Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens

Background The role of the RNA polymerase sigma factor RpoN in regulation of gene expression in Geobacter sulfurreducens was investigated to better understand transcriptional regulatory networks as part of an effort to develop regulatory modules for genome-scale in silico models, which can predict the physiological responses of Geobacter species during groundwater bioremediation or electricity production. Results An rpoN deletion mutant could not be obtained under all conditions tested. In order to investigate the regulon of the G. sulfurreducens RpoN, an RpoN over-expression strain was made in which an extra copy of the rpoN gene was under the control of a taclac promoter. Combining both the microarray transcriptome analysis and the computational prediction revealed that the G. sulfurreducens RpoN controls genes involved in a wide range of cellular functions. Most importantly, RpoN controls the expression of the dcuB gene encoding the fumarate/succinate exchanger, which is essential for cell growth with fumarate as the terminal electron acceptor in G. sulfurreducens. RpoN also controls genes, which encode enzymes for both pathways of ammonia assimilation that is predicted to be essential under all growth conditions in G. sulfurreducens. Other genes that were identified as part of the RpoN regulon using either the computational prediction or the microarray transcriptome analysis included genes involved in flagella biosynthesis, pili biosynthesis and genes involved in central metabolism enzymes and cytochromes involved in extracellular electron transfer to Fe(III), which are known to be important for growth in subsurface environment or electricity production in microbial fuel cells. The consensus sequence for the predicted RpoN-regulated promoter elements is TTGGCACGGTTTTTGCT. Conclusion The G. sulfurreducens RpoN is an essential sigma factor and a global regulator involved in a complex transcriptional network controlling a variety of cellular processes

Bioinformatic analysis of gene regulation in Geobacter sulfurreducens

Background Geobacteraceae are a family of microorganisms from the delta subdivision of Proteobacteria. They have potential for environmental bioremediation and electricity generation. In this presentation, we describe our recent bioinformatic analyses of gene regulation in Geobacter sulfurreducens, a model representative of this family. Results and conclusion We have developed an online database, GSEL (Geobacter Sequence Elements), which compiles regulatory information for G. sulfurreducens. We have recently completed the development of a new, significantly expanded and updated, relational version 2 of the GSEL database and its accompanying online query system, which compiles manually curated information on operon organization and transcription regulatory elements in the genome of G. sulfurreducens. GSEL v. 2 incorporates a graphical browser and provides significantly expanded search capabilities. It also includes new information on predicted and/or experimentally validated genome regulatory sites and provides links to information from microarray experiments stored in public gene expression databases, and to original publications describing how particular regulatory interactions were identified. Using sequence and gene expression analyses, we investigated target genes and promoters regulated by RpoN, an alternative RNA polymerase sigma factor, which regulates a variety of important cellular processes in G. sulfurreducens. Our current studies are focusing on an investigation of several transcription regulatory systems involved in RpoN-dependent regulatory pathways. We have investigated target regulatory sites for an enhancer binding protein, PilR, which participates in RpoN-dependent transcriptional regulation of the pilA gene encoding structural pilin. We predicted multiple PilR-regulated sites upstream of operons related to biosynthesis, assembly, and function of pili and flagella, type II secretory pathways, and cell wall biogenesis. We also investigated sequence changes and molecular classification of the TetR family of transcriptional regulators. In G. sulfurreducens, we identified RpoN-regulated promoters upstream of several operons containing tetR family genes. The genome of G. sulfurreducens contains nine tetR family genes. Some of them are located upstream of operons encoding functionally important c-type cytochromes. In order to better understand the roles of TetR family members in the ability of Geobacteraceae to participate in electron transfer, we investigated phylogenetic relationships among TetR proteins in Geobacteraceae and in other microbial species. We identified their conserved and variable domains, which may be important for the diversity of their functional roles, and classified them into subgroups based on sequence similarities

Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens

BACKGROUND: In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated. RESULTS: The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III). CONCLUSION: This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens

A Mendelian Trait for Olfactory Sensitivity Affects Odor Experience and Food Selection

SummaryHumans vary in acuity to many odors [1–4], with variation within olfactory receptor (OR) genes contributing to these differences [5–9]. How such variation also affects odor experience and food selection remains uncertain [10], given that such effects occur for taste [11–15]. Here we investigate β-ionone, which shows extreme sensitivity differences [4, 16, 17]. β-ionone is a key aroma in foods and beverages [18–21] and is added to products in order to give a pleasant floral note [22, 23]. Genome-wide and in vitro assays demonstrate rs6591536 as the causal variant for β-ionone odor sensitivity. rs6591536 encodes a N183D substitution in the second extracellular loop of OR5A1 and explains >96% of the observed phenotypic variation, resembling a monogenic Mendelian trait. Individuals carrying genotypes for β-ionone sensitivity can more easily differentiate between food and beverage stimuli with and without added β-ionone. Sensitive individuals typically describe β-ionone in foods and beverages as “fragrant” and “floral,” whereas less-sensitive individuals describe these stimuli differently. rs6591536 genotype also influences emotional associations and explains differences in food and product choices. These studies demonstrate that an OR variant that influences olfactory sensitivity can affect how people experience and respond to foods, beverages, and other products

