Thermotoga species transcription on glucose and a mix of polysaccharides
Ontology highlight
ABSTRACT: Hyperthermophilic bacteria of the genus Thermotoga are known to utilize a wide range of simple and complex polysaccharides. T. maritima's transcriptional response to a variety of mono- and poly-saccharides was previously studied to assign functions to genes involved in carbohydrate uptake and utilization. To compare and contrast closely-related members of the Thermotoga genus, a four-species microarray was developed by expanding a whole genome T. maritima array to include unique genes from three other species (T. neapolitana, T. petrophila, and T. sp. RQ2). This multi-species array was used to investigate the diversity of the genus, specifically the response of each of the four species to a mixture of polysaccharides (galactomannan, glucomannan, xylan, pectin, lichenan, and carboxymethyl cellulose). RNA derived from glucose-grown cultures (glu) was compared to RNA derived from polysaccharide-grown cultures (poly) using a dye swap setup.
Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications. Six dye-flip experiments were conducted using C. saccharolyticus genomic DNA as the reference in each dye-flip, and one of six different Caldicellulosiruptor spp. as a tester in each dye-flip
Project description:Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO2 and H2. C. bescii, C. kronotskyensis and C. saccharolyticus solubilized 38%, 36% and 29% (by weight) of unpretreated switchgrass (5 g/l), repectively, which was about half of the concentration of crystalline cellulose (Avicel, 5 g/l) that was solubilized under the same conditions. The lower yields with C. saccharolyticus were unexpected, given that its genome encodes the same GH9-GH48 multi-domain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing Cellulose to switchgrass showed that many carbohydrate ABC transporters and multi-domain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagella biosynthesis were up-regulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation. A dye swap was completed with each of three Caldicellulosiruptor species: C. bescii, C. kronotskyensis and C. saccharolyticus growing on cellulose and switchgrass. Half of the RNA sample for one condition was labeled with Cy3 and the other half Cy5. The two differentially labeled samples were run on two different slides and analyzed to investigate differences in transcription during growth on cellulose and switchgrass.
Project description:Although proteins and peptides encoded in small open reading frames (ORFs < 100 AA) in microbial genomes can play critical roles as toxins, bacteriocins, transcriptional regulators, signaling molecules, and chaperones, many such ORFs remain annotated as M-bM-^@M-^\hypothetical proteinsM-bM-^@M-^]. In the genome of the hyperthermophilic bacterium, Thermotoga maritima, nearly 2/3 of the 167 small ORFs have no known function. As a strategy for investigating the potential significance of specific small ORFs, growth conditions that could trigger the expression of genes encoding small ORFs were sought. A defined medium supplemented with either 1 or 5 g/L yeast extract was used to track transcriptional response during the transition from exponential to stationary phase. RNA derived from exponential (1E) and stationary (1S) phase cultures grown on 1 g/l yeast extract was compared to RNA derived from exponential (5E) and stationary (5S) phase cultures grown on 5 g/l yeast extract using a four-slide loop design.
Project description:Hyperthermophilic bacteria of the genus Thermotoga are known to utilize a wide range of simple and complex polysaccharides. T. maritima's transcriptional response to a variety of mono- and poly-saccharides was previously studied to assign functions to genes involved in carbohydrate uptake and utilization. To compare and contrast closely-related members of the Thermotoga genus, a four-species microarray was developed by expanding a whole genome T. maritima array to include unique genes from three other species (T. neapolitana, T. petrophila, and T. sp. RQ2). This multi-species array was used to investigate the diversity of the genus, specifically the response of each of the four species to a mixture of polysaccharides (galactomannan, glucomannan, xylan, pectin, lichenan, and carboxymethyl cellulose).
