TCDB: the Transporter Classification Database for membrane transport protein analyses and information

The Transporter Classification Database (TCDB) is a web accessible, curated, relational database containing sequence, classification, structural, functional and evolutionary information about transport systems from a variety of living organisms. TCDB is a curated repository for factual information compiled from >10 000 references, encompassing ∼3000 representative transporters and putative transporters, classified into >400 families. The transporter classification (TC) system is an International Union of Biochemistry and Molecular Biology approved system of nomenclature for transport protein classification. TCDB is freely accessible at . The web interface provides several different methods for accessing the data, including step-by-step access to hierarchical classification, direct search by sequence or TC number and full-text searching. The functional ontology that underlies the database structure facilitates powerful query searches that yield valuable data in a quick and easy way. The TCDB website also offers several tools specifically designed for analyzing the unique characteristics of transport proteins. TCDB not only provides curated information and a tool for classifying newly identified membrane proteins, but also serves as a genome transporter-annotation tool

The Transporter Classification Database: recent advances

The Transporter Classification Database (TCDB), freely accessible at http://www.tcdb.org, is a relational database containing sequence, structural, functional and evolutionary information about transport systems from a variety of living organisms, based on the International Union of Biochemistry and Molecular Biology-approved transporter classification (TC) system. It is a curated repository for factual information compiled largely from published references. It uses a functional/phylogenetic system of classification, and currently encompasses about 5000 representative transporters and putative transporters in more than 500 families. We here describe novel software designed to support and extend the usefulness of TCDB. Our recent efforts render it more user friendly, incorporate machine learning to input novel data in a semiautomatic fashion, and allow analyses that are more accurate and less time consuming. The availability of these tools has resulted in recognition of distant phylogenetic relationships and tremendous expansion of the information available to TCDB users

Large Direct Repeats Flank Genomic Rearrangements between a New Clinical Isolate of Francisella tularensis subsp. tularensis A1 and Schu S4

Francisella tularensis subspecies tularensis consists of two separate populations A1 and A2. This report describes the complete genome sequence of NE061598, an F. tularensis subspecies tularensis A1 isolated in 1998 from a human with clinical disease in Nebraska, United States of America. The genome sequence was compared to Schu S4, an F. tularensis subspecies tularensis A1a strain originally isolated in Ohio in 1941. It was determined that there were 25 nucleotide polymorphisms (22 SNPs and 3 indels) between Schu S4 and NE061598; two of these polymorphisms were in potential virulence loci. Pulsed-field gel electrophoresis analysis demonstrated that NE061598 was an A1a genotype. Other differences included repeat sequences (n = 11 separate loci), four of which were contained in coding sequences, and an inversion and rearrangement probably mediated by insertion sequences and the previously identified direct repeats I, II, and III. Five new variable-number tandem repeats were identified; three of these five were unique in NE061598 compared to Schu S4. Importantly, there was no gene loss or gain identified between NE061598 and Schu S4. Interpretation of these data suggests there is significant sequence conservation and chromosomal synteny within the A1 population. Further studies are needed to determine the biological properties driving the selective pressure that maintains the chromosomal structure of this monomorphic pathogen

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology

Type III Secretion System Genes of Dickeya dadantii 3937 Are Induced by Plant Phenolic Acids

Background: Dickeya dadantii is a broad-host range phytopathogen. D. dadantii 3937 (Ech3937) possesses a type III secretion system (T3SS), a major virulence factor secretion system in many Gram-negative pathogens of plants and animals. In Ech3937, the T3SS is regulated by two major regulatory pathways, HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. Although the plant apoplast environment, low pH, low temperature, and absence of complex nitrogen sources in media have been associated with the induction of T3SS genes of phytobacteria, no specific inducer has yet been identified. Methodology/Principal Findings: In this work, we identified two novel plant phenolic compounds, o-coumaric acid (OCA) and t-cinnamic acid (TCA), that induced the expression of T3SS genes dspE (a T3SS effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) in vitro. Assays by qRT-PCR showed higher amounts of mRNA of hrpL (a T3SS alternative sigma factor) and rsmB (an untranslated regulatory RNA), but not hrpS (a s 54-enhancer binding protein) of Ech3937 when these two plant compounds were supplemented into minimal medium (MM). However, promoter activity assays using flow cytometry showed similar promoter activities of hrpN in rsmB mutant Ech148 grown in MM and MM supplemented with these phenolic compounds. Compared with MM alone, only slightly higher promoter activities of hrpL were observed in bacterial cells grown in MM supplemented with OCA/TCA. Conclusion/Significance: The induction of T3SS expression by OCA and TCA is moderated through the rsmB-Rsm

Genomics of Aerobic Cellulose Utilization Systems in Actinobacteria

Cellulose degrading enzymes have important functions in the biotechnology industry, including the production of biofuels from lignocellulosic biomass. Anaerobes including Clostridium species organize cellulases and other glycosyl hydrolases into large complexes known as cellulosomes. In contrast, aerobic actinobacteria utilize systems comprised of independently acting enzymes, often with carbohydrate binding domains. Numerous actinobacterial genomes have become available through the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. We identified putative cellulose-degrading enzymes belonging to families GH5, GH6, GH8, GH9, GH12, GH48, and GH51 in the genomes of eleven members of the actinobacteria. The eleven organisms were tested in several assays for cellulose degradation, and eight of the organisms showed evidence of cellulase activity. The three with the highest cellulase activity were Actinosynnema mirum, Cellulomonas flavigena, and Xylanimonas cellulosilytica. Cellobiose is known to induce cellulolytic enzymes in the model organism Thermobifida fusca, but only Nocardiopsis dassonvillei showed higher cellulolytic activity in the presence of cellobiose. In T. fusca, cellulases and a putative cellobiose ABC transporter are regulated by the transcriptional regulator CelR. Nine organisms appear to use the CelR site or a closely related binding site to regulate an ABC transporter. In some, CelR also regulates cellulases, while cellulases are controlled by different regulatory sites in three organisms. Mining of genome data for cellulose degradative enzymes followed by experimental verification successfully identified several actinobacteria species which were not previously known to degrade cellulose as cellulolytic organisms

Carbon Metabolism of Enterobacterial Human Pathogens Growing in Epithelial Colorectal Adenocarcinoma (Caco-2) Cells

Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens

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

Complete Genome Sequence of Francisella tularensis Subspecies holarctica FTNF002-00

Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD23 deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997–1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also ∼5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1–1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H+ antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities