Formate-driven efficient pyruvate biosynthesis in Vibrio natriegens

Two-dimensional gel electrophoresis (2-DE) is currently unequalled in its capacity to separate individual components of complex protein mixtures, making it the method of choice for monitoring global changes in gene expression in response to defined conditions (Guerreiro et al, 1997). Using 2-DE we have examined the global changes in Rhizobium gene expression which occur in response to specific Tn5-induced mutations that lead to defective symbiotic phenotypes. Exopolysaccharides (EPS) of Rhizobium leguminosarum have been implicated to be involved in a complex series of interactions which lead to the establishment of an effective Rhizobium -legume symbiosis, but the precise role of EPS in this interaction is not well defined. The mutant ANU437, a derivative of R. l. bv. trifolli strain ANU794, contains a Tn5 insertion in pssA (also called pss4), produces very low levels of acidic exopolysaccharide and induces delayed formation of small non-nitrogen-fixing nodules on various clover species (Rolfe et al, 1996). Using 2-DE we have shown ANU437 to exhibit multiple differences in gene expression when compared with its wild-type parent. Such pleiotrophies would have been difficult to show via other methodologies. Twenty-one proteins in ANU437 were either newly-induced or up-regulated in their level of synthesis. Six of these proteins have been characterised by N-terminal sequencing, all were novel proteins except for one which showed a high similarity to a phosphogluconate dehydrogenase. These results have shown the PSSA protein, in addition to being a glycosyltransferase, may be directly or indirectly involved in influencing the expression of a complex series of genes whose levels of expression may be the actual contributing factor to the ineffective nodulation observed with exo− mutants.KeywordsBiological Nitrogen FixationComplex SeriesRhizobium LeguminosarumPhosphogluconate DehydrogenaseMultiple DifferenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

BackgroundRhodospirillum centenum is a photosynthetic member of the Gram-negative Azospirillum clade members of which exhibit a complex developmental life-cycle featuring morphologically distinct cell types. Under periods of nutrient deprivation, replicative vegetative cells differentiate into metabolically dormant cysts that survive harsh environmental stresses such as desiccation. Encystment involves a multi-stage developmental process that includes the rounding of cells, production of large intracellular storage granules of poly-hydroxybutyrate (PHB) and the excretion of a protective exopolysaccharide coating that envelops dormant cysts.ResultsTo study the process of cyst development, we performed RNA-seq studies on cells that were induced to undergo cyst development. To assay for temporal changes in gene expression, RNA was extracted at 4, 24, 48, 72, 96 hours during development and subjected to deep sequence analysis. These results show that 812 genes exhibit log2 ≥ 1.5-fold changes in expression over a 96 hour cyst induction period demonstrating large global changes in gene expression during cyst development.ConclusionsNotable changes in expression occurred in numerous genes involved in cell wall and lipid biosynthesis, metabolic enzymes, and numerous regulatory genes such as histidine kinases and transcription factors. Many genes involved in protein synthesis and DNA replication were also significantly reduced during late stages of cyst development. Genes previously identified by genetic screens as being critical for cyst development also exhibited changes of expression during cyst induction. This study provides the first transcriptome profile of global changes in gene expression that occur during development of cysts in a Gram-negative species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1250-9) contains supplementary material, which is available to authorized users.

[This corrects the article DOI: 10.1371/journal.pone.0221519.].

The mouse is a widely used animal model for studying human reproduction. Although global gene expression changes associated with human uterine receptivity have been determined by independent groups, the same studies in the mouse are scarce. The extent of similarities/differences between mice and humans on uterine receptivity at the molecular level remains to be determined. In the present study, we analyzed global gene expression changes in receptive uterus on day 4 of pregnancy compared to non-receptive uterus on day 3 of pregnancy in mice. A total of 541 differentially expressed genes were identified, of which 316 genes were up-regulated and 225 genes were down-regulated in receptive uterus compared to non-receptive uterus. Gene ontology and gene network analysis highlighted the activation of inflammatory response in the receptive uterus. By analyzing the promoter sequences of differentially expressed genes, we identified 12 causal transcription factors. Through connectivity map (CMap) analysis, we revealed several compounds with potential anti-receptivity activity. Finally, we performed a cross-species comparison against human uterine receptivity from a published dataset. Our study provides a valuable resource for understanding the molecular mechanism underlying uterine receptivity in mice.

