Central Carbohydrate Metabolism Research Articles

Genomic Reconstruction of Carbohydrate Utilization Capacities in Microbial-Mat Derived Consortia

Two nearly identical unicyanobacterial consortia (UCC) were previously isolated from benthic microbial mats that occur in a heliothermal saline lake in northern Washington State. Carbohydrates are a primary source of carbon and energy for most heterotrophic bacteria. Since CO2 is the only carbon source provided, the cyanobacterium must provide a source of carbon to the heterotrophs. Available genomic sequences for all members of the UCC provide opportunity to investigate the metabolic routes of carbon transfer between autotroph and heterotrophs. Here, we applied a subsystem-based comparative genomics approach to reconstruct carbohydrate utilization pathways and identify glycohydrolytic enzymes, carbohydrate transporters and pathway-specific transcriptional regulators in 17 heterotrophic members of the UCC. The reconstructed metabolic pathways include 800 genes, near a one-fourth of which encode enzymes, transporters and regulators with newly assigned metabolic functions resulting in discovery of novel functional variants of carbohydrate utilization pathways. The in silico analysis revealed the utilization capabilities for 40 carbohydrates and their derivatives. Two Halomonas species demonstrated the largest number of sugar catabolic pathways. Trehalose, sucrose, maltose, glucose, and beta-glucosides are the most commonly utilized saccharides in this community. Reconstructed regulons for global regulators HexR and CceR include central carbohydrate metabolism genes in the members of Gammaproteobacteria and Alphaproteobacteria, respectively. Genomics analyses were supplemented by experimental characterization of metabolic phenotypes in four isolates derived from the consortia. Measurements of isolate growth on the defined medium supplied with individual carbohydrates confirmed most of the predicted catabolic phenotypes. Not all consortia members use carbohydrates and only a few use complex polysaccharides suggesting a hierarchical carbon flow from cyanobacteria to each heterotroph. In summary, the genomics-based identification of carbohydrate utilization capabilities provides a basis for future experimental studies of carbon flow in UCC.

Screening and transcriptomic analysis of Crypthecodinium cohnii mutants with high growth and lipid content using the acetyl-CoA carboxylase inhibitor sethoxydim.

The heterotrophic microalga Crypthecodinium cohnii is well known for its lipid accumulation, with a high proportion of docosahexaenoic acid (DHA). In this study, we report a novel screening approach to obtain mutants of C. cohnii with high growth and lipid content using the acetyl-CoA carboxylase (ACCase) inhibitor sethoxydim. C. cohnii mutants were generated using atmospheric and room-temperature plasma (ARTP) and then screened for two rounds in media supplemented with sethoxydim. These efforts led to the identification of mutant M-1-2, which had 24.32% higher growth and 7.05% higher lipid content than the wild type, demonstrating the effectiveness of the sethoxydim-based screening. Consistently, the M-1-2 mutant displayed a 16.15% increase in ACCase enzymatic activity and 1.53-fold upregulation of its ACCase-encoding gene based on comparative ACCase activity analysis and transcriptomic analysis, respectively. In addition, transcriptomic analysis showed that transcripts involved in fatty acid biosynthesis, energy, central carbohydrate, and amino acid metabolism were upregulated in the mutant compared to the wild type.

Genome-Guided Insights into the Plant Growth Promotion Capabilities of the Physiologically Versatile Bacillus aryabhattai Strain AB211.

