Publications

Phototrophic biofilm assembly in microbial-mat-derived unicyanobacterial consortia: model systems for the study of autotroph-heterotroph interactions
Energy and Material Processing

Abstract (Expand)

Microbial autotroph-heterotroph interactions influence biogeochemical cycles on a global scale, but the diversity and complexity of natural systems and their intractability to in situ manipulation make … it challenging to elucidate the principles governing these interactions. The study of assembling phototrophic biofilm communities provides a robust means to identify such interactions and evaluate their contributions to the recruitment and maintenance of phylogenetic and functional diversity over time. To examine primary succession in phototrophic communities, we isolated two unicyanobacterial consortia from the microbial mat in Hot Lake, Washington, characterizing the membership and metabolic function of each consortium. We then analyzed the spatial structures and quantified the community compositions of their assembling biofilms. The consortia retained the same suite of heterotrophic species, identified as abundant members of the mat and assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes. Autotroph growth rates dominated early in assembly, yielding to increasing heterotroph growth rates late in succession. The two consortia exhibited similar assembly patterns, with increasing relative abundances of members from Bacteroidetes and Alphaproteobacteria concurrent with decreasing relative abundances of those from Gammaproteobacteria. Despite these similarities at higher taxonomic levels, the relative abundances of individual heterotrophic species were substantially different in the developing consortial biofilms. This suggests that, although similar niches are created by the cyanobacterial metabolisms, the resulting webs of autotroph-heterotroph and heterotroph-heterotroph interactions are specific to each primary producer. The relative simplicity and tractability of the Hot Lake unicyanobacterial consortia make them useful model systems for deciphering interspecies interactions and assembly principles relevant to natural microbial communities.

Authors: J. K. Cole, J. R. Hutchison, R. S. Renslow, Y. M. Kim, W. B. Chrisler, H. E. Engelmann, A. C. Dohnalkova, D. Hu, T. O. Metz, J. K. Fredrickson, S. R. Lindemann

Date Published: 7th Apr 2014

Publication Type: Not specified

The epsomitic phototrophic microbial mat of Hot Lake, Washington: community structural responses to seasonal cycling
Energy and Material Processing

Abstract (Expand)

Phototrophic microbial mats are compact ecosystems composed of highly interactive organisms in which energy and element cycling take place over millimeter-to-centimeter-scale distances. Although microbial … mats are common in hypersaline environments, they have not been extensively characterized in systems dominated by divalent ions. Hot Lake is a meromictic, epsomitic lake that occupies a small, endorheic basin in north-central Washington. The lake harbors a benthic, phototrophic mat that assembles each spring, disassembles each fall, and is subject to greater than tenfold variation in salinity (primarily Mg(2+) and SO(2-) 4) and irradiation over the annual cycle. We examined spatiotemporal variation in the mat community at five time points throughout the annual cycle with respect to prevailing physicochemical parameters by amplicon sequencing of the V4 region of the 16S rRNA gene coupled to near-full-length 16S RNA clone sequences. The composition of these microbial communities was relatively stable over the seasonal cycle and included dominant populations of Cyanobacteria, primarily a group IV cyanobacterium (Leptolyngbya), and Alphaproteobacteria (specifically, members of Rhodobacteraceae and Geminicoccus). Members of Gammaproteobacteria (e.g., Thioalkalivibrio and Halochromatium) and Deltaproteobacteria (e.g., Desulfofustis) that are likely to be involved in sulfur cycling peaked in summer and declined significantly by mid-fall, mirroring larger trends in mat community richness and evenness. Phylogenetic turnover analysis of abundant phylotypes employing environmental metadata suggests that seasonal shifts in light variability exert a dominant influence on the composition of Hot Lake microbial mat communities. The seasonal development and organization of these structured microbial mats provide opportunities for analysis of the temporal and physical dynamics that feed back to community function.

Authors: S. R. Lindemann, J. J. Moran, J. C. Stegen, R. S. Renslow, J. R. Hutchison, J. K. Cole, A. C. Dohnalkova, J. Tremblay, K. Singh, S. A. Malfatti, F. Chen, S. G. Tringe, H. Beyenal, J. K. Fredrickson

Date Published: 13th Nov 2013

Publication Type: Not specified

Comparative Genomics of DtxR Family Regulons for Metal Homeostasis in Archaea
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

The DtxR family consists of metal-dependent transcription factors (DtxR-TFs) that regulate the expression of genes involved in metal homeostasis in the cell. The majority of characterized DtxR-TFs … belong to Bacteria. In the current work, we applied a comparative genomics approach to predict DNA-binding sites and reconstruct regulons for DtxR-TFs in Archaea. As a result, we inferred 575 candidate binding sites for 139 DtxR-TFs in 77 genomes from 15 taxonomic orders. Novel DNA motifs of archaeal DtxR-TFs that have a common palindromic structure were classified into 10 distinct groups. By combining functional regulon reconstructions with phylogenetic analysis, we selected 28 DtxR-TF clades and assigned them metal specificities and regulator names. The reconstructed FetR (ferrous iron), MntR (manganese), and ZntR (zinc) regulons largely contain known or putative metal uptake transporters from the FeoAB, NRAMP, ZIP, and TroA families. A novel family of putative iron transporters (named Irt), including multiple FetR-regulated paralogs, was identified in iron-oxidizing Archaea from the Sulfolobales order. The reconstructed DtxR-TF regulons were reconciled with available transcriptomics data in Archaeoglobus, Halobacterium, and Thermococcus spp.

Authors: S. A. Leyn, D. A. Rodionov

Date Published: 17th Nov 2014

Publication Type: Not specified

Genomic distribution of B-vitamin auxotrophy and uptake transporters in environmental bacteria from the Chloroflexi phylum
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Bacteria from the Chloroflexi phylum are dominant members of phototrophic microbial mat communities in terrestrial thermal environments. Vitamins of B group are key intermediates (precursors) in the … biosynthesis of indispensable enzyme cofactors driving numerous metabolic processes in all forms of life. A genomics-based reconstruction and comparative analysis of respective biosynthetic and salvage pathways and riboswitch regulons in over 20 representative Chloroflexi genomes revealed a widespread auxotrophy for some of the vitamins. The most prominent predicted phenotypic signature, auxotrophy for vitamins B1 and B7 was experimentally confirmed for the best studied model organism Chloroflexus aurantiacus. These observations along with identified candidate genes for the respective uptake transporters pointed to B vitamin cross-feeding as an important aspect of syntrophic metabolism in microbial communities. Inferred specificities of homologous substrate-binding components of ABC transporters for vitamins B1 (ThiY) and B2 (RibY) were verified by thermofluorescent shift approach. A functional activity of the thiamine-specific transporter ThiXYZ from C. aurantiacus was experimentally verified by genetic complementation in E. coli. Expanding the integrative approach, which was applied here for a comprehensive analysis of B-vitamin metabolism in Chloroflexi would allow reconstruction of metabolic interdependencies in microbial communities.

Authors: I. A. Rodionova, X. Li, A. E. Plymale, K. Motamedchaboki, A. E. Konopka, , , A. L. Osterman, D. A. Rodionov

Date Published: 28th Oct 2014

Publication Type: Not specified

Inference of interactions in cyanobacterial-heterotrophic co-cultures via transcriptome sequencing
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella putrefaciens … W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph-heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic-heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.