Orobanche foetida Poiret is the main constraint facing faba bean crop in Tunisia. Indeed, in heavily infested fields with this parasitic plant, yield losses may reach 90%, and the recent estimation of the infested area is around 80,000 ha. Identifying genes involved in the Vicia faba/O. foetida interaction is crucial for the development of effective faba bean breeding programs. However, there is currently no available information on the transcriptome of faba bean responding to O. foetida parasitism. In this study, we employed RNA sequencing to explore the global gene expression changes associated with compatible and incompatible V. faba/O. foetida interactions. In this perspective, two faba bean varieties (susceptible and resistant) were examined at the root level across three stages of O. foetida development (Before Germination (BG), After Germination (AG) and Tubercule Stage (TS)). Our analyses presented an exploration of the transcriptomic profile, including comprehensive assessments of differential gene expression and Gene Ontology (GO) enrichment analyses. Specifically, we investigated key pathways revealing the complexity of molecular responses to O. foetida attack. In this study, we detected differential gene expression of pathways associated with secondary metabolites: flavonoids, auxin, thiamine, and jasmonic acid. To enhance our understanding of the global changes in V. faba response to O. foetida, we specifically examined WRKY genes known to play a role in plant host-parasitic plant interactions. Furthermore, considering the pivotal role of parasitic plant seed germination in this interaction, we investigated genes involved in the orobanchol biosynthesis pathway. Interestingly, we detected the gene expression of VuCYP722C homolog, coding for a key enzyme involved in orobanchol biosynthesis, exclusively in the susceptible host. Clearly, this study enriches our understanding of the V. faba/O. foetida interaction, shedding light on the main differences between susceptible and resistant faba bean varieties during O. foetida infestation at the gene expression level.

Protein kinase C (PKC) isozymes are key signal transducers involved in normal physiology and disease and have been widely implicated in cancer progression. Despite our extensive knowledge of the signaling pathways regulated by PKC isozymes and their effectors, there is essentially no information on how individual members of the PKC family regulate gene transcription. Here, we report the first PKC isozyme-specific analysis of global gene expression by microarray using RNAi depletion of diacylglycerol/phorbol ester-regulated PKCs. A thorough analysis of this microarray data revealed unique patterns of gene expression controlled by PKCα, PKCδ, and PKCε, which are remarkably different in cells growing in serum or in response to phorbol ester stimulation. PKCδ is the most relevant isoform in controlling the induction of genes by phorbol ester stimulation, whereas PKCε predominantly regulates gene expression in serum. We also established that two PKCδ-regulated genes, FOSL1 and BCL2A1, mediate the apoptotic effect of phorbol esters or the chemotherapeutic agent etoposide in prostate cancer cells. Our studies offer a unique opportunity for establishing novel transcriptional effectors for PKC isozymes and may have significant functional and therapeutic implications.

Estrogen induction of uterine wet weight provides an excellent model to investigate relationships between changes in global gene expression and well-characterized physiological responses. In this study, time course microarray GeneChip data were analyzed using a novel approach to identify temporal changes in uterine gene expression following treatment of immature ovariectomized C57BL/6 mice with 0.1 mg/kg 17alpha-ethynylestradiol. Functional gene annotation information from public databases facilitated the association of changes in gene expression with physiological outcomes, which allowed detailed mechanistic inferences to be drawn regarding cell cycle control and proliferation, transcription and translation, structural tissue remodeling, and immunologic responses. These systematic approaches confirm previously established responses, identify novel estrogen-regulated transcriptional effects, and disclose the coordinated activation of multiple modes of action that support the uterotrophic response elicited by estrogen. In particular, it was possible to elucidate the physiological significance of the dramatic induction of arginase, a classic estrogenic response, by elucidating its mechanistic relevance and delineating the role of arginine and ornithine utilization in the estrogen-stimulated induction of uterine wet weight.

BackgroundDocosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels.Methods and FindingsTwelve baboons were divided into three formula groups: Control, with no DHA-ARA; “L”, LCPUFA, with 0.33%DHA-0.67%ARA; “L3”, LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner.ConclusionsThese data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA.