Bacillus aryabhattai AB211 is a plant growth promoting, Gram-positive firmicute, isolated from the rhizosphere of tea (Camellia sinensis), one of the oldest perennial crops and a major non-alcoholic beverage widely consumed all over the world. The whole genome of B. aryabhattai AB211 was sequenced, annotated and evaluated with special focus on genomic elements related to plant microbe interaction. It’s genome sequence reveals the presence of a 5,403,026 bp chromosome. A total of 5226 putative protein-coding sequences, 16 rRNA, 120 tRNA, 8 ncRNAs, 58 non-protein coding genes, and 11 prophage regions were identified. Genome sequence comparisons between strain AB211 and other related environmental strains of B. aryabhattai, identified about 3558 genes conserved among all B. aryabhattai genomes analyzed. Most of the common genes involved in plant growth promotion activities were found to be present within core genes of all the genomes used for comparison, illustrating possible common plant growth promoting traits shared among all the strains of B. aryabhattai. Besides the core genes, some genes were exclusively identified in the genome of strain AB211. Functional annotation of the genes predicted in the strain AB211 revealed the presence of genes responsible for mineral phosphate solubilization, siderophores, acetoin, butanediol, exopolysaccharides, flagella biosynthesis, surface attachment/biofilm formation, and indole acetic acid production, most of which were experimentally verified in the present study. Genome analysis and experimental evidence suggested that AB211 has robust central carbohydrate metabolism implying that this bacterium can efficiently utilize the root exudates and other organic materials as an energy source. Genes for the production of peroxidases, catalases, and superoxide dismutases, that confer resistance to oxidative stresses in plants were identified in AB211 genome. Besides these, genes for heat shock tolerance, cold shock tolerance, glycine-betaine production, and antibiotic/heavy metal resistance that enable bacteria to survive biotic/abiotic stress were also identified. Based on the genome sequence information and experimental evidence as presented in this study, strain AB211 appears to be metabolically diverse and exhibits tremendous potential as a plant growth promoting bacterium.

Crosstalk of two-component signal transduction systems in regulating central carbohydrate and energy metabolism during autotrophic and photomixotrophic growth of Synechocystis sp. PCC 6803.

Unicellular model cyanobacterium Synechocystis sp. PCC 6803 has received considerable attention as a sustainable energy resource because of its photosynthetic machinery. However, two-component signal transduction systems (TCSTSs) in regulating central carbohydrate and energy metabolism of cyanobacteria are still poorly understood due to their diversity and functional complication. In this study, by comparing the growth of knockout mutants of 44 response regulators (RRs) of TCSTSs in Synechocystis, several RR mutants demonstrating differential growth patterns were identified under auto- or photomixotrophic conditions. However, in spite of no growth difference observed for the remaining RR mutants, liquid chromatography-mass spectrometry based metabolomic profile analysis showed that a widespread crosstalk of TCSTSs in regulating central carbohydrate and energy metabolism of Synechocystis was identified, while most of them showed diverse patterns during different trophic types or growth stages. Furthermore, an integrative analysis between evolutionary relationships and metabolomic profiles revealed some pairs of paralogous RRs with highly functional convergence, suggesting the possible conserved functions of Synechocystis TCSTSs during evolution. This study laid an important basis for understanding the function of TCSTSs in photosynthetic cyanobacteria.

Cell wall synthesis and central carbohydrate metabolism are interconnected by the SNF1/Mig1 pathway in Kluyveromyces lactis

The trimeric AMP-activated kinase complex (AMPK) is conserved from yeast to humans and is best known for its role in balancing energy metabolism. Additional functions, including the regulation of cell wall biosynthesis, have been proposed for the SNF1 complex, the baker’s yeast homolog of AMPK. We here demonstrate that this function is conserved in the Crabtree-negative milk yeast Kluyveromyces lactis. Deletion mutants in the genes encoding the subunits of the trimeric complex (Klsnf1, Klgal83, Klsnf4) displayed increased sensitivities towards cell wall stress agents and a mutant lacking the kinase subunit had a thinner cell wall in transmission electron micrographs as compared to wild type. Epistasis analyses demonstrated that the KlSNF1 complex acts in parallel to cell wall integrity (CWI) signaling and stress sensitivities of Klsnf1 deletions can be suppressed by additional deletions in glycolytic genes (KlPFK1, KlPFK2, KlPGI1) or by a Klmig1 mutant. Western blot analyses of an HA-tagged KlMig1p revealed its phosphorylation on ethanol medium similar to its S. cerevisiae ortholog, but a substantial amount of protein remained phosphorylated even with high glucose concentrations. Application of cell wall stress shifted this equilibrium towards the non-phosphorylated fraction of KlMig1p. We conclude that KlMig1p may exert a negative regulatory function on cell wall biosynthesis.

