Carbohydrate Metabolism in Bacteria: Alternative Specificities in ADP-Glucose Pyrophosphorylases Open Novel Metabolic Scenarios and Biotechnological Tools

We explored the ability of ADP-glucose pyrophosphorylase (ADP-Glc PPase) from different bacteria to use glucosamine (GlcN) metabolites as a substrate or allosteric effectors. The enzyme from the actinobacteria Kocuria rhizophila exhibited marked and distinctive sensitivity to allosteric activation by GlcN-6P when producing ADP-Glc from glucose-1-phosphate (Glc-1P) and ATP. This behavior is also seen in the enzyme from Rhodococcus spp., the only one known so far to portray this activation. GlcN-6P had a more modest effect on the enzyme from other Actinobacteria (Streptomyces coelicolor), Firmicutes (Ruminococcus albus), and Proteobacteria (Agrobacterium tumefaciens) groups. In addition, we studied the catalytic capacity of ADP-Glc PPases from the different sources using GlcN-1P as a substrate when assayed in the presence of their respective allosteric activators. In all cases, the catalytic efficiency of Glc-1P was 1–2 orders of magnitude higher than GlcN-1P, except for the unregulated heterotetrameric protein (GlgC/GgD) from Geobacillus stearothermophilus. The Glc-1P substrate preference is explained using a model of ADP-Glc PPase from A. tumefaciens based on the crystallographic structure of the enzyme from potato tuber. The substrate-binding domain localizes near the N-terminal of an α-helix, which has a partial positive charge, thus favoring the interaction with a hydroxyl rather than a charged primary amine group. Results support the scenario where the ability of ADP-Glc PPases to use GlcN-1P as an alternative occurred during evolution despite the enzyme being selected to use Glc-1P and ATP for α-glucans synthesis. As an associated consequence in such a process, certain bacteria could have improved their ability to metabolize GlcN. The work also provides insights in designing molecular tools for producing oligo and polysaccharides with amino moieties

Understanding the Regulation of Metabolic Enzyme Acetylation in E. Coli

Global protein acetylation is a newly discovered phenomenon in bacteria. Of the more than 250 acetylations reported in E. coli, many are of metabolic enzymes. Thus, acetylation could represent a novel posttranslational mechanism of metabolic control. Yet, almost nothing is known about the regulation of these acetylations or of their metabolic outcomes. Here, we report that the cAMP receptor protein (CRP) regulates protein acetylation in E. coli and provide evidence that protein acetylation modulates the flux of carbon through central metabolism. When we grew cells in mixed amino acids supplemented with glucose and cAMP, global protein acetylation increased in a CRP-dependent manner and several of the acetylated proteins were central metabolic enzymes. Much of this CRP-mediated acetylation required activation region 1 (AR1), a surface patch that allows CRP to interact with RNA polymerase. A second surface patch (AR2) also was involved, albeit to a lesser degree. These results raise the possibility that CRP might regulate the transcription of a protein acetyltransferase. Indeed, a recent report suggested that CRP might regulate transcription of the protein acetyltransferase YfiQ (also known as Pat) by a mechanism that would require AR2. We further obtained bioinformatic evidence that supports the hypothesis that CRP also could regulate yfiQ transcription in an AR1-dependent manner. Since CRP regulates metabolism, we asked if YfiQ could influence metabolism. Using Phenotype MicroArray analysis, we found that a yfiQ null mutant exhibits a distinctive defect during growth on gluconeogenic carbon sources and a distinct advantage during growth on a carbon source that bypasses the need for gluconeogenesis. In vitro acetylation assays identified four substrates of YfiQ. Three YfiQ substrates were the strictly irreversible glycolytic enzymes PfkA, PfkB, and LpdA. The fourth was CRP itself. We thus hypothesize that CRP activates yfiQ transcription, whose protein product acetylates a subset of metabolic enzymes, altering their function and shifting the balance between glycolysis and gluconeogenesis. We further propose that YfiQ acetylates CRP. Efforts to determine how this acetylation affects the CRP-dependent transcriptome are underway

Metabolic engineering and modelling of Escherichia coli for the production of succinate

