108 research outputs found

    LUD, a new protein domain associated with lactate utilization.

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    BackgroundA novel highly conserved protein domain, DUF162 [Pfam: PF02589], can be mapped to two proteins: LutB and LutC. Both proteins are encoded by a highly conserved LutABC operon, which has been implicated in lactate utilization in bacteria. Based on our analysis of its sequence, structure, and recent experimental evidence reported by other groups, we hereby redefine DUF162 as the LUD domain family.ResultsJCSG solved the first crystal structure [PDB:2G40] from the LUD domain family: LutC protein, encoded by ORF DR_1909, of Deinococcus radiodurans. LutC shares features with domains in the functionally diverse ISOCOT superfamily. We have observed that the LUD domain has an increased abundance in the human gut microbiome.ConclusionsWe propose a model for the substrate and cofactor binding and regulation in LUD domain. The significance of LUD-containing proteins in the human gut microbiome, and the implication of lactate metabolism in the radiation-resistance of Deinococcus radiodurans are discussed

    Unravelling anaerobic metabolisms in hypersaline sediment

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    The knowledge on the microbial diversity inhabiting hypersaline sediments is still limited. In particular, existing data about anaerobic hypersaline archaea and bacteria are scarce and refer to a limited number of genera. The approach to obtain existing information has been almost exclusively attempting to grow every organism in axenic culture on the selected electron acceptor with a variety of electron donors. Here, a different approach has been used to interrogate the microbial community of submerged hypersaline sediment of Salitral Negro, Argentina, aiming at enriching consortia performing anaerobic respiration of different electron acceptor compounds, in which ecological associations can maximize the possibilities of successful growth. Growth of consortia was demonstrated on all offered electron acceptors, including fumarate, nitrate, sulfate, thiosulfate, dimethyl sulfoxide, and a polarized electrode. Halorubrum and Haloarcula representatives are here shown for the first time growing on lactate, using fumarate or a polarized electrode as the electron acceptor; in addition, they are shown also growing in sulfate-reducing consortia. Halorubrum representatives are for the first time shown to be growing in nitrate-reducing consortia, probably thanks to reduction of N2O produced by other consortium members. Fumarate respiration is indeed shown for the first time supporting growth of Halanaeroarchaeum and Halorhabdus belonging to the archaea, as well as growth of Halanaerobium, Halanaerobaculum, Sporohalobacter, and Acetohalobium belonging to the bacteria. Finally, evidence is presented suggesting growth of nanohaloarchaea in anaerobic conditions.Fil: Solchaga, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Busalmen, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Nercessian, Debora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

    The complete genome sequence of DSM 755

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    Comparative genomics and proteomics of Eubacterium maltosivorans : functional identification of trimethylamine methyltransferases and bacterial microcompartments in a human intestinal bacterium with a versatile lifestyle

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    Eubacterium maltosivorans YIT is a human intestinal isolate capable of acetogenic, propionogenic and butyrogenic growth. Its 4.3-Mb genome sequence contains coding sequences for 4227 proteins, including 41 different methyltransferases. Comparative proteomics of strain YIT showed the Wood-Ljungdahl pathway proteins to be actively produced during homoacetogenic growth on H-2 and CO2 while butyrogenic growth on a mixture of lactate and acetate significantly upregulated the production of proteins encoded by the recently identified lctABCDEF cluster and accessory proteins. Growth on H-2 and CO2 unexpectedly induced the production of two related trimethylamine methyltransferases. Moreover, a set of 16 different trimethylamine methyltransferases together with proteins for bacterial microcompartments were produced during growth and deamination of the quaternary amines, betaine, carnitine and choline. Growth of strain YIT on 1,2-propanediol generated propionate with propanol and induced the formation of bacterial microcompartments that were also prominently visible in betaine-grown cells. The present study demonstrates that E. maltosivorans is highly versatile in converting low-energy fermentation end-products in the human gut into butyrate and propionate whilst being capable of preventing the formation of the undesired trimethylamine by converting betaine and other quaternary amines in bacterial microcompartments into acetate and butyrate.Peer reviewe

    Sugar metabolism in archaea: Analyses of allosteric regulation of pyruvate kinases and degradation of the deoxy sugar L-rhamnose.

