; six (T. pallidum, T. paraluiscuniculi, T. pedis, T. denticola, T. putidum and T. brennaborense) are pathogens of either humans or animals, and T. caldarium and T. succinifaciens are avirulent species, the former being thermophilic. All ten species have a repertoire of transport proteins that assists them in residing in their respective ecological niches. For instance, oral pathogens use transport proteins that take up nutrients uniquely present in their ecosystem; they also encode multiple multidrug/macromolecule exporters that protect against antimicrobials and aid in biofilm formation. Proteins of termite gut symbionts convert cellulose into other sugars that can be metabolized by the host. As often observed for pathogens and symbionts, several of these treponemes have reduced genome sizes, and their small genomes correlate with their dependencies on the host. Overall, the transportomes of T. pallidum and other pathogens have a conglomerate of parasitic lifestyle-assisting proteins. For example, a T. pallidum repeat protein (TprK) mediates immune evasion; outer membrane proteins (OMPs) allow nutrient uptake and end product export, and several ABC transporters catalyze sugar uptake, considered pivotal to parasitic lifestyles. Taken together, the results of this study yield new information that may help open new avenues of treponeme research.

The ImpX transporters of the drug/metabolite transporter superfamily were first proposed to transport riboflavin (RF; vitamin B2) based on findings of a cis-regulatory RNA element responding to flavin mononucleotide (an FMN riboswitch). Bdellovibrio exovorous JSS has a homolog belonging to this superfamily. It has 10 TMSs and shows 30% identity to the previously characterized ImpX transporter from Fusobacterium nucleatum. However, the ImpX homolog is not regulated by an FMN-riboswitch. In order to test the putative function of the ImpX homolog from B. exovorous (BexImpX), we cloned and heterologously expressed its gene. We used functional complementation, growth inhibition experiments, direct uptake experiments and inhibition studies, suggesting a high degree of specificity for RF uptake. The EC50 for growth with RF was estimated to be in the range 0.5-1 µM, estimated from the half-maximal RF concentration supporting the growth of a RF auxotrophic Escherichia coli strain, but the Khalf for RF uptake was 20 µM. Transport experiments suggested that the energy source is the proton motive force but that NaCl stimulates uptake. Thus, members of the ImpX family members are capable of RF uptake, not only in RF prototrophic species such as F.  nucleatum, but also in the B2 auxotrophic species, B. exovorous.

The multicomponent phosphoenolpyruvate (PEP)-dependent sugar-transporting phosphotransferase system (PTS) in Escherichia coli takes up sugar substrates from the medium and concomitantly phosphorylates them, releasing sugar phosphates into the cytoplasm. We have recently provided evidence that many of the integral membrane PTS permeases interact with the fructose PTS (FruA/FruB) [1]. However, the biochemical and physiological significance of this finding was not known. We have carried out molecular genetic/biochemical/physiological studies that show that interactions of the fructose PTS often enhance, but sometimes inhibit the activities of other PTS transporters many fold, depending on the target PTS system under study. Thus, the glucose (Glc), mannose (Man), mannitol (Mtl) and N-acetylglucosamine (NAG) permeases exhibit enhanced in vivo sugar transport and sometimes in vitro PEP-dependent sugar phosphorylation activities while the galactitol (Gat) and trehalose (Tre) systems show inhibited activities. This is observed when the fructose system is induced to high levels and prevented when the fruA/fruB genes are deleted. Overexpression of the fruA and/or fruB genes in the absence of fructose induction during growth also enhances the rates of uptake of other hexoses. The β-galactosidase activities of man, mtl, and gat-lacZ transcriptional fusions and the sugar-specific transphosphorylation activities of these enzyme transporters were not affected either by frustose induction or by fruAB overexpression, showing that the rates of synthesis of the target PTS permeases were not altered. We thus suggest that specific protein-protein interactions within the cytoplasmic membrane regulate transport in vivo (and sometimes the PEP-dependent phosphorylation activities in vitro) of PTS permeases in a physiologically meaningful way that may help to provide a hierarchy of preferred PTS sugars. These observations appear to be applicable in principle to other types of transport systems as well.