Authors: A. S. Beliaev, , M. Serres, Hans Bernstein, B. E. Linggi, , N. G. Isern, , L. A. Kucek, E. A. Hill, G. E. Pinchuk, D. A. Bryant, , , A. Konopka

Date Published: 29th Apr 2014

Publication Type: Not specified

In situ analysis of oxygen consumption and diffusive transport in high-temperature acidic iron-oxide microbial mats
Energy and Material Processing

Abstract (Expand)

The role of dissolved oxygen as a principal electron acceptor for microbial metabolism was investigated within Fe(III)-oxide microbial mats that form in acidic geothermal springs of Yellowstone National … Park (USA). Specific goals of the study were to measure and model dissolved oxygen profiles within high-temperature (65-75 degrees C) acidic (pH = 2.7-3.8) Fe(III)-oxide microbial mats, and correlate the abundance of aerobic, iron-oxidizing Metallosphaera yellowstonensis organisms and mRNA gene expression levels to Fe(II)-oxidizing habitats shown to consume oxygen. In situ oxygen microprofiles were obtained perpendicular to the direction of convective flow across the aqueous phase/Fe(III)-oxide microbial mat interface using oxygen microsensors. Dissolved oxygen concentrations dropped from approximately 50-60 muM in the bulk-fluid/mat surface to below detection (< 0.3 muM) at a depth of approximately 700 mum ( approximately 10% of the total mat depth). Net areal oxygen fluxes into the microbial mats were estimated to range from 1.4-1.6 x 10(-4) mumol cm(-2) s(-1) . Dimensionless parameters were used to model dissolved oxygen profiles and establish that mass transfer rates limit the oxygen consumption. A zone of higher dissolved oxygen at the mat surface promotes Fe(III)-oxide biomineralization, which was supported using molecular analysis of Metallosphaera yellowstonensis 16S rRNA gene copy numbers and mRNA expression of haem Cu oxidases (FoxA) associated with Fe(II)-oxidation.

Authors: Hans Bernstein, J. P. Beam, M. A. Kozubal, R. P. Carlson, W. P. Inskeep

Date Published: 21st Mar 2013

Publication Type: Not specified

Conservation of transcription start sites within genes across a bacterial genus
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, … we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5'-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function. Importance: The first step of gene expression is the initiation of transcription from promoters, which have been traditionally thought to be located upstream of genes. Recently, studies showed that in diverse bacteria, promoters are often located inside genes. It has not been clear if these unexpected promoters are important to the organism or if they result from transcriptional noise. Here, we identify and examine promoters in eight related bacterial species. Promoters that lie within genes on the sense strand are often conserved as locations and in their sequences. Furthermore, these promoters often affect the bacterium's growth. Thus, many of these unexpected promoters are likely functional. Fewer promoters that lie within genes on the antisense strand are conserved, but the conserved ones seem to drive the expression of nearby genes.

Authors: W. Shao, M. N. Price, A. M. Deutschbauer, , A. P. Arkin

Date Published: 6th Jul 2014

Publication Type: Not specified

Genome Sequence of the Thermophilic Cyanobacterium Thermosynechococcus sp. Strain NK55a
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

The genome of the unicellular cyanobacterium Thermosynechococcus sp. strain NK55a, isolated from the Nakabusa hot spring, Nagano Prefecture, Japan, comprises a single, circular, 2.5-Mb chromosome. The … genome is predicted to contain 2,358 protein-encoding genes, including genes for all typical cyanobacterial photosynthetic and metabolic functions. No genes encoding hydrogenases or nitrogenase were identified.

Authors: S. Stolyar, , V. Thiel, L. P. Tomsho, N. Pinel, , , , S. Haruta, S. C. Schuster, D. A. Bryant,

Date Published: 1st Feb 2014

Publication Type: Not specified

Live cell chemical profiling of temporal redox dynamics in a photoautotrophic cyanobacterium
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Protein reduction-oxidation (redox) modification is an important mechanism that allows microorganisms to sense environmental changes and initiate cellular responses. We have developed a quantitative … chemical probe approach for live cell labeling and imaging of proteins that are sensitive to redox modifications. We utilize this in vivo strategy to identify 176 proteins undergoing approximately 5-10-fold dynamic redox change in response to nutrient limitation and subsequent replenishment in the photoautotrophic cyanobacterium Synechococcus sp. PCC 7002. We detect redox changes in as little as 30 s after nutrient perturbation and oscillations in reduction and oxidation for 60 min following the perturbation. Many of the proteins undergoing dynamic redox transformations participate in the major components for the production (photosystems and electron transport chains) or consumption (Calvin-Benson cycle and protein synthesis) of reductant and/or energy in photosynthetic organisms. Thus, our in vivo approach reveals new redox-susceptible proteins and validates those previously identified in vitro.