Activation of hepatic stellate cells (HSC) to a myofibroblast-like phenotype is the pivotal event in liver fibrosis. In uninjured liver, HSC are quiescent and non-dividing, but upon liver injury these cells undergo a dramatic change in phenotype which generates activated myofibroblast-like HSC. The change in phenotype is underpinned by a global change in gene expression with hundreds of genes being up- or downregulated. Molecular events that orchestrate changes in gene expression take place at the level of chromatin packaging which is altered through three main processes: histone modifications, DNA methylation, and silencing by non-coding RNAs. The present review focuses on the epigenetic regulation of HSC activation.

The CBF signal pathway is responsible for a significant portion of plant responses to low temperature and freezing. Overexpression of CBF genes in model organisms such as Arabidopsis thaliana enhances abiotic stress tolerance but also reduces growth. In addition to these effects, overexpression of the peach (Prunus persica [L.] Batsch) CBF1 gene in transgenic apple (Malus x domestica Borkh.) line T166 also results in early entry into and late exit from dormancy. Although the regulation of dormancy-induction and dormancy-release occur while the CBF regulon is operative in perennial, woody plants, how overexpression of CBF1 affects these dormancy-related changes in gene expression is incompletely understood. The objective of the present study was to characterize global changes in gene expression in peach CBF1-overexpressing and non-transformed apple bark tissues at different states of dormancy via RNA-seq. RNA-seq bioinformatics data was confirmed by RT-qPCR on a number of genes. Results indicate that the greatest number of significantly differentially expressed genes (DEGs) occurred in April when dormancy release and bud break normally occur but are delayed in Line T166. Genes involved in storage and inactivation of auxin, GA, and cytokinin were generally upregulated in T166 in April, while those for biosynthesis, uptake or signal transduction were generally downregulated in T166. Genes for cell division and cambial growth were also downregulated in T166 relative to the non-transformed line. These data suggest that overexpression of the peach CBF1 gene impacts growth hormone homeostasis and as a result the activation of growth in the spring, and most likely growth cessation in the fall as well.

Plants respond to the proximity of neighboring vegetation by elongating to prevent shading. Red-depleted light reflected from neighboring vegetation triggers a shade avoidance response leading to a dramatic change in plant architecture. These changes in light quality are detected by the phytochrome family of photoreceptors. We analyzed global changes in gene expression over time in wild-type, phyB mutant, and phyA phyB double mutant seedlings of Arabidopsis in response to simulated shade. Using pattern fitting software, we identified 301 genes as shade responsive with patterns of expression corresponding to one of various physiological response modes. A requirement for a consistent pattern of expression across 12 chips in this way allowed more subtle changes in gene expression to be considered meaningful. A number of previously characterized genes involved in light and hormone signaling were identified as shade responsive, as well as several putative, novel shade-specific signal transduction factors. In addition, changes in expression of genes in a range of pathways associated with elongation growth and stress responses were observed. The majority of shade-responsive genes demonstrated antagonistic regulation by phyA and phyB in response to shade following the pattern of many physiological responses. An analysis of promoter elements of genes regulated in this way identified conserved promoter motifs potentially important in shade regulation.

Integrative analysis of DNA methylation and mRNA expression during differentiation of umbilical cord blood derived mononuclear cells to endothelial cells

BackgroundActinobacteria form a major bacterial phylum that includes numerous human pathogens. Actinobacteria are primary contributors to carbon cycling and also represent a primary source of industrial high value products such as antibiotics and biopesticides. Consistent with other members of the actinobacterial phylum, Saccharopolyspora erythraea undergo a transitional switch. This switch is characterized by numerous metabolic and morphological changes.ResultsWe performed RNA sequencing to analyze the transcriptional changes that occur during growth of Saccharopolyspora erythraea in batch culture. By sequencing RNA across the fermentation time course, at a mean coverage of 4000X, we found the vast majority of genes to be prominently expressed, showing that we attained close to saturating sequencing coverage of the transcriptome. During the metabolic switch, global changes in gene expression influence the metabolic machinery of Saccharopolyspora erythraea, resetting an entirely novel gene expression program. After the switch, global changes include the broad repression of half the genes regulated by complex transcriptional mechanisms. Paralogous transposon clusters, delineate these transcriptional programs. The new transcriptional program is orchestrated by a bottleneck event during which mRNA levels are severely restricted by targeted mRNA degradation.ConclusionsOur results, which attained close to saturating sequencing coverage of the transcriptome, revealed unanticipated transcriptional complexity with almost one third of transcriptional content originating from un-annotated sequences. We showed that the metabolic switch is a sophisticated mechanism of transcriptional regulation capable of resetting and re-synchronizing gene expression programs at extraordinary speed and scale.