Metabolic model of central carbon and energy metabolisms of growing Arabidopsis thaliana in relation to sucrose translocation.

BackgroundSucrose translocation between plant tissues is crucial for growth, development and reproduction of plants. Systemic analysis of these metabolic and underlying regulatory processes allow a detailed understanding of carbon distribution within the plant and the formation of associated phenotypic traits. Sucrose translocation from ‘source’ tissues (e.g. mesophyll) to ‘sink’ tissues (e.g. root) is tightly bound to the proton gradient across the membranes. The plant sucrose transporters are grouped into efflux exporters (SWEET family) and proton-symport importers (SUC, STP families). To better understand regulation of sucrose export from source tissues and sucrose import into sink tissues, there is a need for a metabolic model that takes in account the tissue organisation of Arabidopsis thaliana with corresponding metabolic specificities of respective tissues in terms of sucrose and proton production/utilization. An ability of the model to operate under different light modes (‘light’ and ‘dark’) and correspondingly in different energy producing modes is particularly important in understanding regulatory modules.ResultsHere, we describe a multi-compartmental model consisting of a mesophyll cell with plastid and mitochondrion, a phloem cell, as well as a root cell with mitochondrion. In this model, the phloem was considered as a non-growing transport compartment, the mesophyll compartment was considered as both autotrophic (growing on CO2 under light) and heterotrophic (growing on starch in darkness), and the root was always considered as heterotrophic tissue dependent on sucrose supply from the mesophyll compartment. In total, the model includes 413 balanced compounds interconnected by 400 transformers. The structured metabolic model accounts for central carbon metabolism, photosynthesis, photorespiration, carbohydrate metabolism, energy and redox metabolisms, proton metabolism, biomass growth, nutrients uptake, proton gradient generation and sucrose translocation between tissues. Biochemical processes in the model were associated with gene-products (742 ORFs). Flux Balance Analysis (FBA) of the model resulted in balanced carbon, nitrogen, proton, energy and redox states under both light and dark conditions. The main H+-fluxes were reconstructed and their directions matched with proton-dependent sucrose translocation from ‘source’ to ‘sink’ under any light condition.ConclusionsThe model quantified the translocation of sucrose between plant tissues in association with an integral balance of protons, which in turn is defined by operational modes of the energy metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0868-3) contains supplementary material, which is available to authorized users.

Complete genome sequence of plant growth-promoting bacterium Leifsonia xyli SE134, a possible gibberellin and auxin producer

Leifsonia xyli SE134 is a potential plant growth-promoting bacterium isolated from a soil in Daegu, Republic of Korea, which produces large amounts of gibberellin (GA) and indole acetic acid (IAA). In this study, we sequenced the complete genome of L. xyli SE134 by the Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. The genome of L. xyli SE134 contains a single chromosome that is 3,596,761bp in length, with 70.2% G+C content. The genome contains 3466 protein-coding genes (CDSs) and 51 rRNA- and 46 tRNA-coding genes. By genomic analysis, we identified genes that are potentially involved in plant growth promotion such as genes participating in indole-3-acetic acid (IAA) biosynthesis, siderophore, and trehalose production. L. xyli SE134 also harbours genes for central carbohydrate metabolism, indicating that it can utilise the root exudates with other organic materials as an energy source. Furthermore, the SE134 genome is equipped with various kinds of genes for adaptation to plant surfaces, e.g. defence against desiccation, nutrient deficiencies, and oxidative stress, and a large proportion of genes related to secretion mechanisms and signalling. The genetic information provided here may help to expand this bacterium’s biotechnological potential and to further improve its plant growth-promoting characteristics.

