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27 Publications visible to you, out of a total of 27
Geoarchaeota: a new candidate phylum in the Archaea from high-temperature acidic iron mats in Yellowstone National Park
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Geothermal systems in Yellowstone National Park (YNP) provide an outstanding opportunity to understand the origin and evolution of metabolic processes necessary for life in extreme environments including … low pH, high temperature, low oxygen and elevated concentrations of reduced iron. Previous phylogenetic studies of acidic ferric iron mats from YNP have revealed considerable diversity of uncultivated and undescribed archaea. The goal of this study was to obtain replicate de novo genome assemblies for a dominant archaeal population inhabiting acidic iron-oxide mats in YNP. Detailed analysis of conserved ribosomal and informational processing genes indicates that the replicate assemblies represent a new candidate phylum within the domain Archaea referred to here as 'Geoarchaeota' or 'novel archaeal group 1 (NAG1)'. The NAG1 organisms contain pathways necessary for the catabolism of peptides and complex carbohydrates as well as a bacterial-like Form I carbon monoxide dehydrogenase complex likely used for energy conservation. Moreover, this novel population contains genes involved in the metabolism of oxygen including a Type A heme copper oxidase, a bd-type terminal oxidase and a putative oxygen-sensing protoglobin. NAG1 has a variety of unique bacterial-like cofactor biosynthesis and transport genes and a Type3-like CRISPR system. Discovery of NAG1 is critical to our understanding of microbial community structure and function in extant thermophilic iron-oxide mats of YNP, and will provide insight regarding the evolution of Archaea in early Earth environments that may have important analogs active in YNP today.

Authors: M. A. Kozubal, , R. d. Jennings, Z. J. Jay, S. G. Tringe, D. B. Rusch, J. P. Beam, L. A. McCue, W. P. Inskeep

Date Published: 15th Nov 2012

Publication Type: Not specified

Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one … of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

Authors: L. Galeazzi, P. Bocci, A. Amici, L. Brunetti, S. Ruggieri, , S. Reed, A. L. Osterman, D. A. Rodionov, L. Sorci, N. Raffaelli

Date Published: 27th Sep 2011

Publication Type: Not specified

Control of proteobacterial central carbon metabolism by the HexR transcriptional regulator: a case study in Shewanella oneidensis
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated the HexR transcriptional factor as … a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of the Cra regulator characteristic of Enterobacteriales. In this study, we combined a large scale comparative genomic reconstruction of HexR-controlled regulons in 87 species of Proteobacteria with the detailed experimental analysis of the HexR regulatory network in the Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1 to 2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa was confirmed as a HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6-phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatic analysis and experimentally verified by changed gene expression pattern in S. oneidensis DeltahexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using [(13)C]lactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).

Authors: S. A. Leyn, X. Li, Q. Zheng, P. S. Novichkov, S. Reed, , , C. Yang, A. L. Osterman, D. A. Rodionov

Date Published: 17th Aug 2011

Publication Type: Not specified

Genome-wide protein localization prediction strategies for gram negative bacteria
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

BACKGROUND: Genome-wide prediction of protein subcellular localization is an important type of evidence used for inferring protein function. While a variety of computational tools have been developed … for this purpose, errors in the gene models and use of protein sorting signals that are not recognized by the more commonly accepted tools can diminish the accuracy of their output. RESULTS: As part of an effort to manually curate the annotations of 19 strains of Shewanella, numerous insights were gained regarding the use of computational tools and proteomics data to predict protein localization. Identification of the suite of secretion systems present in each strain at the start of the process made it possible to tailor-fit the subsequent localization prediction strategies to each strain for improved accuracy. Comparisons of the computational predictions among orthologous proteins revealed inconsistencies in the computational outputs, which could often be resolved by adjusting the gene models or ortholog group memberships. While proteomic data was useful for verifying start site predictions and post-translational proteolytic cleavage, care was needed to distinguish cellular versus sample processing-mediated cleavage events. Searches for lipoprotein signal peptides revealed that neither TatP nor LipoP are designed for identification of lipoprotein substrates of the twin arginine translocation system and that the +2 rule for lipoprotein sorting does not apply to this Genus. Analysis of the relationships between domain occurrence and protein localization prediction enabled identification of numerous location-informative domains which could then be used to refine or increase confidence in location predictions. This collective knowledge was used to develop a general strategy for predicting protein localization that could be adapted to other organisms. CONCLUSION: Improved localization prediction accuracy is not simply a matter of developing better computational algorithms. It also entails gathering key knowledge regarding the host architecture and translocation machinery and associated substrate recognition via experimentation and integration of diverse computational analyses from many proteins and, where possible, that are derived from different species within the same genus.