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    In der vorliegenden Arbeit wurden zwei Fragestellungen zum Zuckerstoffwechseln in Archaeen untersucht. Zum einen wurde die allosterische Regulation von archaeellen Pyruvat Kinasen (PKs) sowie deren Evolution analysiert. Zum anderen wurde der Abbau des Desoxy-Zuckers L-Rhamnose in halophilen und hyperthermophilen Archaeen untersucht: PKs aus Bacteria und Eukarya werden durch Fructose-1,6-Bisphosphat oder AMP allosterisch aktiviert. In dieser Arbeit wurde gezeigt, dass 3-Phosphoglycerat (3PG) ein neuartiger allosterischer Aktivator der PKs von hyperthermophilen Archaeen der Ordnung Thermoproteales ist. Außerdem wurde ein 3PG-Bindemotiv identifiziert, mit dem es möglich ist Vorhersagen über die allosterische Aktivierbarkeit von PKs mit 3PG zu treffen. Für die PKs aus hyperthermophilen methanogenen Archaeen wurde eine allosterische Aktivierung durch den klassischen Aktivator AMP nachgewiesen. Im zweiten Teil dieser Arbeit wurde gezeigt, dass das halophile Euryarchaeon Haloferax volcanii Rhamnose über einen ABC-Transporter aufnimmt und oxidativ über einen Diketo-Hydrolase Weg zu Pyruvat und L-Lactat abbaut. Die Transkription der Gene der Rhamnose-Aufnahme und des –Abbaus werden durch den Transkriptionsregulator RhcR aktiviert. Ein Intermediat des Rhamnose-Abbaus wurde als Signalmolekül von RhcR identifiziert. Die Untersuchungen umfassen die Charakterisierung der entsprechenden Enyzme, Transkriptionsanalysen der beteiligten Gene sowie Wachstumsexperimente mit Knockout Mutanten. Weiterhin wurde gezeigt, dass der Diketo-Hydrolase Weg auch von den hyperthermophilen Crenarchaeota Vulcanisaeta distributa und Sulfolobus solfataricus für den Abbau von Rhamnose verwendet wird.In this thesis two questions concerning the sugar metabolism in archaea have been analysed. First, the allosteric regulation of archaeal pyruvate kinases (PKs) and their evolution were studied. Second, the degradation of the deoxy sugar L-rhamnose in halophilic and hyperthermophilic archaea was analysed: PKs from bacteria and eukarya are allosteric enzymes activated by fructose-1,6-bisphosphate or AMP. In this work, it is shown that PKs from hyperthermophilic archaea of the order Thermoproteales are allosterically activated by the novel effector 3-phosphoglycerate (3PG). A 3PG-binding motif could be identified, which allows to predict the allosteric regulation of PKs by 3PG. PKs from hyperthermophilic methanoarchaea were found to be activated by AMP, the classical activator from bacteria. In the second part of this work, it is shown that the halophilic euryarchaeon Haloferax volcanii takes up rhamnose by an ABC transporter and degrades rhamnose oxidatively to pyruvate and L-lactate via the diketo-hydrolase pathway. The genes involved in rhamnose uptake and degradation form a gene cluster, whose transcription is regulated by the transcriptional activator RhcR. An intermediate of rhamnose degradation was identified as the inducer molecule of RhcR. The study includes characterization of the respective enzymes, transcriptional analyses of involved genes and growth experiments with knockout mutants. Further, it is shown that the hyperthermophilic crenarchaeota Vulcanisaeta distributa and Sulfolobus solfataricus also degrades rhamnose via the diketo-hydrolase pathway

    Sulfate-Reducing Bacteria That Produce Exopolymers Thrive in the Calcifying Zone of a Hypersaline Cyanobacterial Mat

