Transcriptome analysis reveals response regulator SO2426-mediated gene expression in Shewanella oneidensis MR-1 under chromate challenge

BACKGROUND: Shewanella oneidensis MR-1 exhibits diverse metal ion-reducing capabilities and thus is of potential utility as a bioremediation agent. Knowledge of the molecular components and regulatory mechanisms dictating cellular responses to heavy metal stress, however, remains incomplete. In a previous work, the S. oneidensis so2426 gene, annotated as a DNA-binding response regulator, was demonstrated to be specifically responsive at both the transcript and protein levels to acute chromate [Cr(VI)] challenge. To delineate the cellular function of SO2426 and its contribution to metal stress response, we integrated genetic and physiological approaches with a genome-wide screen for target gene candidates comprising the SO2426 regulon. RESULTS: Inactivation of so2426 by an in-frame deletion resulted in enhanced chromate sensitivity and a reduced capacity to remove extracellular Cr(VI) relative to the parental strain. Time-resolved microarray analysis was used to compare transcriptomic profiles of wild-type and SO2426-deficient mutant S. oneidensis under conditions of chromate exposure. In total, 841 genes (18% of the arrayed genome) were up- or downregulated at least twofold in the Δso2426 mutant for at least one of six time-point conditions. Hierarchical cluster analysis of temporal transcriptional profiles identified a distinct cluster (n = 46) comprised of co-ordinately regulated genes exhibiting significant downregulated expression (p < 0.05) over time. Thirteen of these genes encoded proteins associated with transport and binding functions, particularly those involved in Fe transport and homeostasis (e.g., siderophore biosynthetic enzymes, TonB-dependent receptors, and the iron-storage protein ferritin). A conserved hypothetical operon (so1188-so1189-so1190), previously identified as a potential target of Fur-mediated repression, as well as a putative bicyclomycin resistance gene (so2280) and cation efflux family protein gene (so2045) also were repressed in the so2426 deletion mutant. Furthermore, the temporal expression profiles of four regulatory genes including a cpxR homolog were perturbed in the chromate-challenged mutant. CONCLUSION: Our findings suggest a previously unrecognized functional role for the response regulator SO2426 in the activation of genes required for siderophore-mediated Fe acquisition, Fe storage, and other cation transport mechanisms. SO2426 regulatory function is involved at a fundamental molecular level in the linkage between Fe homeostasis and the cellular response to chromate-induced stress in S. oneidensis

Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge

BACKGROUND: Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI) [Cr(VI)], the proteome of aerobically grown, Cr(VI)-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB) media and minimal (M9L) media containing lactate as the sole carbon source. RESULTS: Growth studies demonstrated that F1 sensitivity to Cr(VI) was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose >> acetate) observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI) in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI) conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates) tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI) conditions. CONCLUSIONS: Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion transport/metabolism, and amino acid transport/metabolism. These proteins might potentially serve as indicators of chromate stress in natural microbial communities

Glycolate Oxidase Isozymes Are Coordinately Controlled by GLO1 and GLO4 in Rice

Glycolate oxidase (GLO) is a key enzyme in photorespiratory metabolism. Four putative GLO genes were identified in the rice genome, but how each gene member contributes to GLO activities, particularly to its isozyme profile, is not well understood. In this study, we analyzed how each gene plays a role in isozyme formation and enzymatic activities in both yeast cells and rice tissues. Five GLO isozymes were detected in rice leaves. GLO1 and GLO4 are predominately expressed in rice leaves, while GLO3 and GLO5 are mainly expressed in the root. Enzymatic assays showed that all yeast-expressed GLO members except GLO5 have enzymatic activities. Further analyses suggested that GLO1, GLO3 and GLO4 interacted with each other, but no interactions were observed for GLO5. GLO1/GLO4 co-expressed in yeast exhibited the same isozyme pattern as that from rice leaves. When either GLO1 or GLO4 was silenced, expressions of both genes were simultaneously suppressed and most of the GLO activities were lost, and consistent with this observation, little GLO isozyme protein was detected in the silenced plants. In contrast, no observable effect was detected when GLO3 was suppressed. Comparative analyses between the GLO isoforms expressed in yeast and the isozymes from rice leaves indicated that two of the five isozymes are homo-oligomers composed of either GLO1 or GLO4, and the other three are hetero-oligomers composed of both GLO1 and GLO4. Our current data suggest that GLO isozymes are coordinately controlled by GLO1 and GLO4 in rice, and the existence of GLO isozymes and GLO molecular and compositional complexities implicate potential novel roles for GLO in plants

Biomass in the manufacture of industrial products—the use of proteins and amino acids

The depletion in fossil feedstocks, increasing oil prices, and the ecological problems associated with CO2 emissions are forcing the development of alternative resources for energy, transport fuels, and chemicals: the replacement of fossil resources with CO2 neutral biomass. Allied with this, the conversion of crude oil products utilizes primary products (ethylene, etc.) and their conversion to either materials or (functional) chemicals with the aid of co-reagents such as ammonia and various process steps to introduce functionalities such as -NH2 into the simple structures of the primary products. Conversely, many products found in biomass often contain functionalities. Therefore, it is attractive to exploit this to bypass the use, and preparation of, co-reagents as well as eliminating various process steps by utilizing suitable biomass-based precursors for the production of chemicals. It is the aim of this mini-review to describe the scope of the possibilities to generate current functionalized chemical materials using amino acids from biomass instead of fossil resources, thereby taking advantage of the biomass structure in a more efficient way than solely utilizing biomass for the production of fuels or electricity