Kohlenhydratstoffwechsel in Archaea–Besonderheiten und Herausforderung

Archaea exhibit unique features especially with respect to their central carbohydrate metabolism characterized by many unusual pathways and enzymes with specific energetic and regulatory properties. This probably contributes to “metabolic thermoadaptation” towards high temperature environments in which many archaea reside. The unusual metabolic and adaptive properties of Archaea also possess a great potential for biotechnology, which becomes more and more accessible by systems biology approaches.

The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes.

Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental advantage for methanosarcinales when carbon sources are scarce. Also, the understanding of the central carbohydrate metabolism in methanosarcinales may help to optimize methane production.

Potato tuber expression of Arabidopsis WRINKLED1 increase triacylglycerol and membrane lipids while affecting central carbohydrate metabolism.

SummaryTuber and root crops virtually exclusively accumulate storage products in the form of carbohydrates. An exception is yellow nutsedge (Cyperus esculentus) in which tubers have the capacity to store starch and triacylglycerols (TAG) in roughly equal amounts. This suggests that a tuber crop can efficiently handle accumulation of energy dense oil. From a nutritional as well as economic aspect, it would be of interest to utilize the high yield capacity of tuber or root crops for oil accumulation similar to yellow nutsedge. The transcription factor WRINKLED1 from Arabidopsis thaliana, which in seed embryos induce fatty acid synthesis, has been shown to be a major factor for oil accumulation. WRINKLED1 was expressed in potato (Solanum tuberosum) tubers to explore whether this factor could impact tuber metabolism. This study shows that a WRINKLED1 transcription factor could induce triacylglycerol accumulation in tubers of transformed potato plants grown in field (up to 12 nmol TAG/mg dry weight, 1% of dry weight) together with a large increase in polar membrane lipids. The changes in metabolism further affected starch accumulation and composition concomitant with massive increases in sugar content.

Transcriptional Regulation of Carbohydrate Utilization Pathways in the Bifidobacterium Genus.

Bifidobacteria, which represent common commensals of mammalian gut, are believed to have positive effects on human health. The influence of certain non-digestible carbohydrates (and their use as so-called prebiotics) on growth and metabolic activity of bifidobacteria is of increasing interest; however, mechanisms of transcriptional control of carbohydrate metabolism are poorly understood in these species. We used a comparative genomics approach to reconstruct carbohydrate utilization pathways and transcriptional regulons in 10 Bifidobacterium genomes. Analysis of regulatory gene regions revealed candidate DNA motifs and reconstructed regulons for 268 transcription factors from the LacI, ROK, DeoR, AraC, GntR, and TetR families that form 64 orthologous groups of regulators. Most of the reconstructed regulons are local and control specific catabolic pathways for host- and diet-derived glycans and monosaccharides. Mosaic distributions of many of these local regulators across Bifidobacterium species correlate with distribution of corresponding catabolic pathways. In contrast, the maltose, galactose, sucrose, and fructose regulons, as well as a novel global LacI-family regulator that is predicted to control the central carbohydrate metabolism and arabinose catabolism genes, are universally present in all 10 studied bifidobacteria. A novel group of TetR-family regulators presumably controls the glucoside and galactoside utilization pathways. Paralogs of the ribose repressor RbsR control the pyrimidine nucleoside utilization genes. Multiple paralogs of the maltose regulator MalR co-regulate large sets of genes involved in maltodextrin utilization. The inferred metabolic regulons provide new insights on diverse carbohydrate utilization networks in bifidobacteria that can be employed in metabolic modeling, phenotype prediction and the rational development of novel prebiotics.

MID Max: LC-MS/MS Method for Measuring the Precursor and Product Mass Isotopomer Distributions of Metabolic Intermediates and Cofactors for Metabolic Flux Analysis Applications.