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    Calcifying microbial mats in hypersaline environments are important model systems for the study of the earliest ecosystems on Earth that started to appear more than three billion years ago and have been preserved in the fossil record as laminated lithified structures known as stromatolites. It is believed that sulfate-reducing bacteria play a pivotal role in the lithification process by increasing the saturation index of calcium minerals within the mat. Strain L21-Syr-ABT was isolated from anoxic samples of a several centimeters-thick microbialite-forming cyanobacterial mat of a hypersaline lake on the Kiritimati Atoll (Kiribati, Central Pacific). The novel isolate was assigned to the family Desulfovibrionaceae within the Deltaproteobacteria. Available 16S rRNA-based population surveys obtained from discrete layers of the mat indicate that the occurrence of a species-level clade represented by strain L21-Syr-ABT is restricted to a specific layer of the suboxic zone, which is characterized by the presence of aragonitic spherulites. To elucidate a possible function of this sulfate-reducing bacterium in the mineral formation within the mat a comprehensive phenotypic characterization was combined with the results of a comparative genome analysis. Among the determined traits of strain L21-Syr-ABT, several features were identified that could play a role in the precipitation of calcium carbonate: (i) the potential deacetylation of polysaccharides and consumption of substrates such as lactate and sulfate could mobilize free calcium; (ii) under conditions that favor the utilization of formate and hydrogen, the alkalinity engine within the mat is stimulated, thereby increasing the availability of carbonate; (iii) the production of extracellular polysaccharides could provide nucleation sites for calcium mineralization. In addition, our data suggest the proposal of the novel species and genus Desulfohalovibrio reitneri represented by the type strain L21-Syr-ABT (=DSM 26903T = JCM 18662T)

    Identification and characterisation of rumen bacteria with prominent roles in the ruminal metabolism of forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Microbiology and Genetics) at Massey University, Palmerston North, New Zealand

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    Foigures 1.2, 1.4, 1.5 & 1.6 are re-used with permission.This thesis documents the characterisation of two groups of rumen bacteria that are both prominent in forage-fed ruminants, with the aim to better understand their roles in ruminal metabolism. The first group, referred to as the R-7 group, has in recent years been shown to be one of the most abundant rumen bacterial groups, though the few isolated representative strains available were uncharacterised. Two strains of the group included in the Hungate1000 culture collection, R-7 and WTE2008, were selected for characterisation. To facilitate phylogenetic analyses of this group, the complete genomes of an additional three previously isolated R-7 group strains were sequenced. Genomic, phylogenetic and phenotypic characterisation of R-7 and WTE2008 demonstrated that despite their 16S rRNA gene sequences sharing 98.6-99.0% nucleotide identity, their genome-wide average nucleotide identity of 84% assigned them as separate species of a novel genus and family of the proposed order ‘Christensenellales’ using the Genome Taxonomy Database. Phenotypic characterisation showed that the strains were identical in morphology, and both possessed the ability to degrade plant cell wall polysaccharides xylan and pectin, but not cellulose. Acetate, ethanol, hydrogen and lactate were produced by both strains, though R-7 produced greater amounts of hydrogen than WTE2008, which instead produced more lactate. Based on these analyses, it is proposed that R-7 and WTE2008 belong to separate species (Aristaeella gen. nov. hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively) of a newly proposed family (Aristaeellaceae fam. nov.). The second bacterial group of interest, due to their dominant role in ruminal propionate production, was the Prevotella 1 group, following analyses of metatranscriptome datasets of rumen microbial communities of lucerne-fed sheep for dominant community members that express propionate pathway genes from succinate. Screening of 14 strains spanning the diversity of Prevotella 1 found that all except one P. brevis strain produced propionate in a cobalamin (vitamin B12)-dependent manner. To better understand the pathway and regulation of propionate production from succinate, a comparative multi-omics approach was used to test the hypothesis that propionate production is regulated by a cobalamin-binding riboswitch. Scanning of a completed genome assembly of Prevotella ruminicola KHP1 identified four ‘cobalamin’ family riboswitches. However, the riboswitches were not in close proximity to genes putatively involved in converting succinate to propionate, nor were these genes arranged in a single operon. Comparative genomics of the 14 screened strains found that all strains possessed all homologues of candidate propionate pathway genes identified in the KHP1 genome. However, the 13 propionate-producing strains possessed a putative transporter and three subunits encoding a putative methylmalonyl-CoA decarboxylase upstream but antisense to two genes encoding methylmalonyl-CoA mutase subunits, whereas the non-producing strain did not. Comparative transcriptomics and proteomics of KHP1 cultures in the presence and absence of cobalamin demonstrated that some gene candidates were upregulated by cobalamin at the transcriptome level, including co-located genes annotated as phosphate butyryltransferase and butyrate kinase, despite the strain not producing butyrate, suggesting that propionate production may occur via propionyl phosphate. However, only both subunits of methylmalonyl-CoA mutase showed greater transcript and protein abundances in the presence of cobalamin. These results show that while some propionate pathway candidate genes were differentially expressed between cobalamin treatments, they did not appear to be under direct control of a cobalamin-binding riboswitch. This study has contributed to our understanding of the roles of both Aristaeellaceae fam. nov. and Prevotella 1 in ruminal metabolism