Current climate issues and the ongoing depletion of oil reserves have led to an increased attention for biobased production processes. Not only the production of bio-energy but also biochemicals have gained interest. Recent reports of the US department of energy and the GROWTH program of the European commission review a comprehensive list of chemicals that can be produced via biological processes and which may be of great importance to sustain a green chemical industry in the future. Succinate is one of those biochemicals. Today, this compound is synthesised via maleic anhydride, which is produced by a petrochemical production process. The conditions which a biological production processes have to meet to be economically viable are quite strict. Such a process has to obtain a yield of 0.88 g/g, a rate between 1.8 and 2.5 g/l/h and a titer around 80 g/l. None of the available (reported) processes reach either of these values. In most cases the rate and the titer are still a problem. To optimise succinate production via metabolic engineering, first a mutation strategy has to be developed. This strategy can then be applied to a suitable production host. The choice of this host has nowadays become less important due to the recent developments in genetic engineering and synthetic biology. These developments allow the introduction or altering of almost every cellular function. What has become important is the availability of information on the potential host and its genetic accessibility. E. coli is therefore still an excellent host for the development of production processes. Since its isolation vast amounts of information have been gathered and several biological databases are devoted to it. Moreover, almost each cellular function has been modified. However, E. coli does not naturally produce succinate in large amounts. It will have to undergo some genetic modifications to overproduce this chemical. Which modifications are needed can be uncovered in silico. A functional and comparative genomics analyses of natural producing and non-producing strains revealed which genes and reactions may influence succinate production. The optimal biochemical route towards succinate is then uncovered via stoichiometric network analysis. For this analysis, elementary flux modes was combined with partial least squares regression. Both tools resulted in the identification of optimal biochemical production routes for several substrates and allowed to evaluate how reactions that do not naturally occur in E. coli may affect the succinate yield. The transport reaction is one of the reactions that could be identified by the EFM-PLS model. E. coli possesses both succinate import as well as export proteins. However, export is normally only active under anaerobic conditions and import under aerobic conditions. Therefore, the import protein was knocked out and the export protein was expressed with an artificial promoter. These modifications led to an increased succinate yield and production rate, but also revealed alternative import proteins. An analysis of the phenotype of mutant strains in these alternative importers did however not lead to increases in succinate yield. These mutations influenced biomass yield and growth rate. A second route that was identified in the stoichiometric network analysis was the glyoxylate route. This route correlated positively with succinate production and is strongly regulated by the transcription factors ArcA and IclR. In order to gain more insight into the synergy that may exist between both regulators, knock outs in both genes were studied under chemostat and batch conditions. This analysis revealed a synergetic effect between both proteins on the biomass yield. A strain in which both arcA and iclR are knocked out showed a biomass yield that approached the maximal theoretical yield. The single knock out strains did not have such an outspoken phenotype. Finally, several mutations were introduced and evaluated for succinate production and byproduct formation. The formation of acetate was studied in detail to uncover alternative acetate formation reactions. First, the known reactions, acetate kinase, phospho-acetyltransferase and pyruvate oxidase were knocked out. This resulted in a significant decrease in acetate production but not in the total elimination. Several alternatives such as citrate lyase and acetate CoA-transferase were evaluated, but without success. The remaining acetate formation reactions could not be identified. Succinate dehydrogenase can be seen as one of the most crucial enzymes for succinate production. This enzyme converts succinate into fumarate and therefore has to be knocked out to increase production. Strains that possess a succinate dehydrogenase deletion immediately show an increased production. However, pyruvate becomes one of the main byproducts. Several enzymes influence pyruvate production. The most important enzymes in the context of succinate production are PEP carboxykinase, oxaloacetate decarboxylase, malic enzyme, PEP carboxylase, and citrate synthase. The three former reactions are gluconeogenic reactions that can form futile cycles. Deletions in these genes resulted in an increase in biomass yield due to a more energy efficient metabolism, but does not increase succinate yield. Point mutations in PEP carboxylase and citrate synthase increased the flux towards the TCA cycle. The flux ratio between the glyoxylate pathway and the reductive and oxidative TCA cycle can be influenced by these enzymes. The activity of the reductive TCA is however strongly dependent on the availability of reduced equivalents. To modulate this availability a point mutation was introduced in FNR, an anaerobic transcription factor that activates the reductive TCA and represses the electron transport chain. Although none of the developed strains are economically viable yet, many of the mutations that have been introduced show great promise for future improvements. In fact, the next steps in strain development should not be to identify new targets to modify, but rather to fine tune activities of the routes towards succinate in such a way that the theoretical yields can be approached with sufficiently high rates

Streptomyces cell-free systems for natural product discovery and engineering

Bacteria are a major microbial source of natural products, which are encoded within so-called biosynthetic gene clusters (BGCs). This highlight discusses the emergence of native s cell-free systems as a new tool to accelerate the study of the fundamental chemistry and biology of natural product biosynthesis from these bacteria. Cell-free systems provide a prototyping platform to study plug-and-play reactions in microscale reactions. So far, s cell-free systems have been used to rapidly characterise gene expression regulation, access secondary metabolite biosynthetic enzymes, and catalyse cell-free transcription, translation, and biosynthesis of example natural products. With further progress, we anticipate the development of more complex systems to complement existing experimental tools for the discovery and engineering of natural product biosynthesis from and related high G + C (%) bacteria

The Role of the Glyoxylate Cycle in the Pathogenesis of Mycobacterium tuberculosis

