Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless, Mgl activity is not essential for growth of these mutants, since deletion of this transporter did not affect the growth rate; the activities of the remaining transporters seem to be sufficient. Under conditions of glucose limitation, mgl is upregulated 23-fold compared to levels for growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose uptake at micromolar concentrations, the glucose as well as mannose PTS might be sufficient for growth at this relatively low dilution rate.     

细菌磷酸转移酶系统(PTS)的组成与功能研究进展

细菌磷酸烯醇丙酮酸(phosphoenolpyruvate,PEP)-磷酸转移酶系统(phosphotransferase system,PTS)广泛存在于细菌、真菌和一些古细菌中,但不存在于动植物中。PTS由酶I (EI)、组氨酸磷酸载体蛋白(HPr或NPr)和酶II复合物等磷酸转移酶组成,既具有催化转运功能,又具有非常广泛的调节功能。PTS主要是通过磷酸级联反应将各种糖及其衍生物进行磷酸化然后运输到胞内。其不仅参与碳、氮中心代谢,调节铁、钾稳态,调控某些病原体的毒力,还能介导应激反应。在这些不同的调节过程中,信号由PTS组分的磷酸化状态提供,而该磷酸化状态根据PTS底物的可用性和细胞代谢状态的变化而变化。本文对细菌中磷酸转移酶系统的组成和调控网络进行综述,以期为PTS的整体调控机制及其对细菌整体代谢影响的研究提供参考依据。     

Membrane transport metabolons

In this review evidence from a wide variety of biological systems is presented for the genetic, functional, and likely physical association of membrane transporters and the enzymes that metabolize the transported substrates. This evidence supports the hypothesis that the dynamic association of transporters and enzymes creates functional membrane transport metabolons that channel substrates typically obtained from the extracellular compartment directly into their cellular metabolism. The immediate modification of substrates on the inner surface of the membrane prevents back-flux through facilitated transporters, increasing the efficiency of transport. In some cases products of the enzymes are themselves substrates for the transporters that efflux the products in an exchange or antiport mechanism. Regulation of the binding of enzymes to transporters and their mutual activities may play a role in modulating flux through transporters and entry of substrates into metabolic pathways. Examples showing the physical association of transporters and enzymes are provided, but available structural data is sparse. Genetic and functional linkages between membrane transporters and enzymes were revealed by an analysis of Escherichia coli operons encoding polycistronic mRNAs and provide a list of predicted interactions ripe for further structural studies. This article supports the view that membrane transport metabolons are important throughout Nature in organisms ranging from bacteria to humans.     

Membrane transport metabolons

In this review evidence from a wide variety of biological systems is presented for the genetic, functional, and likely physical association of membrane transporters and the enzymes that metabolize the transported substrates. This evidence supports the hypothesis that the dynamic association of transporters and enzymes creates functional membrane transport metabolons that channel substrates typically obtained from the extracellular compartment directly into their cellular metabolism. The immediate modification of substrates on the inner surface of the membrane prevents back-flux through facilitated transporters, increasing the efficiency of transport. In some cases products of the enzymes are themselves substrates for the transporters that efflux the products in an exchange or antiport mechanism. Regulation of the binding of enzymes to transporters and their mutual activities may play a role in modulating flux through transporters and entry of substrates into metabolic pathways. Examples showing the physical association of transporters and enzymes are provided, but available structural data is sparse. Genetic and functional linkages between membrane transporters and enzymes were revealed by an analysis of Escherichia coli operons encoding polycistronic mRNAs and provide a list of predicted interactions ripe for further structural studies. This article supports the view that membrane transport metabolons are important throughout Nature in organisms ranging from bacteria to humans.     

Polyamines and membrane transporters

In recent years, our understanding of the importance of membrane transporters (MTs) in the disposition of and response to drugs has increased significantly. MTs are proteins that regulate the transport of endogenous molecules and xenobiotics across the cell membrane. In mammals, two super-families have been identified: ATP-binding cassette (ABC) and solute carrier (SLC) transporters. There is evidence that MTs might mediate polyamines (PA) transport. PA are ubiquitous polycations which are found in all living cells. In mammalian cells, three major PA are synthesised: putrescine, spermidine and spermine; whilst the decarboxylated arginine (agmatine) is not produced by mammals but is synthesised by plants and bacteria. In addition, research in the PA field suggests that PA are transported into cells via a specific transporter, the polyamine transport system(s) (PTS). Although the PTS has not been fully defined, there is evidence that some of the known MTs might be involved in PA transport. In this mini review, eight SLC transporters will be reviewed and their potential to mediate PA transport in human cells discussed. These transporters are SLC22A1, SLC22A2, SLC22A3, SLC47A1, SLC7A1, SLC3A2, SLC12A8A, and SLC22A16. Preliminary data from our laboratory have revealed that SLC22A1 might be involved in the PA uptake; in addition to one member of ABC superfamily (MDR1 protein) might also mediate the efflux of polyamine like molecules.     

Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species

Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria.     

Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host

A common mechanism for high affinity carbohydrate uptake in microbial species is the phosphoenolpyruvate‐dependent phosphotransferase system (PTS). This system consists of a shared component, EI, which is required for all PTS transport, and numerous carbohydrate uptake transporters. In Vibrio cholerae, there are 13 distinct PTS transporters. Due to genetic redundancy within this system, the carbohydrate specificity of each of these transporters is not currently defined. Here, using multiplex genome editing by natural transformation (MuGENT), we systematically dissect PTS transport in V. cholerae. Specifically, we generated a mutant strain that lacks all 13 PTS transporters, and from this strain, we created a panel of mutants where each expresses a single transporter. Using this panel, we have largely defined the carbohydrate specificities of each PTS transporter. In addition, this analysis uncovered a novel glucose transporter. We have further defined the mechanism of this transporter and characterized its regulation. Using our 13 PTS transporter mutant, we also provide the first clear evidence that carbohydrate transport by the PTS is not essential during infection in an infant mouse model of cholera. In summary, this study shows how multiplex genome editing can be used to rapidly dissect complex biological systems and genetic redundancy in microbial systems.     

Metal transport across biomembranes: emerging models for a distinct chemistry

Transition metals are essential components of important biomolecules, and their homeostasis is central to many life processes. Transmembrane transporters are key elements controlling the distribution of metals in various compartments. However, due to their chemical properties, transition elements require transporters with different structural-functional characteristics from those of alkali and alkali earth ions. Emerging structural information and functional studies have revealed distinctive features of metal transport. Among these are the relevance of multifaceted events involving metal transfer among participating proteins, the importance of coordination geometry at transmembrane transport sites, and the presence of the largely irreversible steps associated with vectorial transport. Here, we discuss how these characteristics shape novel transition metal ion transport models.     

P5B-ATPases in the mammalian polyamine transport system and their role in disease

Polyamines (PAs) are physiologically relevant molecules that are ubiquitous in all organisms. The vitality of PAs to the healthy functioning of a cell is due to their polycationic nature causing them to interact with a vast plethora of cellular players and partake in numerous cellular pathways. Naturally, the homeostasis of such essential molecules is tightly regulated in a strictly controlled interplay between intracellular synthesis and degradation, uptake from and secretion to the extracellular compartment, as well as intracellular trafficking. Not surprisingly, dysregulated PA homeostasis and signaling are implicated in multiple disorders, ranging from cancer to neurodegeneration; leading many to propose rectifying the PA balance as a potential therapeutic strategy. Despite being well characterized in bacteria, fungi and plants, the molecular identity and properties of the PA transporters in animals are poorly understood. This review brings together the current knowledge of the cellular function of the mammalian PA transport system (PTS). We will focus on the role of P5B-ATPases ATP13A2-5 which are PA transporters in the endosomal system that have emerged as key players in cellular PA uptake and organelle homeostasis. We will discuss recent breakthroughs on their biochemical and structural properties as well as their implications for disease and therapy.     

Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes

Here, we present a comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms. These organisms include four Gram-positive bacteria, seven Gram-negative bacteria, two spirochetes, one cyanobacterium and four archaea. Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families, including four families of channel proteins, four families of primary carriers, 54 families of secondary carriers, six families of group translocators, and eight unclassified families. These families are analyzed in terms of the paralogous and orthologous relationships of their protein members, the substrate specificities of their constituent transporters and their distributions in each of the 18 organisms studied. The families vary from large superfamilies with hundreds of represented members, to small families with only one or a few members. The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H(+) and Na(+) pumps in the cell. The phosphotransferase system is less prevalent in the analyzed bacteria than previously thought (only six of 14 bacteria transport sugars via this system) and is completely lacking in archaea and eukaryotes. Escherichia coli is shown to be exceptionally broad in its transport capabilities and therefore, at a membrane transport level, does not appear representative of the bacteria thus far sequenced. Archaea and spirochetes exhibit fewer proteins with multiple transmembrane segments and fewer net transporters than most bacteria. These results provide insight into the relevance of transport to the overall physiology of prokaryotes.     

Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.     

The dihydroxyacetone kinase of Escherichia coli utilizes a phosphoprotein instead of ATP as phosphoryl donor

The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N- and C-terminal halves of the ATP-dependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI- and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-specific transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.     

Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein-ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure-function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino‐acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequence-based methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.     

Sugar transporters in higher plants--a diversity of roles and complex regulation

Sugar-transport proteins play a crucial role in the cell-to-cell and long-distance distribution of sugars throughout the plant. In the past decade, genes encoding sugar transporters (or carriers) have been identified, functionally expressed in heterologous systems, and studied with respect to their spatial and temporal expression. Higher plants possess two distinct families of sugar carriers: the disaccharide transporters that primarily catalyse sucrose transport and the monosaccharide transporters that mediate the transport of a variable range of monosaccharides. The tissue and cellular expression pattern of the respective genes indicates their specific and sometimes unique physiological tasks. Some play a purely nutritional role and supply sugars to cells for growth and development, whereas others are involved in generating osmotic gradients required to drive mass flow or movement. Intriguingly, some carriers might be involved in signalling. Various levels of control regulate these sugar transporters during plant development and when the normal environment is perturbed. This article focuses on members of the monosaccharide transporter and disaccharide transporter families, providing details about their structure, function and regulation. The tissue and cellular distribution of these sugar transporters suggests that they have interesting physiological roles.     

Comparative molecular biological analysis of membrane transport genes in organisms

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H⁺ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na⁺ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H⁺ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of ptsO in carbon-mediated inhibition of the Pu promoter belonging to the pWW0 Pseudomonas putida plasmid

An investigation was made into the role of the ptsO gene in carbon source inhibition of the Pu promoter belonging to the Pseudomonas putida upper TOL (toluene degradation) operon. ptsO is coexpressed with ptsN, the loss of which is known to render Pu unresponsive to glucose. Both ptsN and ptsO, coding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family proteins IIA(Ntr) and NPr, respectively, have been mapped adjacent to the rpoN gene of P. putida. The roles of these two genes in the responses of Pu to glucose were monitored by lacZ reporter technology with a P. putida strain engineered with all regulatory elements in monocopy gene dosage. In cells lacking ptsO, Pu activity seemed to be inhibited even in the absence of glucose. A functional relationship with ptsN was revealed by the phenotype of a double ptsN ptsO mutant that was equivalent to the phenotype of a mutant with a single ptsN disruption. Moreover, phosphorylation of the product of ptsO seemed to be required for C inhibition of Pu, since an H15A change in the NPr sequence that prevents phosphorylation of this conserved amino acid residue did not restore the wild-type phenotype. A genomic search for proteins able to phosphorylate ptsO revealed the presence of two open reading frames, designated ptsP and mtp, with the potential to encode PTS type I enzymes in P. putida. However, neither an insertion in ptsP nor an insertion in mtp resulted in a detectable change in inhibition of Pu by glucose. These results indicate that some PTS proteins have regulatory functions in P. putida that are independent of their recognized role in sugar transport in other bacteria.     

Transport proteins (carriers) of mitochondria

Mitochondria are subcellular structures essential to the aerobic eukaryotic cell. Their role extends much beyond their basic reactions of oxidative phosphorylation. It encompasses the steps critical for cellular metabolic pathways, for apoptosis, and for other processes such as antiviral signaling. This short review is limited to transport proteins (carriers) that catalyze the transport of metabolites across the inner mitochondrial membrane and thus link metabolic pathway reactions in the cytosol and the mitochondrial matrix. Such transport must minimally affect the electrochemical proton gradient essential for oxidative phosphorylation (chemiosmotic mechanism of oxidative phosphorylation). Many of these transport proteins belong to a family of membrane proteins, and the major part of this review will consider their structures and functions. First studies of these transporters were carried out with intact mitochondria and with inhibitors that appeared transporter-specific. Such an inhibitor was then utilized in the first purification of one of these transporter proteins. Its substrate-specificity was then established after functionally active incorporation into liposomes. Questions about copurification of other transporters and thus a definitive identification of transported substrate with the purified protein were resolved definitively only after heterologous expression in bacteria, most generally as inclusion bodies, and followed by reconstitution in liposomes. Site-specific mutations permitted the identification of amino acids essential to their transport function. These mutagenesis studies then also helped interpret human diseases with mutations in these transport proteins. The high-resolution structure of a member of this transporter protein family dramatically advanced these studies. It raised new questions because this structure complexed with a high-affinity inhibitor showed a monomeric protein, while purification and inhibitor stoichiometry studies suggest a functional homodimeric transport protein. Remaining key questions need to address: the homodimeric nature of the transporters, details of their transport mechanism, and the functional identification of many members of this family whose existence has only been suggested from genomic data.