Microcystis blooms occur worldwide and threaten aquatic ecosystems and human health. Sublethal effects on early developmental stages of fish are largely unknown, and research has mainly focused on microcystin toxins (such as MC-LR) rather than Microcystis cells. We exposed (96 h) zebrafish larvae to purified MC-LR (0-1000 μg/L) or lyophilized Microcystis aeruginosa containing 4.5 μg/L MC-LR and evaluated changes in global gene expression (Affymetrix GeneChip zebrafish genome arrays). Significant changes in gene expression (≥ 1.7-fold change, p < 0.0001) were determined with Rosetta Resolver 7.0, and ontology analysis was conducted with the DAVID bioinformatics tool. The number of differentially expressed genes relative to control increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR in mammals and older life stages of fish, as well as genes unique to larval zebrafish. Up-regulation of vitellogenin genes (vtg) (19.2-fold to >100-fold on arrays; 619.3-fold confirmed by quantitative PCR) was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR. Up-regulation of vtg indicates exposure to estrogenic substance(s) and suggests that Microcystis may be a natural source of environmental estrogens. Concerns about effects of Microcystis blooms may extend beyond those associated with the microcystin toxin.

Development of pancreatic ductal adenocarcinoma (PDAC) follows an increasingly well-defined series of genetic events in the pancreatic epithelium. Less well understood are the steps orchestrating the exuberant desmoplasia that accompanies every case of PDAC. This accumulation of stromal components arises from and is maintained by the reciprocal interactions between genetically altered tumor cells and adjacent stroma. Pancreatic stellate cells (PSCs) are myofibroblast-like cells activated by tissue damage and implicated in wound healing. Recent work suggests that PSCs are key primary drivers of the desmoplastic reaction that characterizes the disease, possibly through their established secretory capacity. We wished to explore the paracrine effects of PSC secreted factors on gene expression in pancreatic cancer cells. To investigate the global gene expression changes induced by stromal factors, PDAC cell lines were grown in “a-stromal” or “stromal” conditions. The addition of structural and diffusible PSC-derived factors significantly altered expression of thousands of genes in the cancer genome, suggesting that these factors exert genome-wide effects on the epigenetic state to so broadly affect transcription level. Stromal inputs lead to broad hyperacetylation of histones in the PDAC epigenome, particularly on gene-activating lysines on histone tails. These epigenetic events coincided with induction of genes implicated in anabolic metabolism, the glycosylation pathway, and inflammation. The gene expression changes induced by stromal factors overlap with those previously identified following oncogenic Kras, suggesting functional complementarity between cell-autonomous and microenvironmental cues characteristic of PDAC. As epigenome modification results in changes in gene expression through the functions of readers, we tested a small molecule inhibitor of a family of epigenetic readers for the capacity to inhibit gene expression changes downstream of stromal factors. Paracrine induction of pro-growth and inflammatory genes was inhibited by this small molecule, suggesting that acetyl-lysine readers serve as key transducers of stromal inputs to drive alterations in gene expression in pancreatic cancer cells. Inhibition of acetyl-lysine sensing reduced pancreatic tumor growth and associated inflammation in vivo, with significant reduction in expression of genes implicated in macromolecule biosynthesis and tumor-permissive immune regulation. This work supports further investigation of epigenetic modulators, in combination with targeted drugs or immunotherapeutic approaches, for the treatment of PDAC. Citation Format: Mara H. Sherman, Eric Collisson, Michael Downes, Ronald M. Evans. Paracrine regulation of the pancreatic cancer epigenome. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A42.