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the K half of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB >10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in Escherichia coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the β-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

Our laboratory has developed bioinformatic strategies for identifying distant phylogenetic relationships and characterizing families and superfamilies of transport proteins. Results using these tools suggest that the Anoctamin Superfamily of cation and anion channels, as well as lipid scramblases, includes three functionally characterized families: the Anoctamin (ANO), Transmembrane Channel (TMC) and Ca2+-permeable Stress-gated Cation Channel (CSC) families; as well as four families of functionally uncharacterized proteins, which we refer to as the Anoctamin-like (ANO-L), Transmembrane Channel-like (TMC-L), and CSC-like (CSC-L1 and CSC-L2) families. We have constructed protein clusters and trees showing the relative relationships among the seven families. Topological analyses suggest that the members of these families have essentially the same topologies. Comparative examination of these homologous families provides insight into possible mechanisms of action, indicates the currently recognized organismal distributions of these proteins, and suggests drug design potential for the disease-related channel proteins.

The histidine-phosphorylatable phosphocarrier protein (HPr) is an essential component of the sugar-transporting phosphotransferase system (PTS) in many bacteria. Recent interactome findings suggested that HPr interacts with several carbohydrate-metabolizing enzymes, but whether HPr plays a regulatory role was unclear. Here, we provide evidence that HPr interacts with a large number of proteins in Escherichia coli We demonstrate HPr-dependent allosteric regulation of the activities of pyruvate kinase (PykF, but not PykA), phosphofructokinase (PfkB, but not PfkA), glucosamine-6-phosphate deaminase (NagB), and adenylate kinase (Adk). HPr is either phosphorylated on a histidyl residue (HPr-P) or non-phosphorylated (HPr). PykF is activated only by non-phosphorylated HPr, which decreases the PykF Khalf for phosphoenolpyruvate by 10-fold (from 3.5 to 0.36 mm), thus influencing glycolysis. PfkB activation by HPr, but not by HPr-P, resulted from a decrease in the Khalf for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria.

Galdieria sulphuraria and Cyanidioschyzon merolae are thermo-acidophilic unicellular red algal cousins capable of living in volcanic environments, although the former can additionally thrive in the presence of toxic heavy metals. Bioinformatic analyses of transport systems were carried out on their genomes, as well as that of the mesophilic multicellular red alga Chondrus crispus (Irish moss). We identified transport proteins related to the metabolic capabilities, physiological properties, and environmental adaptations of these organisms. Of note is the vast array of transporters encoded in G. sulphuraria capable of importing a variety of carbon sources, particularly sugars and amino acids, while C. merolae and C. crispus have relatively few such proteins. Chondrus crispus may prefer short chain acids to sugars and amino acids. In addition, the number of encoded proteins pertaining to heavy metal ion transport is highest in G. sulphuraria and lowest in C. crispus. All three organisms preferentially utilize secondary carriers over primary active transporters, suggesting that their primary source of energy derives from electron flow rather than substrate-level phosphorylation. Surprisingly, the percentage of inorganic ion transporters encoded in C. merolae more closely resembles that of C. crispus than G. sulphuraria, but only C. crispus appears to signal via voltage-gated cation channels and possess a Na+ /K+ -ATPase and a Na+ exporting pyrophosphatase. The results presented in this report further our understanding of the metabolic potential and toxic compound resistances of these three organisms.

Escherichia coli is a genetically diverse species that can be pathogenic, probiotic, commensal, or a harmless laboratory strain. Pathogenic strains of E. coli cause urinary tract infections, diarrhea, hemorrhagic colitis, and pyelonephritis, while the two known probiotic E. coli strains combat inflammatory bowel disease and play a role in immunomodulation. Salmonella enterica, a close relative of E. coli, includes two important pathogenic serovars, Typhi and Typhimurium, causing typhoid fever and enterocolitis in humans, respectively, with the latter strain also causing a lethal typhoid fever-like disease in mice. In this study, we identify the transport systems and their substrates within seven E. coli strains: two probiotic strains, two extracellular pathogens, two intracellular pathogens, and K-12, as well as the two intracellular pathogenic S. enterica strains noted above. Transport systems characteristic of each probiotic or pathogenic species were thus identified, and the tabulated results obtained with all of these strains were compared. We found that the probiotic and pathogenic strains generally contain more iron-siderophore and sugar transporters than E. coli K-12. Pathogens have increased numbers of pore-forming toxins, protein secretion systems, decarboxylation-driven Na+ exporters, electron flow-driven monovalent cation exporters, and putative transporters of unknown function compared to the probiotic strains. Both pathogens and probiotic strains encode metabolite transporters that reflect their intracellular versus extracellular environments. The results indicate that the probiotic strains live extracellularly. It seems that relatively few virulence factors can convert a beneficial or commensal microorganism into a pathogen. Taken together, the results reveal the distinguishing features of these strains and provide a starting point for future engineering of beneficial enteric bacteria.

The flagellar system in Escherichia coli K12 is expressed under the control of the flhDC-encoded master regulator FlhDC. Transposition of insertion sequence (IS) elements to the upstream flhDC promoter region up-regulates transcription of this operon, resulting in a more rapid motility. Wang and Wood (ISME J 2011;5:1517-1525) provided evidence that insertion of IS5 into upstream activating sites occurs at higher rates in semi-solid agar media in which swarming behaviour is allowed as compared with liquid or solid media where swarming cannot occur. We confirm this conclusion and show that three IS elements, IS1, IS3 and IS5, transpose to multiple upstream sites within a 370 bp region of the flhDC operon control region. Hot spots for IS insertion correlate with positions of stress-induced DNA duplex destabilization (SIDD). We show that IS insertion occurs at maximal rates in 0.24 % agar, with rates decreasing dramatically with increasing or decreasing agar concentrations. In mixed cultures, we show that these mutations preferentially arise from the wild-type parent at frequencies of up to 3×10-3 cell-1 day-1 when the inoculated parental and co-existing IS-activated mutant cells are entering the stationary growth phase. We rigorously show that the apparent increased mutation frequencies cannot be accounted for by increased swimming or by increased growth under the selective conditions used. Thus, our data are consistent with the possibility that appropriate environmental conditions, namely those that permit but hinder flagellar rotation, result in the activation of a mutational pathway that involves IS element insertion upstream of the flhDC operon.

Connexins or innexins form gap junctions, while claudins and occludins form tight junctions. In this study, statistical data, derived using novel software, indicate that these four junctional protein families and eleven other families of channel and channel auxiliary proteins are related by common descent and comprise the Tetraspan (4 TMS) Junctional Complex (4JC) Superfamily. These proteins all share similar 4 transmembrane α-helical (TMS) topologies. Evidence is presented that they arose via an intragenic duplication event, whereby a 2 TMS-encoding genetic element duplicated tandemly to give 4 TMS proteins. In cases where high resolution structural data were available, the conclusion of homology was supported by conducting structural comparisons. Phylogenetic trees reveal the probable relationships of these 15 families to each other. Long homologues containing fusions to other recognizable domains as well as internally duplicated or fused domains are reported. Large "fusion" proteins containing 4JC domains proved to fall predominantly into family-specific patterns as follows: (1) the 4JC domain was N-terminal; (2) the 4JC domain was C-terminal; (3) the 4JC domain was duplicated or occasionally triplicated and (4) mixed fusion types were present. Our observations provide insight into the evolutionary origins and subfunctions of these proteins as well as guides concerning their structural and functional relationships.

We introduce the value of information obtained by comparing alternative crystal forms of the same sub-state (of outward open UlaA, our example protein), which is found in the same lattice configuration but different space groups. We compare instability estimates obtained using this new method (alternative crystal forms) with temperature factors. Using a transport assay result, we correlate observations for two homologous secondary structure elements, and show that the alternative states method for obtaining instability estimates provide differentiating information about an important and immobilized mid-TMS region. The data presented in this article are related to the article entitled "The V-motifs facilitate the substrate capturing step of the PTS elevator mechanism" (A. Vastermark, A. Driker, J. Weng, X. Li, J. Wang, M.H. Saier Jr., 2016).