The analytical challenges to acquire accurate isotopic data of intracellular metabolic intermediates for stationary, nonstationary, and dynamic metabolic flux analysis (MFA) are numerous. This work presents MID Max, a novel LC-MS/MS workflow, acquisition, and isotopomer deconvolution method for MFA that takes advantage of additional scan types that maximizes the number of mass isotopomer distributions (MIDs) that can be acquired in a given experiment. The analytical method was found to measure the MIDs of 97 metabolites, corresponding to 74 unique metabolite-fragment pairs (32 precursor spectra and 42 product spectra) with accuracy and precision. The compounds measured included metabolic intermediates in central carbohydrate metabolism and cofactors of peripheral metabolism (e.g., ATP). Using only a subset of the acquired MIDs, the method was found to improve the precision of flux estimations and number of resolved exchange fluxes for wild-type E. coli compared to traditional methods and previously published data sets.

Functional Classification of Uncultured "Candidatus Caldiarchaeum subterraneum" Using the Maple System.

In this study, the metabolic and physiological potential evaluator system based on Kyoto Encyclopedia of Genes and Genomes (KEGG) functional modules was employed to establish a functional classification of archaeal species and to determine the comprehensive functions (functionome) of the previously uncultivated thermophile “Candidatus Caldiarchaeum subterraneum” (Ca. C. subterraneum). A phylogenetic analysis based on the concatenated sequences of proteins common among 142 archaea and 2 bacteria, and among 137 archaea and 13 unicellular eukaryotes suggested that Ca. C. subterraneum is closely related to thaumarchaeotic species. Consistent with the results of the phylogenetic analysis, clustering and principal component analyses based on the completion ratio patterns for all KEGG modules in 79 archaeal species suggested that the overall metabolic and physiological potential of Ca. C. subterraneum is similar to that of thaumarchaeotic species. However, Ca. C. subterraneum possessed almost no genes in the modules required for nitrification and the hydroxypropionate–hydroxybutyrate cycle for carbon fixation, unlike thaumarchaeotic species. However, it possessed all genes in the modules required for central carbohydrate metabolism, such as glycolysis, pyruvate oxidation, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle, as well as multiple sets of sugar and branched chain amino acid ABC transporters. These metabolic and physiological features appear to support the predominantly aerobic character of Ca. C. subterraneum, which lives in a subsurface thermophilic microbial mat community with a heterotrophic lifestyle.

Characterization of three putative xylulose 5-phosphate/fructose 6-phosphate phosphoketolases in the cyanobacterium Anabaena sp. PCC 7120.

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) is a key enzyme in the central carbohydrate metabolism in heterofermentative bacteria, in which enzymatic property of Xfps is well characterized. This is not the case in other microbes. The cyanobacterium Anabaena sp. PCC 7120 possesses three putative genes encoding Xfp, all1483, all2567, and alr1850. We purified three putative Xfps as recombinant proteins. The results of gel filtration indicated that these proteins form homomultimer complex. All1483 and All2567 showed phosphoketolase activity, whereas Alr1850 did not show the activity. Kinetic analyses demonstrated that substrates, fructose 6-phosphate and inorganic phosphate, are cooperatively bound to enzymes positively and negatively, respectively.

Comparative metagenomics demonstrating different degradative capacity of activated biomass treating hydrocarbon contaminated wastewater

This study demonstrates the diverse degradative capacity of activated biomass, when exposed to different levels of total dissolved solids (TDS) using a comparative metagenomics approach. The biomass was collected at two time points to examine seasonal variations. Four metagenomes were sequenced on Illumina Miseq platform and analysed using MG-RAST. STAMP tool was used to analyse statistically significant differences amongst different attributes of metagenomes. Metabolic pathways related to degradation of aromatics via the central and peripheral pathways were found to be dominant in low TDS metagenome, while pathways corresponding to central carbohydrate metabolism, nitrogen, organic acids were predominant in high TDS sample. Seasonal variation was seen to affect catabolic gene abundance as well as diversity of the microbial community. Degradation of model compounds using activated sludge demonstrated efficient utilisation of single aromatic ring compounds in both samples but cyclic compounds were not efficiently utilised by biomass exposed to high TDS.