Project description:The closely related Coffea arabica cultivars ‘Tall Mokka’ and ‘Typica’, with excellent flavor, but differing distinctively in the size of aerial organs, branching pattern and branch numbers. Differential gene expression analysis of shoot tips of arabica coffee cultivars 'Tall Mokka' and 'Typica' were done using Potato cDNA microarray as cross-species platform. Using cross-species microarray hybridization, we identified a prolyl oligopeptidase (CaPOP) gene as differentially expressed between the shoot tips of ‘Tall Mokka’ and ‘Typica’. Isolation and sequencing of POP genes from coffee identified three paralogs, CaPOP1, CaPOP2 and CaPOP3. All three genes were present in both cultivars, which suggest that differences in the expression of CaPOP are caused by factor(s) regulating the transcription of CaPOPs. CaPOP1 differs in sequence from CaPOP2 primarily in having two large deletions in the promoter region. CaPOP genes are homologous to arabidopsis At1g20380, encoding a post-proline cleaving enzyme that acts on substrates shorter than 30 amino acids. Ectopic expression of CaPOP1 under its native promoter in transgenic arabidopsis resulted in more secondary branches than in the wild type. This is the first study to successfully isolate CaPOP genes and characterize their expression in the developing tissues of coffee. This study also identified a novel role for prolyl oligopeptidase in control of branching. Eight coffee trees of 'Typica' ('K') and six trees of 'Tall Mokka' ('M') cultivar were used in this study. The trees were equally divided into two groups 'A' and 'B' for each cultivar ('MA','MB', 'KA' and 'KB') and treated as biological replicates. Eight two channel microarray hybridizations were done in following pairs: MA x KA, MA x KB, MB x KA, MB x KB and dye swap replicate of each pair. Summary: Two-sample experiment: Tall Mokka vs. Typica . 8 Hybridizations. 2 Biological replicates per sample. 1 Dye swap per array.
Project description:Thermomacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, a M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supra-normal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to up-regulation of 55 genes. Genome re-sequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism or transport. These mutations included 7 non-synonymous substitutions, 4 insertions and 1 deletion. One of the insertion mutations mapped to pseudogene, Msed_1517, and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that include the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula was naturally lacking this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low affinity high velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated spontaneous arsenate resistant mutants derived from CuR1 all underwent mutation in pitA and non-selectively became copper resistant. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. The study comprises 5 samples, described in detail below. WT_CuR1: Differential transcriptional response of Metallosphaera sedula DSM 5348, WT, to the supra-normal copper resistant spontaneous Metallosphaera sedula mutant, CuR1 under normal growth conditions. This experiment was done to analyze the differential transcription of WT cells compared with CuR1 cells at mid log phase. WT-15_CuR1-15: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 15 minutes post copper challenge. The copper cultures were harvested 15 minutes after the shock. WT-60_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 60 minutes post copper challenge. The copper cultures were harvested 60 minutes after the shock. WT-15_WT-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively. CuR1-15_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula CuR1 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively.
Project description:Co-immunoprecipitation of the maize pentatricopeptide protein PPR4 and extraction of bound RNA. RNA is labelled and hybridized to a maize chloroplast genome tiling array in order to identify target RNA species of PPR4.
Project description:Thermoacidophilic archaea are found in heavy metal-rich environments and, in some cases, these microorganisms are causative agents of metal mobilization through cellular processes related to their bioenergetics. Given the nature of their habitats, these microorganisms must deal with the potentially toxic effect of heavy metals. Here, we show that two thermoacidophilic Metallosphaera species with nearly identical (99.99%) genomes differed significantly in their sensitivity and reactivity to uranium. M. prunae, isolated from a smoldering heap on a uranium mine in Thuringen, Germany, could be viewed as a M-bM-^@M-^\spontaneous mutantM-bM-^@M-^] of M. sedula, an isolate from Pisciarelli solfatara near Naples, Italy. M. prunae tolerated U3O8 and U(VI) to a much greater extent than M. sedula. Within 15 minutes following exposure to M-bM-^@M-^\U(VI) shockM-bM-^@M-^], M. sedula, and not M. prunae, exhibited transcriptomic features associated with severe stress response. Furthermore, within 15 minutes post-U(VI) shock, M. prunae, and not M. sedula, showed evidence of substantial degradation of cellular RNA. This suggested that transcriptional and translational processes were aborted as a dynamic mechanism for resisting U toxicity; by 60 minutes post-U(VI) shock, RNA integrity in M. prunae recovered, and known modes for heavy metal resistance were activated. In addition, M. sedula rapidly oxidized solid U3O8 to soluble U(VI) for bioenergetic purposes, a chemolithoautotrophic feature not previously reported. M. prunae, however, did not solubilize solid U3O8 to any significant extent, thereby not exacerbating U(VI) toxicity. These results point to uranium extremophily as an adaptive, rather than intrinsic, feature for Metallosphaera species, driven by environmental factors. The study comprises 9 Samples, described in detail below. MprAU_MseAU: Transcriptional analysis of the response of Metallosphaera prunae (Mpr) and Metallosphaera sedula(Mse) to chemolithoautotrophic conditions (0.1 wt% Uranium octaoxide with CO2 supplementation in headspace). This experiment was done to identify the key terminal oxidases which responded to a Uranium oxide while doing inter-species comparison between Mpr and Mse. Transcriptional response of the terminal oxidase clusters proved that certain key genes play a role in the vastly different physiologies of these two species. MprN_MprU60: Transcriptional analysis of the response of Metallosphaera prunae (Mpr) to 60 min of Uranium shock. This experiment was done to analyze the differential transcription of Mpr cells challenged with 1 mM uranyl acetate shock (U shock) compared to normal growth. The Uranium cultures were harvested 60 min after the shock. MprN_MseN: Differential transcription of Metallosphaera species under normal growth conditions. This experiment was done to analyze the differential transcription of Mpr cells compared with Mse cells at mid log phase. MprN_MprU3h: Transcriptional response of Metallosphaera prunae (Mpr) to 3h of Uranium shock compared to normal growth. This experiment was done to analyze the differential transcription of Mpr cells challenged with 1 mM uranyl acetate shock (U shock) . The Uranium cultures were harvested 3 h after the shock. MseN_MseU15: Transcriptional response of Metallosphaera sedula (Mse) to 15 min of Uranium shock. This experiment was done to analyze the differential transcription of Mse cells challenged with 1 mM uranyl acetate shock (U shock) compared to normal growth. The Uranium cultures were harvested 15 min after the shock. MseN_MseU60: Transcriptional response of Metallosphaera sedula to 60 min of Uranium shock. Mse cells were grown upto mid log phase after which the cells were subjected to U shock and harvested 60 min later. Biological repeats were done for both experimental conditions. MseN_MseU3h: Transcriptional response of Metallosphaera sedula (Mse) to 3h of Uranium shock compared to normal growth. This experiment was done to analyze the differential transcription of Mse cells challenged with 1 mM uranyl acetate shock (U shock) . The Uranium cultures were harvested 3 h after the shock. MseU15_MseU60: Transcriptional response of Metallosphaera sedula to 15 min of Uranium shock compared with 60 min of Uranium shock. This experiment was done to analyze the differential transcription of Mse cells challenged with 1 mM uranyl acetate shock (U shock) . The Uranium cultures were harvested 15 min and 60 min after the shock. MprU3h_MseU3h: Differential transcription of Metallosphaera cells under Uranium shock. This experiment was done to analyze the differential transcription of Metallosphaera sedula (Mse) and Metallosphaera prunae (Mpr) challenged with 1 mM uranyl acetate.
Project description:This work describes the development of the first microarray detection system that simultaneously identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. 34 probes that reproducibly detected multiple Legionella species with high specificity were included in the array.
Project description:The AUXIN BINDING PROTEIN 1 (ABP1) is an essential plant protein involved in the control of growth and development all along the plant life. This protein was initially identified on its capacity to bind the phytohormone auxin. Binding of auxin to ABP1 was shown to enhance cell expansion of leaf cells. Conversely, the functional inactivation of ABP1 was demonstrated to severely impair cell expansion in shoot tissues. To date, the mechanism by which ABP1 controls cell expansion is still poorly understood. ABP1 was reported to affect expression of various auxin regulated genes but little is known on broader effects of ABP1 on gene expression. Here we have investigated the role of ABP1 in the control of cell expansion using dark grown hypocotyls by analyzing changes in gene expression resulting from ABP1 inactivation. Experiments were performed using the control transgenic line Alc-GUS and the SS12K9, which express GUS and ABP1, respectively, in response to ethanol treatment. Three biological repeats were prepared. RNA samples were labelled with Cy5 and Cy3 as indicated below. Samples from the SS12K9 line were compared to the corresponding control line.