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

Transposon-mediated "directed" mutations occur at higher frequencies when beneficial than when detrimental and relieve the stress that causes them. The first and best-studied example involves regulation of Insertion Sequence-5 (IS5) insertion into a specific activating site upstream of the glycerol utilization operon in Escherichia coli, glpFK. This event promotes high level expression of the glpFK operon, allowing glycerol utilization in wild type cells under inhibitory conditions. The phosphoenolpyruvate-dependent, sugar transporting, phosphotransferase system (PTS) influences this process by regulating cytoplasmic glycerol-3-phosphate and cyclic AMP concentrations. Insertion frequencies are determined by IS5-specific tetranucleotide target sequences in stress-induced (DNA) duplex destabilization (SIDD) structures counteracted by two DNA binding proteins, GlpR and Crp which directly inhibit insertion, responding to cytoplasmic glycerol-3-phosphate and cyclic AMP, respectively. Expression of the E. coli master regulator of flagellar gene control, flhDC, is subject to activation by IS elements by a directed mechanism, and zinc-induced transposon-mediated zinc resistance has been demonstrated in Cupriavidus metallidurans. The use of DNA conformation and DNA binding proteins to control transposon hopping also occurs in eukaryotes.

The membrane attack complex/perforin (MACPF) superfamily consists of a diverse group of proteins involved in bacterial pathogenesis and sporulation as well as eukaryotic immunity, embryonic development, neural migration and fruiting body formation. The present work shows that the evolutionary relationships between the members of the superfamily, previously suggested by comparison of their tertiary structures, can also be supported by analyses of their primary structures. The superfamily includes the MACPF family (TC 1.C.39), the cholesterol-dependent cytolysin (CDC) family (TC 1.C.12.1 and 1.C.12.2) and the pleurotolysin pore-forming (pleurotolysin B) family (TC 1.C.97.1), as revealed by expansion of each family by comparison against a large protein database, and by the comparisons of their hidden Markov models. Clustering analyses demonstrated grouping of the CDC homologues separately from the 12 MACPF subfamilies, which also grouped separately from the pleurotolysin B family. Members of the MACPF superfamily revealed a remarkably diverse range of proteins spanning eukaryotic, bacterial, and archaeal taxonomic domains, with notable variations in protein domain architectures. Our strategy should also be helpful in putting together other highly divergent protein families.

Insertion sequence elements (IS elements) are proposed to play major roles in shaping the genetic and phenotypic landscapes of prokaryotic cells. Recent evidence has raised the possibility that environmental stress conditions increase IS hopping into new sites, and often such hopping has the phenotypic effect of relieving the stress. Although stress-induced targeted mutations have been reported for a number of E. coli genes, the glpFK (glycerol utilization) and the cryptic bglGFB (β-glucoside utilization) systems are among the best characterized where the effects of IS insertion-mediated gene activation are well-characterized at the molecular level. In the glpFK system, starvation of cells incapable of utilizing glycerol leads to an IS5 insertion event that activates the glpFK operon, and enables glycerol utilization. In the case of the cryptic bglGFB operon, insertion of IS5 (and other IS elements) into a specific region in the bglG upstream sequence has the effect of activating the operon in both growing cells, and in starving cells. However, a major unanswered question in the glpFK system, the bgl system, as well as other examples, has been why the insertion events are promoted at specific locations, and how the specific stress condition (glycerol starvation for example) can be mechanistically linked to enhanced insertion at a specific locus. In this paper, we show that a specific DNA structural feature (superhelical stress-induced duplex destabilization, SIDD) is associated with "stress-induced" IS5 insertion in the glpFK, bglGFB, flhDC, fucAO and nfsB systems. We propose a speculative mechanistic model that links specific environmental conditions to the unmasking of an insertional hotspot in the glpFK system. We demonstrate that experimentally altering the predicted stability of a SIDD element in the nfsB gene significantly impacts IS5 insertion at its hotspot.

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

We propose that the alternative crystal forms of outward open UlaA (which are experimental, not simulated, and contain the substrate in the cavity) can be used to interpret/validate the MD results from MalT (the substrate capture step, which involves the mobile second TMSs of the V-motifs, TMSs 2 and 7). Since the crystal contacts are the same between the two alternative crystal forms of outward open UlaA, the striking biological differences noted, including rearranged hydrogen bonds and salt bridge coordination, are not attributable to crystal packing differences. Using transport assays, we identified G58 and G286 as essential for normal vitamin C transport, but the comparison of alternative crystal forms revealed that these residues to unhinge TMS movements from substrate-binding side chains, rendering the mid-TMS regions of homologous TMSs 2 and 7 relatively immobile. While the TMS that is involved in substrate binding in MalT is part of the homologous bundle that holds the two separate halves of the transport assembly (two proteins) together, an unequal effect of the two knockouts was observed for UlaA where both V-motifs are free from such dimer interface interactions.