According to the World Health Organization a third of the world\u27s population is infected with Mycobacterium tuberculosis. The unparalleled success of M. tuberculosis as a pathogen reflects the bacterium\u27s extraordinary ability to persist in its host in spite of eliciting a robust immune response. Currently available treatment is inadequate and drug resistance is rapidly spreading. New antibiotics are desperately needed. The substrates and metabolic pathways utilized by pathogens during infection are largely unknown and represent an under-exploited area of investigation. Uniquely, evolution of the genus Mycobacterium has involved extensive duplication of fatty acid metabolism genes, including two homologs encoding prokaryotic- and eukaryotic-like isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL). The glyoxylate cycle is employed by cells when fatty acids are the main carbon source available. Here, we show that these enzymes are jointly required by M. tuberculosis for growth on fatty acids and for virulence in experimental infections. Although deletion of icll or icl2 had little impact on replication of M. tuberculosis in macrophages and mice, deletion of both genes abrogated intracellular growth, and resulted in rapid bacterial clearance from the lungs. A dual-specificity ICL inhibitor similarly blocked replication of M. tuberculosis on fatty acids in vitro and in macrophages. The absence of ICL orthologs in mammals, and recent findings implicating the glyoxylate pathway in the virulence of other bacterial and fungal pathogens makes this metabolic pathway an attractive novel target for drug development

Research into cancer metabolomics: towards a clinical metamorphosis

The acknowledgement that metabolic reprogramming is a central feature of cancer has generated high expectations for major advances in both diagnosis and treatment of malignancies through addressing metabolism. These have so far only been partially fulfilled, with only a few clinical applications. However, numerous diagnostic and therapeutic compounds are currently being evaluated in either clinical trials or pre-clinical models and new discoveries of alterations in metabolic genes indicate future prognostic or other applicable relevance. Altogether, these metabolic approaches now stand alongside other available measures providing hopes for the prospects of metabolomics in the clinic. Here we present a comprehensive overview of both ongoing and emerging clinical, pre-clinical and technical strategies for exploiting unique tumour metabolic traits, highlighting the current promises and anticipations of research in the field

A novel role for the transcriptional modulator NusA in DNA repair/damage tolerance pathways in Escherichia coli

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.Includes bibliographical references.All organisms must contend with the consequences of DNA damage, induced by a variety of both endogenous and exogenous sources. Mechanisms of DNA repair and DNA damage tolerance are crucial for cellular survival after DNA damage. Translesion DNA synthesis (TLS) is one such mechanism of DNA damage tolerance which utilizes a specialized translesion DNA polymerase capable of catalyzing DNA synthesis on imperfect templates. There are two TLS polymerases present in Escherichia coli encoded by the dinB (Pol IV) and umuDC (Pol V) gene products. While TLS polymerases provide a variety of benefits to the cell, it is important that they are properly regulated as they have reduced fidelity on undamaged DNA compared to replicative DNA polymerases. Here I present evidence that the essential transcriptional modulator NusA associates with TLS polymerases in E. coli both physically, as noted for DinB, and genetically, with DinB and the umuDC gene products. Mutation of nusA renders cells sensitive to DNA damaging agents and produces phenotypes reminiscent of mutants with altered DNA processing. Moreover, I report that the nusAll mutation completely eliminates the formation of adaptive mutants, revealing that nusA+ function is required for cells to adapt and mutate in response to stress. Though the phenomenon of adaptive mutagenesis also requires dinB+, my data suggest that the role for nusA in adaptive mutagenesis extends beyond an interaction with DinB.(cont.) Furthermore, I report that NusA in addition to having a role in transcription elongation is also important for promoting survival after DNA damage. Phenotypes of nusA mutants are more exaggerated than those of TLS polymerase mutants. Genetic interactions of nusA+ with the nucleotide excision repair pathway suggest that nusA+ may play a role in a new class of NusA-dependent transcription coupled repair. Moreover, I have isolated RNA polymerase mutants with altered ability to survive after DNA damage, and this altered ability is absolutely dependent on nusA+ and uvrA+. The completion of translesion DNA synthesis requires both the insertion of a nucleotide opposite the adducted template base and extension from that position by several subsequent nucleotide additions. We present evidence that DinB is specialized to perform strikingly proficient extension after insertion opposite an N2-dG lesion. Our data indicate that cellular survival is coupled to completion of TLS and regulation of these precise steps in vivo is genetically complex and involves the toxin-antitoxin module MazEF and the iron import protein TonB.by Susan E. Cohen.Ph.D

AMPK in Pathogens

During host–pathogen interactions, a complex web of events is crucial for the outcome of infection. Pathogen recognition triggers powerful cellular signaling events that is translated into the induction and maintenance of innate and adaptive host immunity against infection. In opposition, pathogens employ active mechanisms to manipulate host cell regulatory pathways toward their proliferation and survival. Among these, subversion of host cell energy metabolism by pathogens is currently recognized to play an important role in microbial growth and persistence. Extensive studies have documented the role of AMP-activated protein kinase (AMPK) signaling, a central cellular hub involved in the regulation of energy homeostasis, in host–pathogen interactions. Here, we highlight the most recent advances detailing how pathogens hijack cellular metabolism by suppressing or increasing the activity of the host energy sensor AMPK. We also address the role of lower eukaryote AMPK orthologues in the adaptive process to the host microenvironment and their contribution for pathogen survival, differentiation, and growth. Finally, we review the effects of pharmacological or genetic AMPK modulation on pathogen growth and persistence.CIHR -Canadian Institutes of Health Researc