Authors: N. C. Sadler, M. R. Melnicki, M. H. Serres, E. D. Merkley, , , , , E. M. Zink, S. Datta, R. D. Smith, A. S. Beliaev, A. Konopka,

Date Published: 6th Nov 2013

Publication Type: Not specified

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

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

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

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

Changes in translational efficiency is a dominant regulatory mechanism in the environmental response of bacteria
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

To understand how cell physiological state affects mRNA translation, we used Shewanella oneidensis MR-1 grown under steady state conditions at either 20% or 8.5% O2. Using a combination of quantitative … proteomics and RNA-Seq, we generated high-confidence data on >1000 mRNA and protein pairs. By using a steady state model, we found that differences in protein-mRNA ratios were primarily due to differences in the translational efficiency of specific genes. When oxygen levels were lowered, 28% of the proteins showed at least a 2-fold change in expression. Transcription levels were sp. significantly altered for 26% of the protein changes; translational efficiency was significantly altered for 46% and a combination of both was responsible for the remaining 28%. Changes in translational efficiency were significantly correlated with the codon usage pattern of the genes and measurable tRNA pools changed in response to altered O2 levels. Our results suggest that changes in the translational efficiency of proteins, in part due to altered tRNA pools, is a major determinant of regulated alterations in protein expression levels in bacteria.

Authors: , B. J. Webb Robertson, , M. H. Serres, B. E. Linggi, J. T. Aldrich, , , ,

Date Published: 21st Oct 2013

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

Diel metabolomics analysis of a hot spring chlorophototrophic microbial mat leads to new hypotheses of community member metabolisms
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Dynamic environmental factors such as light, nutrients, salt, and temperature continuously affect chlorophototrophic microbial mats, requiring adaptive and acclimative responses to stabilize composition … and function. Quantitative metabolomics analysis can provide insights into metabolite dynamics for understanding community response to such changing environmental conditions. In this study, we quantified volatile organic acids, polar metabolites (amino acids, glycolytic and citric acid cycle intermediates, nucleobases, nucleosides, and sugars), wax esters, and polyhydroxyalkanoates, resulting in the identification of 104 metabolites and related molecules in thermal chlorophototrophic microbial mat cores collected over a diel cycle in Mushroom Spring, Yellowstone National Park. A limited number of predominant taxa inhabit this community and their functional potentials have been previously identified through metagenomic and metatranscriptomic analyses and in situ metabolisms, and metabolic interactions among these taxa have been hypothesized. Our metabolomics results confirmed the diel cycling of photorespiration (e.g., glycolate) and fermentation (e.g., acetate, propionate, and lactate) products, the carbon storage polymers polyhydroxyalkanoates, and dissolved gasses (e.g., H2 and CO2) in the waters overlying the mat, which were hypothesized to occur in major mat chlorophototrophic community members. In addition, we have formulated the following new hypotheses: (1) the morning hours are a time of biosynthesis of amino acids, DNA, and RNA; (2) photo-inhibited cells may also produce lactate via fermentation as an alternate metabolism; (3) glycolate and lactate are exchanged among Synechococcus and Roseiflexus spp.; and (4) fluctuations in many metabolite pools (e.g., wax esters) at different times of day result from species found at different depths within the mat responding to temporal differences in their niches.