Escherichia coli cells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required activator of the glycerol utilization operon, glpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can insert into the upstream regulatory region of the operon to activate the glpFK promoter and enable glycerol utilization. GlpR, which represses glpFK transcription, binds to the glpFK upstream region near the site of IS5 insertion and inhibits insertion. By adding cAMP to the culture medium in ΔcyaA cells, we here show that the cAMP-Crp complex, which also binds to the glpFK upstream regulatory region, inhibits IS5 hopping into the activating site. Control experiments showed that the frequencies of mutations in response to cAMP were independent of parental cell growth rate and the selection procedure. These findings led to the prediction that glpFK-activating IS5 insertions can also occur in wild-type (Crp+) cells under conditions that limit cAMP production. Accordingly, we found that IS5 insertion into the activating site in wild-type cells is elevated in the presence of glycerol and a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. The resultant IS5 insertion mutants arising in this minimal medium become dominant constituents of the population after prolonged periods of growth. The results show that DNA binding transcription factors can reversibly mask a favored transposon target site, rendering a hot spot for insertion less favored. Such mechanisms could have evolved by natural selection to overcome environmental adversity.

Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they all have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity arose in Leptospira correlating to progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles.

The vast majority of well studied transmembrane channels, secondary carriers, primary active transporters and group translocators are believed to have arisen vis intragenic duplication events from simple channel-forming peptides with just 1-3 transmembrane α-helical segments, found ubiquitously in nature. Only a few established channel-forming proteins appear to have evolved via other pathways. The proposed pathway for the evolutionary appearance of the five types of transport proteins involved intragenic duplication of transmembrane pore-forming peptide-encoding genes, giving rise to channel proteins. These gave rise to single protein secondary carriers which upon superimposition of addition protein domains and proteins, including energy-coupling proteins and extracytoplasmic receptors, gave rise to multidomain, multicomponent carriers, primary active transporters and group translocators. Some of the largest and best characterized superfamilies of these transmembrane transport proteins are discussed from topological and evolutionary standpoints.

In this issue of Structure, McCoy et al. (2016) describe the 2.55-Å X-ray structure of the outward-facing occluded conformation of the Bacillus cereus maltose transporter MalT. This structure represents the penultimate piece needed to complete the picture of the transport cycle of the glucose superfamily of membrane-spanning EIIC components.

The Transporter Classification Database (TCDB; http://www.tcdb.org) is a freely accessible reference database for transport protein research, which provides structural, functional, mechanistic, evolutionary and disease/medical 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). It consists of more than 10,000 non-redundant transport systems with more than 11 000 reference citations, classified into over 1000 transporter families. Transporters in TCDB can be single or multi-component systems, categorized in a functional/phylogenetic hierarchical system of classes, subclasses, families, subfamilies and transport systems. TCDB also includes updated software designed to analyze the distinctive features of transport proteins, extending its usefulness. Here we present a comprehensive update of the database contents and features and summarize recent discoveries recorded in TCDB.

We have designed a freely accessible program, PhyST, which allows the automated characterization of any family of homologous proteins within the Transporter Classification Database. The program performs an NCBI-PSI-BLAST search and reports (1) the average protein sequence length with standard deviations, (2) the average predicted number of transmembrane segments, (3) the total number of homologues retrieved, (4) a quantitative list of all source phyla, and (5) potential fusion proteins of sizes considerably exceeding the average size of the proteins retrieved. We have applied this program to 58 families of holins, and the results are presented. The results show that holins are very rarely fused to other protein domains, suggesting that holins form transmembrane pores as homooligomers without the participation of other proteins or protein domains.