Influence of frost damage on the sugars and sugar alcohol composition in quince (Cydonia oblonga Mill.) floral nectar

Cold stress adversely affects growth and productivity, and triggers a series of morphological, physiological, biochemical and molecular changes in plants. Since sugars are present in all floral nectars in greater amounts than any other constituent, the aim of this study was to examine how frost exposure changes sugar metabolism and how it affects on the content of sugar components in the nectar of quince. Three quince cultivars (‘Vranjska’, ‘Triumph’ and ‘Leskovacka’) were investigated in this study. The contents of sugars (glucose, fructose, sucrose, trehalose, maltose, isomaltose, rhamnose, arabinose, ribose, melezitose, raffinose, and panose) and sugar alcohols (sorbitol, erythritol, mannitol and galactitol) were analyzed by high performance anion exchange chromatography (HPAEC) with amperometric detection. The results showed that after late spring frosts and irreversible damage of flower parts, the nectar of the three quince cultivars contained elevated levels of fructose, trehalose, arabinose, ribose, rhamnose, raffinose, galactitol and mannitol, indicating an impairment of central carbohydrate metabolism. The ratios between individual sugars, such as the glucose/fructose ratio, were changed in the nectar of damaged flowers in all three quince cultivars. The examined cultivars showed similar sugar response to cold stress. The only exception was ‘Leskovacka’ for the glucose and melezitose pathway, which means that composition of those two sugars changed significantly according to the genotype. The larger are the carbohydrates reserves in different parts of a fruit tree, the higher is the tolerance to any form of frost damage, the results of this study could help in the understanding of how different quince cultivars react to this kind of stress and how they modulate their sugar metabolism.

Whole genome sequencing and analysis of plant growth promoting bacteria isolated from the rhizosphere of plantation crops coconut, cocoa and arecanut.

Coconut, cocoa and arecanut are commercial plantation crops that play a vital role in the Indian economy while sustaining the livelihood of more than 10 million Indians. According to 2012 Food and Agricultural organization's report, India is the third largest producer of coconut and it dominates the production of arecanut worldwide. In this study, three Plant Growth Promoting Rhizobacteria (PGPR) from coconut (CPCRI-1), cocoa (CPCRI-2) and arecanut (CPCRI-3) characterized for the PGP activities have been sequenced. The draft genome sizes were 4.7 Mb (56% GC), 5.9 Mb (63.6% GC) and 5.1 Mb (54.8% GB) for CPCRI-1, CPCRI-2, CPCRI-3, respectively. These genomes encoded 4056 (CPCRI-1), 4637 (CPCRI-2) and 4286 (CPCRI-3) protein-coding genes. Phylogenetic analysis revealed that both CPCRI-1 and CPCRI-3 belonged to Enterobacteriaceae family, while, CPCRI-2 was a Pseudomonadaceae family member. Functional annotation of the genes predicted that all three bacteria encoded genes needed for mineral phosphate solubilization, siderophores, acetoin, butanediol, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinase, phenazine, 4-hydroxybenzoate, trehalose and quorum sensing molecules supportive of the plant growth promoting traits observed in the course of their isolation and characterization. Additionally, in all the three CPCRI PGPRs, we identified genes involved in synthesis of hydrogen sulfide (H2S), which recently has been proposed to aid plant growth. The PGPRs also carried genes for central carbohydrate metabolism indicating that the bacteria can efficiently utilize the root exudates and other organic materials as energy source. Genes for production of peroxidases, catalases and superoxide dismutases that confer resistance to oxidative stresses in plants were identified. Besides these, genes for heat shock tolerance, cold shock tolerance and glycine-betaine production that enable bacteria to survive abiotic stress were also identified.

Comparative genomics and evolution of regulons of the LacI-family transcription factors.