Authors: Young-Mo Kim, Shane Nowack, Millie T. Olsen, Eric D. Becraft, Jason M. Wood, Vera Thiel, Isaac Klapper, Michael Kühl, James K. Fredrickson, Donald A. Bryant, David M. Ward, Thomas O. Metz

Date Published: 17th Apr 2015

Publication Type: Not specified

Comparative genomics of transcriptional regulation of methionine metabolism in Proteobacteria
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known … transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in approximately 200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the metX or metZ genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific and genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria.

Authors: S. A. Leyn, I. A. Suvorova, T. D. Kholina, S. S. Sherstneva, P. S. Novichkov, M. S. Gelfand, D. A. Rodionov

Date Published: 20th Nov 2014

Publication Type: Not specified

Mathematical Modeling of Microbial Community Dynamics: A Methodological Review
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract

Not specified

Authors: Hyun-Seob Song, William Cannon, Alexander Beliaev, Allan Konopka

Date Published: 1st Dec 2014

Publication Type: Not specified

Integrated in silico Analyses of Regulatory and Metabolic Networks of Synechococcus sp. PCC 7002 Reveal Relationships between Gene Centrality and Essentiality
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations … is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as "topologically important." Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as "functionally important" genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.

Authors: H. S. Song, R. S. McClure, Hans Bernstein, C. C. Overall, E. A. Hill, A. S. Beliaev

Date Published: 1st Apr 2015

Publication Type: Not specified

Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin, Yellowstone National Park
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of … chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonensis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with 13C-formaldehyde. Isotopic analysis of incubation-derived CO2 and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.

Authors: J. J. Moran, L. M. Whitmore, N. G. Isern, M. F. Romine, K. M. Riha, W. P. Inskeep, H. W. Kreuzer

Date Published: 19th Mar 2016

Publication Type: Not specified

Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are needed to shorten the gap between characterizing a microbial genome and experimentally … validating bioinformatically predicted functions. Of particular importance are transport mechanisms, which shuttle nutrients such as B vitamins and metabolites across cell membranes and are required for the survival of microbes ranging from members of environmental microbial communities to pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, along with characterization of intracellular enzyme-cofactor associations, are needed to enable a significantly improved understanding of microbial biochemistry and physiology, microbial interactions, and microbial responses to perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular enzyme-cofactor associations through live cell labeling of the filamentous anoxygenic photoheterotroph, Chloroflexus aurantiacus J-10-fl, known to employ mechanisms for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by identifying B vitamin transport and cofactor-dependent interactions required for survival.

Authors: L. N. Anderson, P. K. Koech, A. E. Plymale, E. V. Landorf, A. Konopka, F. R. Collart, M. S. Lipton, M. F. Romine, A. T. Wright

Date Published: 22nd Dec 2015

Publication Type: Not specified

Ecophysiology of an uncultivated lineage of Aigarchaeota from an oxic, hot spring filamentous 'streamer' community
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

The candidate archaeal phylum 'Aigarchaeota' contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced … from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous 'streamer' community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with other community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus 'Calditenuis aerorheumensis' for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.

Authors: J. P. Beam, Z. J. Jay, M. C. Schmid, D. B. Rusch, M. F. Romine, M. Jennings Rde, M. A. Kozubal, S. G. Tringe, M. Wagner, W. P. Inskeep

Date Published: 3rd Jul 2015

Publication Type: Not specified

Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements … are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process.

Authors: Hans Bernstein, M. A. Charania, R. S. McClure, N. C. Sadler, M. R. Melnicki, E. A. Hill, L. M. Markillie, C. D. Nicora, A. T. Wright, M. F. Romine, A. S. Beliaev

Date Published: 4th Nov 2015

Publication Type: Not specified

Identification and Resolution of Microdiversity through Metagenomic Sequencing of Parallel Consortia
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. … Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9x to 2,700x) within the metagenomic data set.