We here report statistical analyses of 76 families of integral outer membrane pore-forming proteins (OMPPs) found in bacteria and eukaryotic organelles. 47 of these families fall into one superfamily (SFI) which segregate into fifteen phylogenetic clusters. Families with members of the same protein size, topology and substrate specificities often cluster together. Virtually all OMPP families include only proteins that form transmembrane pores. Nine such families, all of which cluster together in the SFI phylogenetic tree, contain both α- and β-structures, are multi domain, multi subunit systems, and transport macromolecules. Most other SFI OMPPs transport small molecules. SFII and SFV homologues derive from Actinobacteria while SFIII and SFIV proteins derive from chloroplasts. Three families of actinobacterial OMPPs and two families of eukaryotic OMPPs apparently consist primarily of α-helices (α-TMSs). Of the 71 families of (putative) β-barrel OMPPs, only twenty could not be assigned to a superfamily, and these derived primarily from Actinobacteria (1), chloroplasts (1), spirochaetes (8), and proteobacteria (10). Proteins were identified in which two or three full length OMPPs are fused together. Family characteristic are described and evidence agrees with a previous proposal suggesting that many arose by adjacent β-hairpin structural unit duplications.

Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity was accompanied by progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles.

Transport systems comprise roughly 10% of all proteins in a cell, playing critical roles in many processes. Improving and expanding their classification is an important goal that can affect studies ranging from comparative genomics to potential drug target searches. It is not surprising that different classification systems for transport proteins have arisen, be it within a specialized database, focused on this functional class of proteins, or as part of a broader classification system for all proteins. Two such databases are the Transporter Classification Database (TCDB) and the Protein family (Pfam) database. As part of a long-term endeavor to improve consistency between the two classification systems, we have compared transporter annotations in the two databases to understand the rationale for differences and to improve both systems. Differences sometimes reflect the fact that one database has a particular transporter family while the other does not. Differing family definitions and hierarchical organizations were reconciled, resulting in recognition of 69 Pfam 'Domains of Unknown Function', which proved to be transport protein families to be renamed using TCDB annotations. Of over 400 potential new Pfam families identified from TCDB, 10% have already been added to Pfam, and TCDB has created 60 new entries based on Pfam data. This work, for the first time, reveals the benefits of comprehensive database comparisons and explains the differences between Pfam and TCDB.

The Δ-distance maps can detect local remodeling that is difficult to accurately determine using superimpositions. Transmembrane segments (TMSs) 11 in both LacY and XylE of the major facilitator superfamily uniquely contribute the greatest amount of mobile surface area in the outward-occluded state and undergo analogous movements. The intracellular part of TMS11 moves away from the C-terminal domain and into the substrate cavity during the conformational change from the outward-occluded to the inward-occluded state. A difference was noted between LacY and XylE when they assumed the inward open state after releasing a substrate to the inside in which TMS11 of LacY moved further into the substrate release space, whereas in XylE, TMS11 slightly retracted into the C-terminal domain. Independent movement of the N-terminal half of TMS11 suggests that it is flexible in the middle. Repeat-swapped homology modeling was used to discover that a loop connecting TMSs 10 and 11 in LacY probably moves during the transition between the unavailable outward-open state and the outward-occluded state. TMSs 11 and the other elements displaying a notable domain-independent movement colocalize with the interdomain linker, suggesting that these elements could drive the alternating access movement between the domain halves. Preliminary evidence indicates that analogous movements occur in other members of the major facilitator superfamily.

Integral membrane transport proteins homologous to those found in the Transporter Classification Database (TCDB; www.tcdb.org) were identified and bioinformatically characterized by transporter class, family, and substrate specificity in three ciliates, Paramecium tetraurelia (Para), Tetrahymena thermophila (Tetra), and Ichthyophthirius multifiliis (Ich). In these three organisms, 1,326 of 39,600 proteins (3.4%), 1,017 of 24,800 proteins (4.2%), and 504 out of 8,100 proteins (6.2%) integral membrane transport proteins were identified, respectively. Thus, an inverse relationship was observed between the % transporters identified and the number of total proteins per genome reported. This surprising observation provides insight into the evolutionary process, giving rise to genome reduction following whole genome duplication (as in the case of Para) or during pathogenic association with a host organism (Ich). Of these transport proteins in Para and Tetra, about 41% were channels (more than any other type of organism studied), 31% were secondary carriers (fewer than most eukaryotes) and 26% were primary active transporters, mostly ATP-hydrolysis driven (more than most other eukaryotes). In Ich, the number of channels was selectively reduced by 66%, relative to Para and Tetra. Para has four times more inorganic anion transporters than Tetra, and Ich has nonselectively lost most of these. Tetra and Ich preferentially transport sugars and monocarboxylates while Para prefers di- and tricarboxylates. These observations serve to characterize the transport proteins of these related ciliates, providing insight into their nutrition and metabolism.