    Molecular regulation of the cardiac-enriched acetyl-CoA carboxylase isoform (ACCβ) : a novel target for therapeutic interventions in cardiovascular disease

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    Includes bibliographical references (leaves 130-173).Metabolic remodeling is thought to be an important contributor towards the development of various cardiac pathophysiologic conditions. Therefore, studies attempting to delineate undenying mechanisms driving cardiac metabolic remodeling represent an important initiative toward the development of novel therapeutic interventions. To further investigate the role of metabolic substrate switches in the heart, we focused on a pivotal, rate-limiting step of cardiac fatty acid metabolism i.e. an upstream modulator of long-chain fatty acid importation into the mitochondrion. In the heart, long-chain fatty acids are transported into the mitochondrion by the rate-limiting enzyme, carnitine palmitoyl transferase 1 (CPT1). CPT1 is potently inhibited by malonyl-CoA, the product of the acetylCoA carboxylation reaction that is catalyzed by acetyl-CoA carboxylase (ACC). Recent studies have demonstrated that metabolic fuels such as fatty acids and glucose can function as signaling ligands, directing transcriptional regulation of numerous metabolic genes. However, transcriptional mechanisms directing the gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCβ) are less well understood. Previously, four E-box (CANNTG) sequence motifs were identified on the human ACCβ promoter. Since E-boxes act as binding sites for upstream stimulatory factors (US F), putative glucose-responsive transcriptional modulators, we hypothesized that ACCβ is induced by USF1 in a glucosedependent manner. To investigate this, we began by acutely fasting and subsequently refeeding Balb/C mice with a carbohydrate-enriched diet. Here, high carbohydrate feeding resulted in elevated systemic glucose levels associated with increased cardiac ACCβ gene and protein expression. To further explore these interesting findings, we tranSiently cotransfected neonatal card iom yocytes , H9C2 myoblasts, CV-1 fibroblasts and HepG2 hepatocytes with the full-length and deletion constructs of the human ACC[3 gene promoter together with a putative activator and repressor expression vector, respectively: a) USF1 (glucose-responsive transcription factor) - the rationale that it should elevate ACCβ gene promoter activity in accordance with the glucose-fatty acid cycle, and b) nuclear respiratory factor 1(NRF1) - the hypothesis being that this mitochondrial biogenesis and β-oxidation enhancing modulator would be expected to attenuate ACCβ promoter activity in order to increase fatty acid oxidation capacity

    Characterisation of the interaction between Neisseria meningitidis and human polymorphonuclear leukocytes

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    During infection with Neisseria meningitidis, an important causative agent of bacterial meningitis and septicaemia, the host innate immune system clears bacteria by complement-mediated lysis and phagocytosis. In order to evade phagocytosis, the bacterium expresses a number of surface components, including a polysaccharide capsule and sialylated lipopolysaccharide (LPS). The aim of this project was to investigate the influence of bacterial metabolism and DNA repair on the interaction with polymorphonuclear leukocytes (PMNs). Results demonstrated reduced expression of the polysaccharide capsule and lowered LPS sialylation in a strain (ΔlctP) unable to acquire exogenous lactate, but no effect in a strain unable to utilise glutamate (ΔperM). These changes were associated with increased phagocytosis of fixed bacteria by the human PMN cell line, HL60. Further investigations showed that capsule expression in the ΔlctP strain was restored to wild-type levels following exposure of live bacteria to PMNs. Next, assays were established using primary human PMNs to investigate post-phagocytic events. The wild-type meningococcus survives within PMNs and is able to delay host cell apoptosis. Furthermore, the ΔlctP and ΔperM strains were significantly killed by PMNs, indicating a role for acquisition of both lactate and glutamate for survival in PMNs. Killing was dependent on actin polymerisation and the PMN oxidative burst. The role of the meningococcal Base Excision Repair (BER) DNA repair pathway was also investigated through characterisation of two Apurinic / Apyrimidinic (AP) endonuclease paralogues, NExo and NApe. Both exhibit distinct biochemical functions and are required for resistance against oxidative stress. A strain lacking both these enzymes, which is attenuated in vivo, was significantly killed by human PMNs as a result of the oxidative burst
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