Authors: W. C. Nelson, Y. Maezato, Y. W. Wu, M. F. Romine, S. R. Lindemann

Date Published: 23rd Oct 2015

Publication Type: Not specified

Pyrobaculum yellowstonensis Strain WP30 Respires on Elemental Sulfur and/or Arsenate in Circumneutral Sulfidic Geothermal Sediments of Yellowstone National Park
Energy and Material Processing, Microbial Community Dynamics, Adaptive Responses

Abstract (Expand)

Thermoproteales (phylum Crenarchaeota) populations are abundant in high-temperature (>70 degrees C) environments of Yellowstone National Park (YNP) and are important in mediating the biogeochemical … cycles of sulfur, arsenic, and carbon. The objectives of this study were to determine the specific physiological attributes of the isolate Pyrobaculum yellowstonensis strain WP30, which was obtained from an elemental sulfur sediment (Joseph's Coat Hot Spring [JCHS], 80 degrees C, pH 6.1, 135 muM As) and relate this organism to geochemical processes occurring in situ. Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as an electron acceptor. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58% G+C content), revealing numerous metabolic pathways for the degradation of carbohydrates, amino acids, and lipids. Multiple dimethyl sulfoxide-molybdopterin (DMSO-MPT) oxidoreductase genes, which are implicated in the reduction of sulfur and arsenic, were identified. Pathways for the de novo synthesis of nearly all required cofactors and metabolites were identified. The comparative genomics of P. yellowstonensis and the assembled metagenome sequence from JCHS showed that this organism is highly related ( approximately 95% average nucleotide sequence identity) to in situ populations. The physiological attributes and metabolic capabilities of P. yellowstonensis provide an important foundation for developing an understanding of the distribution and function of these populations in YNP.

Authors: Z. J. Jay, J. P. Beam, A. Dohnalkova, R. Lohmayer, B. Bodle, B. Planer-Friedrich, M. Romine, W. P. Inskeep

Date Published: 19th Jun 2015

Publication Type: Not specified

Predicting Species-Resolved Macronutrient Acquisition during Succession in a Model Phototrophic Biofilm Using an Integrated 'Omics Approach.
Microbial Community Dynamics

Abstract (Expand)

The principles governing acquisition and interspecies exchange of nutrients in microbial communities and how those exchanges impact community productivity are poorly understood. Here, we examine energy … and macronutrient acquisition in unicyanobacterial consortia for which species-resolved genome information exists for all members, allowing us to use multi-omic approaches to predict species' abilities to acquire resources and examine expression of resource-acquisition genes during succession. Metabolic reconstruction indicated that a majority of heterotrophic community members lacked the genes required to directly acquire the inorganic nutrients provided in culture medium, suggesting high metabolic interdependency. The sole primary producer in consortium UCC-O, cyanobacterium Phormidium sp. OSCR, displayed declining expression of energy harvest, carbon fixation, and nitrate and sulfate reduction proteins but sharply increasing phosphate transporter expression over 28 days. Most heterotrophic members likewise exhibited signs of phosphorus starvation during succession. Though similar in their responses to phosphorus limitation, heterotrophs displayed species-specific expression of nitrogen acquisition genes. These results suggest niche partitioning around nitrogen sources may structure the community when organisms directly compete for limited phosphate. Such niche complementarity around nitrogen sources may increase community diversity and productivity in phosphate-limited phototrophic communities.

Authors: S. R. Lindemann, J. M. Mobberley, J. K. Cole, L. M. Markillie, R. C. Taylor, E. Huang, W. B. Chrisler, H. S. Wiley, M. S. Lipton, W. C. Nelson, J. K. Fredrickson, M. F. Romine

Date Published: No date defined

Publication Type: Not specified

Digitoxin metabolism by rat liver microsomes.
Adaptive Responses

Abstract

Not specified

Authors: A. Schmoldt, H. F. Benthe, G. Haberland

Date Published: No date defined

Publication Type: Not specified

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