During 11-12 August 2014, a Protein Bioinformatics and Community Resources Retreat was held at the Wellcome Trust Genome Campus in Hinxton, UK. This meeting brought together the principal investigators of several specialized protein resources (such as CAZy, TCDB and MEROPS) as well as those from protein databases from the large Bioinformatics centres (including UniProt and RefSeq). The retreat was divided into five sessions: (1) key challenges, (2) the databases represented, (3) best practices for maintenance and curation, (4) information flow to and from large data centers and (5) communication and funding. An important outcome of this meeting was the creation of a Specialist Protein Resource Network that we believe will improve coordination of the activities of its member resources. We invite further protein database resources to join the network and continue the dialogue.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) has been shown to control transport, cell metabolism and gene expression. We here present results supporting the novel suggestion that in certain instances it also regulates the mutation rate. Directed mutations are defined as mutations that occur at higher frequencies when beneficial than when neutral or detrimental. To date, the occurrence of directed point mutations has not been documented and confirmed, but a few examples of transposon-mediated directed mutations have been reported. Here we focus on the first and best-studied example of directed mutation, which involves the regulation of insertion sequence-5 hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli. This insertional event specifically activates expression of the glpFK operon, allowing the growth of wild-type cells with glycerol as a carbon source in the presence of nonmetabolizable glucose analogues which normally block glycerol utilization. The sugar-transporting PTS controls this process by regulating levels of cytoplasmic glycerol-3-phosphate and cyclic (c)AMP as established in previous publications. Direct involvement of the glycerol repressor, GlpR, and the cAMP receptor protein, Crp, in the regulation of transposon-mediated directed mutation has been demonstrated.

In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which they subsequently proposed might catalyze sugar transport as well as sugar phosphorylation. What we have learned in the 50 years since its discovery is that, in addition to these primary functions, the PTS serves as a complex protein kinase system that regulates a wide variety of transport, metabolic and mutagenic processes as well as the expression of numerous genes. Recent operon- and genome-sequencing projects have revealed novel PTS protein-encoding genes, many of which have yet to be functionally defined. The current picture of the PTS is that of a complex system with ramifications in all aspects of cellular physiology. Moreover, its mosaic evolutionary history is unusual and intriguing. The PTS can be considered to serve many prokaryotes in capacities of communication and coordination, as do the nervous systems of animals.

Microbial products, released into the bloodstreams of mammals including humans, cross the blood-brain barrier and influence neurodevelopment. They can either promote or alleviate neurological disorders including autism spectrum disorders (ASD). This editorial describes how our microbiota influence our feelings, attitudes and mental states with particular reference to ASD.

The SLC22 family includes organic anion transporters (OATs), organic cation transporters (OCTs) and organic carnitine and zwitterion transporters (OCTNs). These are often referred to as drug transporters even though they interact with many endogenous metabolites and signaling molecules (Nigam, S.K., Nature Reviews Drug Discovery, 14:29-44, 2015). Phylogenetic analysis of SLC22 supports the view that these transporters may have evolved over 450 million years ago. Many OAT members were found to appear after a major expansion of the SLC22 family in mammals, suggesting a physiological and/or toxicological role during the mammalian radiation. Putative SLC22 orthologs exist in worms, sea urchins, flies, and ciona. At least six groups of SLC22 exist. OATs and OCTs form two Major clades of SLC22, within which (apart from Oat and Oct subclades), there are also clear Oat-like, Octn, and Oct-related subclades, as well as a distantly related group we term "Oat-related" (which may have different functions). Based on available data, it is arguable whether SLC22A18, which is related to bacterial drug-proton antiporters, should be assigned to SLC22. Disease-causing mutations, single nucleotide polymorphisms (SNPs) and other functionally analyzed mutations in OAT1, OAT3, URAT1, OCT1, OCT2, OCTN1, and OCTN2 map to the first extracellular domain, the large central intracellular domain, and transmembrane domains 9 and 10. These regions are highly conserved within subclades, but not between subclades, and may be necessary for SLC22 transporter function and functional diversification. Our results not only link function to evolutionarily conserved motifs but indicate the need for a revised sub-classification of SLC22.