Editor:

Date Published: 15th Jun 2011

Publication Type: Not specified

Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

BACKGROUND: Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in bacteria is one of the critical tasks of modern genomics. The Shewanella genus is … comprised of metabolically versatile gamma-proteobacteria, whose lifestyles and natural environments are substantially different from Escherichia coli and other model bacterial species. The comparative genomics approaches and computational identification of regulatory sites are useful for the in silico reconstruction of transcriptional regulatory networks in bacteria. RESULTS: To explore conservation and variations in the Shewanella transcriptional networks we analyzed the repertoire of transcription factors and performed genomics-based reconstruction and comparative analysis of regulons in 16 Shewanella genomes. The inferred regulatory network includes 82 transcription factors and their DNA binding sites, 8 riboswitches and 6 translational attenuators. Forty five regulons were newly inferred from the genome context analysis, whereas others were propagated from previously characterized regulons in the Enterobacteria and Pseudomonas spp.. Multiple variations in regulatory strategies between the Shewanella spp. and E. coli include regulon contraction and expansion (as in the case of PdhR, HexR, FadR), numerous cases of recruiting non-orthologous regulators to control equivalent pathways (e.g. PsrA for fatty acid degradation) and, conversely, orthologous regulators to control distinct pathways (e.g. TyrR, ArgR, Crp). CONCLUSIONS: We tentatively defined the first reference collection of ~100 transcriptional regulons in 16 Shewanella genomes. The resulting regulatory network contains ~600 regulated genes per genome that are mostly involved in metabolism of carbohydrates, amino acids, fatty acids, vitamins, metals, and stress responses. Several reconstructed regulons including NagR for N-acetylglucosamine catabolism were experimentally validated in S. oneidensis MR-1. Analysis of correlations in gene expression patterns helps to interpret the reconstructed regulatory network. The inferred regulatory interactions will provide an additional regulatory constrains for an integrated model of metabolism and regulation in S. oneidensis MR-1.

Authors: D. A. Rodionov, P. S. Novichkov, E. D. Stavrovskaya, I. A. Rodionova, X. Li, M. D. Kazanov, D. A. Ravcheev, A. V. Gerasimova, A. E. Kazakov, G. Y. Kovaleva, E. A. Permina, O. N. Laikova, R. Overbeek, , , A. P. Arkin, I. Dubchak, A. L. Osterman, M. S. Gelfand

Date Published: 15th Jun 2011

Publication Type: Not specified

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in Shewanella oneidensis MR-1
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

BACKGROUND: EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators … Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood. RESULTS: The expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down-regulated at least 2-fold in the mutant, which, showed lower or no reduction of these electron acceptors when compared to the wild type, suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down-regulation of genes implicated in aerobic metabolism. CONCLUSION: In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and, in conjunction with other regulators, fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1. Transcriptomic and sequence analyses of the genes differentially expressed showed that those mostly affected by the mutation belonged to the "Energy metabolism" category, while stress-related genes were indirectly regulated in the mutant possibly as a result of a secondary perturbation (e.g. oxidative stress, starvation). We also conclude based on sequence, physiological and expression analyses that this regulator is more appropriately termed Fnr and recommend this descriptor be used in future publications.

Authors: C. Cruz-Garcia, A. E. Murray, J. L. Rodrigues, J. A. Gralnick, L. A. McCue, , F. E. Loffler, J. M. Tiedje

Date Published: 30th Mar 2011

Publication Type: Not specified

Genomic encyclopedia of sugar utilization pathways in the Shewanella genus
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

BACKGROUND: Carbohydrates are a primary source of carbon and energy for many bacteria. Accurate projection of known carbohydrate catabolic pathways across diverse bacteria with complete genomes constitutes … a substantial challenge due to frequent variations in components of these pathways. To address a practically and fundamentally important challenge of reconstruction of carbohydrate utilization machinery in any microorganism directly from its genomic sequence, we combined a subsystems-based comparative genomic approach with experimental validation of selected bioinformatic predictions by a combination of biochemical, genetic and physiological experiments. RESULTS: We applied this integrated approach to systematically map carbohydrate utilization pathways in 19 genomes from the Shewanella genus. The obtained genomic encyclopedia of sugar utilization includes ~170 protein families (mostly metabolic enzymes, transporters and transcriptional regulators) spanning 17 distinct pathways with a mosaic distribution across Shewanella species providing insights into their ecophysiology and adaptive evolution. Phenotypic assays revealed a remarkable consistency between predicted and observed phenotype, an ability to utilize an individual sugar as a sole source of carbon and energy, over the entire matrix of tested strains and sugars.Comparison of the reconstructed catabolic pathways with E. coli identified multiple differences that are manifested at various levels, from the presence or absence of certain sugar catabolic pathways, nonorthologous gene replacements and alternative biochemical routes to a different organization of transcription regulatory networks. CONCLUSIONS: The reconstructed sugar catabolome in Shewanella spp includes 62 novel isofunctional families of enzymes, transporters, and regulators. In addition to improving our knowledge of genomics and functional organization of carbohydrate utilization in Shewanella, this study led to a substantial expansion of our current version of the Genomic Encyclopedia of Carbohydrate Utilization. A systematic and iterative application of this approach to multiple taxonomic groups of bacteria will further enhance it, creating a knowledge base adequate for the efficient analysis of any newly sequenced genome as well as of the emerging metagenomic data.

Authors: D. A. Rodionov, C. Yang, X. Li, I. A. Rodionova, Y. Wang, A. Y. Obraztsova, O. P. Zagnitko, R. Overbeek, M. F. Romine, S. Reed, J. K. Fredrickson, K. H. Nealson, A. L. Osterman

Date Published: 13th Sep 2010

Publication Type: Not specified

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