Conserved synteny at the protein family level reveals genes underlying Shewanella species’ cold tolerance and predicts their novel phenotypes

© The Authors 2009. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License. The definitive version was published in Functional & Integrative Genomics 10 (2010): 97-110, doi:10.1007/s10142-009-0142-y.Bacteria of the genus Shewanella can thrive in different environments and demonstrate significant variability in their metabolic and ecophysiological capabilities including cold and salt tolerance. Genomic characteristics underlying this variability across species are largely unknown. In this study, we address the problem by a comparison of the physiological, metabolic, and genomic characteristics of 19 sequenced Shewanella species. We have employed two novel approaches based on association of a phenotypic trait with the number of the trait-specific protein families (Pfam domains) and on the conservation of synteny (order in the genome) of the trait-related genes. Our first approach is top-down and involves experimental evaluation and quantification of the species’ cold tolerance followed by identification of the correlated Pfam domains and genes with a conserved synteny. The second, a bottom-up approach, predicts novel phenotypes of the species by calculating profiles of each Pfam domain among their genomes and following pair-wise correlation of the profiles and their network clustering. Using the first approach, we find a link between cold and salt tolerance of the species and the presence in the genome of a Na+/H+ antiporter gene cluster. Other cold-tolerance-related genes include peptidases, chemotaxis sensory transducer proteins, a cysteine exporter, and helicases. Using the bottom-up approach, we found several novel phenotypes in the newly sequenced Shewanella species, including degradation of aromatic compounds by an aerobic hybrid pathway in Shewanella woodyi, degradation of ethanolamine by Shewanella benthica, and propanediol degradation by Shewanella putrefaciens CN32 and Shewanella sp. W3-18-1.This research was supported by the U.S. Department of Energy (DOE) Office of Biological and Environmental Research under the Genomics: GTL Program via the Shewanella Federation consortium

Expression Patterns of Genes Involved in Sugar Metabolism and Accumulation during Apple Fruit Development

Both sorbitol and sucrose are imported into apple fruit from leaves. The metabolism of sorbitol and sucrose fuels fruit growth and development, and accumulation of sugars in fruit is central to the edible quality of apple. However, our understanding of the mechanisms controlling sugar metabolism and accumulation in apple remains quite limited. We identified members of various gene families encoding key enzymes or transporters involved in sugar metabolism and accumulation in apple fruit using homology searches and comparison of their expression patterns in different tissues, and analyzed the relationship of their transcripts with enzyme activities and sugar accumulation during fruit development. At the early stage of fruit development, the transcript levels of sorbitol dehydrogenase, cell wall invertase, neutral invertase, sucrose synthase, fructokinase and hexokinase are high, and the resulting high enzyme activities are responsible for the rapid utilization of the imported sorbitol and sucrose for fruit growth, with low levels of sugar accumulation. As the fruit continues to grow due to cell expansion, the transcript levels and activities of these enzymes are down-regulated, with concomitant accumulation of fructose and elevated transcript levels of tonoplast monosaccharide transporters (TMTs), MdTMT1 and MdTMT2; the excess carbon is converted into starch. At the late stage of fruit development, sucrose accumulation is enhanced, consistent with the elevated expression of sucrose-phosphate synthase (SPS), MdSPS5 and MdSPS6, and an increase in its total activity. Our data indicate that sugar metabolism and accumulation in apple fruit is developmentally regulated. This represents a comprehensive analysis of the genes involved in sugar metabolism and accumulation in apple, which will serve as a platform for further studies on the functions of these genes and subsequent manipulation of sugar metabolism and fruit quality traits related to carbohydrates

Generation and Validation of a Shewanella oneidensis MR-1 Clone Set for Protein Expression and Phage Display

A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro

Detection of transcriptional triggers in the dynamics of microbial growth: application to the respiratorily versatile bacterium Shewanella oneidensis

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nucleic Acids Research 40 (2012): 7132-7149, doi:10.1093/nar/gks467.The capacity of microorganisms to respond to variable external conditions requires a coordination of environment-sensing mechanisms and decision-making regulatory circuits. Here, we seek to understand the interplay between these two processes by combining high-throughput measurement of time-dependent mRNA profiles with a novel computational approach that searches for key genetic triggers of transcriptional changes. Our approach helped us understand the regulatory strategies of a respiratorily versatile bacterium with promising bioenergy and bioremediation applications, Shewanella oneidensis, in minimal and rich media. By comparing expression profiles across these two conditions, we unveiled components of the transcriptional program that depend mainly on the growth phase. Conversely, by integrating our time-dependent data with a previously available large compendium of static perturbation responses, we identified transcriptional changes that cannot be explained solely by internal network dynamics, but are rather triggered by specific genes acting as key mediators of an environment-dependent response. These transcriptional triggers include known and novel regulators that respond to carbon, nitrogen and oxygen limitation. Our analysis suggests a sequence of physiological responses, including a coupling between nitrogen depletion and glycogen storage, partially recapitulated through dynamic flux balance analysis, and experimentally confirmed by metabolite measurements. Our approach is broadly applicable to other systems.Office of Science (BER), U.S. Department of Energy [DE-FG02-07ER64388 to D.S. and DE-FG02- 08ER64511 to M.H.S.]; National Aeronautics and Space Administration, NASA Astrobiology Institute [NNA08CN84A to D.S.]