DNA-binding transcription factors (TFs) are essential components of transcriptional regulatory networks in bacteria. LacI-family TFs (LacI-TFs) are broadly distributed among certain lineages of bacteria. The majority of characterized LacI-TFs sense sugar effectors and regulate carbohydrate utilization genes. The comparative genomics approaches enable in silico identification of TF-binding sites and regulon reconstruction. To study the function and evolution of LacI-TFs, we performed genomics-based reconstruction and comparative analysis of their regulons. For over 1300 LacI-TFs from over 270 bacterial genomes, we predicted their cognate DNA-binding motifs and identified target genes. Using the genome context and metabolic subsystem analyses of reconstructed regulons, we tentatively assigned functional roles and predicted candidate effectors for 78 and 67% of the analyzed LacI-TFs, respectively. Nearly 90% of the studied LacI-TFs are local regulators of sugar utilization pathways, whereas the remaining 125 global regulators control large and diverse sets of metabolic genes. The global LacI-TFs include the previously known regulators CcpA in Firmicutes, FruR in Enterobacteria, and PurR in Gammaproteobacteria, as well as the three novel regulators—GluR, GapR, and PckR—that are predicted to control the central carbohydrate metabolism in three lineages of Alphaproteobacteria. Phylogenetic analysis of regulators combined with the reconstructed regulons provides a model of evolutionary diversification of the LacI protein family. The obtained genomic collection of in silico reconstructed LacI-TF regulons in bacteria is available in the RegPrecise database (http://regprecise.lbl.gov). It provides a framework for future structural and functional classification of the LacI protein family and identification of molecular determinants of the DNA and ligand specificity. The inferred regulons can be also used for functional gene annotation and reconstruction of sugar catabolic networks in diverse bacterial lineages.

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

The metabolism of Archaea, the third domain of life, resembles in its complexity those of Bacteria and lower Eukarya. However, this metabolic complexity in Archaea is accompanied by the absence of many "classical" pathways, particularly in central carbohydrate metabolism. Instead, Archaea are characterized by the presence of unique, modified variants of classical pathways such as the Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway. The pentose phosphate pathway is only partly present (if at all), and pentose degradation also significantly differs from that known for bacterial model organisms. These modifications are accompanied by the invention of "new," unusual enzymes which cause fundamental consequences for the underlying regulatory principles, and classical allosteric regulation sites well established in Bacteria and Eukarya are lost. The aim of this review is to present the current understanding of central carbohydrate metabolic pathways and their regulation in Archaea. In order to give an overview of their complexity, pathway modifications are discussed with respect to unusual archaeal biocatalysts, their structural and mechanistic characteristics, and their regulatory properties in comparison to their classic counterparts from Bacteria and Eukarya. Furthermore, an overview focusing on hexose metabolic, i.e., glycolytic as well as gluconeogenic, pathways identified in archaeal model organisms is given. Their energy gain is discussed, and new insights into different levels of regulation that have been observed so far, including the transcript and protein levels (e.g., gene regulation, known transcription regulators, and posttranslational modification via reversible protein phosphorylation), are presented.

Mutations in SNF1 complex genes affect yeast cell wall strength

The trimeric SNF1 complex from Saccharomyces cerevisiae, a homolog of mammalian AMP-activated kinase, has been primarily implicated in signaling for the utilization of alternative carbon sources to glucose. We here find that snf1 deletion mutants are hypersensitive to different cell wall stresses, such as the presence of Calcofluor white, Congo red, Zymolyase or the glucan synthase inhibitor Caspofungin in the growth medium. They also have a thinner cell wall. Caspofungin treatment triggers the phosphorylation of the catalytic Snf1 kinase subunit at Thr210 and removal of this phosphorylation site by mutagenesis (Snf1-T210A) abolishes the function of Snf1 in cell wall integrity. Deletion of the PFK1 gene encoding the α-subunit of the heterooctameric yeast phosphofructokinase suppresses the cell wall phenotypes of a snf1 deletion, which suggests a compensatory effect of central carbohydrate metabolism. Epistasis analyses with mutants in cell wall integrity (CWI) signaling confirm that the SNF1 complex and the CWI pathway independently affect yeast cell integrity.