Evaluation of oseltamivir prophylaxis regimens for reducing influenza virus infection, transmission and disease severity in a ferret model of household contact

OBJECTIVES: The emergence of the pandemic influenza A(H1N1)pdm09 virus in 2009 saw a significant increase in the therapeutic and prophylactic use of neuraminidase inhibitors (NAIs) to mitigate the impact of this highly transmissible virus. Prior to the pandemic, many countries stockpiled NAIs and developed pandemic plans for the use of antiviral drugs, based on either treatment of high-risk individuals and/or prophylaxis of contacts. However, to date there has been a lack of in vivo models to test the efficacy of treatment or prophylaxis with NAIs, for influenza-infected individuals or exposed contacts, in a household setting. METHODS: A ferret model of household contact was developed to study the efficacy of different prophylaxis regimens in preventing infection in contact ferrets exposed to influenza A(H1N1)pdm09-infected index ferrets. RESULTS: Among the different prophylactic regimens, contact ferrets receiving oseltamivir prophylaxis twice daily showed better outcomes than those receiving oseltamivir once daily. Benefits included a significant delay in the time to secondary infection, lower weight loss and higher activity levels. The treatment of index ferrets at 36 h post-infection did not influence either secondary infection rates or clinical symptoms in exposed contact ferrets. Neither prophylaxis nor treatment prevented infection or reduced the duration of viral shedding, although clinical symptoms did improve in infected animals receiving prophylaxis. CONCLUSIONS: Different oseltamivir prophylaxis regimens did not prevent infections, but consistently resulted in a reduction in symptoms in infected ferrets. However, oseltamivir prophylaxis failed to reduce viral titres, which warrants further investigation in humans.Ding Yuan Oh, Sue Lowther, James M. McCaw, Sheena G. Sullivan, Sook-Kwan Leang, Jessica Haining, Rachel Arkinstall, Anne Kelso, Jodie Mcvernon, Ian G. Barr, Deborah Middleton, Aeron C. Hur

Genome-wide gene regulation of biosynthesis and energy generation by a novel transcriptional repressor in Geobacter species

Geobacter species play important roles in bioremediation of contaminated environments and in electricity production from waste organic matter in microbial fuel cells. To better understand physiology of Geobacter species, expression and function of citrate synthase, a key enzyme in the TCA cycle that is important for organic acid oxidation in Geobacter species, was investigated. Geobacter sulfurreducens did not require citrate synthase for growth with hydrogen as the electron donor and fumarate as the electron acceptor. Expression of the citrate synthase gene, gltA, was repressed by a transcription factor under this growth condition. Functional and comparative genomics approaches, coupled with genetic and biochemical assays, identified a novel transcription factor termed HgtR that acts as a repressor for gltA. Further analysis revealed that HgtR is a global regulator for genes involved in biosynthesis and energy generation in Geobacter species. The hgtR gene was essential for growth with hydrogen, during which hgtR expression was induced. These findings provide important new insights into the mechanisms by which Geobacter species regulate their central metabolism under different environmental conditions

Characteristics of a Widespread Community Cluster of H275Y Oseltamivir-Resistant A(H1N1)pdm09 Influenza in Australia

Background. Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported

Constraint-based modeling analysis of the metabolism of two Pelobacter species

BACKGROUND: Pelobacter species are commonly found in a number of subsurface environments, and are unique members of the Geobacteraceae family. They are phylogenetically intertwined with both Geobacter and Desulfuromonas species. Pelobacter species likely play important roles in the fermentative degradation of unusual organic matters and syntrophic metabolism in the natural environments, and are of interest for applications in bioremediation and microbial fuel cells. RESULTS: In order to better understand the physiology of Pelobacter species, genome-scale metabolic models for Pelobacter carbinolicus and Pelobacter propionicus were developed. Model development was greatly aided by the availability of models of the closely related Geobacter sulfurreducens and G. metallireducens. The reconstructed P. carbinolicus model contains 741 genes and 708 reactions, whereas the reconstructed P. propionicus model contains 661 genes and 650 reactions. A total of 470 reactions are shared among the two Pelobacter models and the two Geobacter models. The different reactions between the Pelobacter and Geobacter models reflect some unique metabolic capabilities such as fermentative growth for both Pelobacter species. The reconstructed Pelobacter models were validated by simulating published growth conditions including fermentations, hydrogen production in syntrophic co-culture conditions, hydrogen utilization, and Fe(III) reduction. Simulation results matched well with experimental data and indicated the accuracy of the models. CONCLUSIONS: We have developed genome-scale metabolic models of P. carbinolicus and P. propionicus. These models of Pelobacter metabolism can now be incorporated into the growing repertoire of genome scale models of the Geobacteraceae family to aid in describing the growth and activity of these organisms in anoxic environments and in the study of their roles and interactions in the subsurface microbial community