Comparative microarrays of Actinoplanes sp. SE50/110 ΔacrA with the Actinoplanes sp. SE50/110 wildtype
ABSTRACT: In order to characterize the transcriptional regulator AcrA, comparative genome wide transcriptome analyses were conducted. Therefore, the wild type Actinoplanes sp. SE50/110 and the mutant ΔacrA were each cultivated in triplicates in minimal medium supplemented with maltose or glucose as single carbon source. RNA samples from the biological replicates were taken from the middle of the growth phase of both strains in each maltose and glucose minimal medium, respectively. RNA was isolated and the three replicates were combined for each strain and condition. For each cultivation condition, the data from two arrays (dye swap) were combined to make statistically reliable conclusions.
Project description:The acarviose metabolite acarbose is an a glucosidase inhibitor produced by Actinoplanes sp. SE50/110. It is medically important because it is used in the treatment of type 2 diabetes. In this work a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out. The associated txt and RAW files were used for two different analyses and publications. While one study focused on a comparative analysis of Actinoplanes sp. SE50/110 to elucidate differences in the proteome cultures that were grown with either maltose or glucose, the other study applied spectral counting and analyzed only the maltose-grown cultures to determine the major proteins and their location in the cell. The txt files for the comparative data are labeled as "heavy_light" and of the spectral counting data as "light". Both datasets were derived from the same RAW files.
Project description:Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. A PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), is localized upstream of the maltase gene amlE (ACSP50_2474), which is organized in an operon with and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. A targeted gene deletion mutant of amlR was constructed by use of CRISPR/Cas9 technology. The transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ∆amlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ∆amlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes, which was shown by RNAseq. We used a pooled library for RNAseq for global pre-screening and validated these results for a total of 25 genes by RT-qPCR. RNA of triplicates of the wildtype and the deletion mutant was isolated from growing cultures and pooled equimolar separately for the wildtype and the deletion mutant (total amount of 2.5 µg RNA each in 26 µL RNase-free water). rRNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina, San Diego, USA). Successful depletion of rRNA was verified by an Agilent RNA 6000 Pico chip in the Bioanalyzer (Agilent, Böblingen, Germany). cDNA libraries were prepared following protocols from Pfeifer-Sancar et al. (2013) and Irla et al. (2015) by use of the TruSeq stranded mRNA kit (Illumina, San Diego, CA, USA). The libraries were quantified by a DNA High Sensitivity Assay chip in the Bioanalyzer (Agilent, Böblingen, Germany) and sequenced on a 2 x 75 nt HiSeq 1500 run (Illumina, San Diego, CA, USA). Sequencing yielded about 10.7 million read pairs for the wildtype library and 10.2 million read pairs for the deletion mutant library, respectively. Raw reads were quality-trimmed using Trimmomatic v0.3.5 (Bolger et al., 2014). Trimmed reads were mapped to the respective reference sequence (GenBank: LT827010.1) using bowtie2 in the paired-end mode (Langmead and Salzberg, 2012), resulting in 20.7 and 19.5 million mappings. ReadXplorer was used for visualization and differential gene expression analysis (Hilker et al., 2016).
Project description:The whole coding RNA of Actinoplanes sp. SE50/110 mutants containing two different integrative vectors (pSET152::PrpsJ:acbC and pGUS::PrpsJ:gusA) were sequenced. Both vectors are integrated via a phiC31 integrase (Bierman et al. 1992) into the genetic locus ACSP50_6589 (former: acpl_6602) (Gren et al. 2016). For expression of the gene of interest (either gusA or acbC) the native promoter of the ribosomal gene rpsJ (ACSP50_0690) was used. The pGUS and pSET152 backbone mainly differ by the integration of a spectinomycin resistance cassette aadA in the pGUS-vector. The cassette is flanked by two T4-terminators to block the transcription of the goi from upstream promoters (Myronovskyi et al. 2011). As shown by this experiment, the two T4-terminators block transcription and prevent read-through efficiently in pGUS, whereas continuous tracks can be observed in pSET152, which extent from int to acbC.
Project description:The promoter structure influences binding and clearance of RNA polymerase and therefore substantially influences expression of a gene. A promoter usually consists of a -10 and a -35-region, an extended -10-motif and A+T-rich upstream promoter elements. Most of these elements are optional, whereas the -10-region is essential (Albersmeier et al. 2017). Knowledge about the transcription start sites (TSS) of genes allows genome-wide localization and determination of the promoter regions. In our group, a special protocol for the amplification of primary transcripts was developed, including the capture of primary transcripts, rewriting them into cDNA (complementary DNA) and amplification in the further course of the protocol (Pfeifer-Sancar et al. 2013). Here, TSS were manually determined with special regard to the heterologous promoters. For each construct, at least one and up to three different TSS were found, leading to the identification of one or several -10-core-hexamers. These were located mostly 6 to 7 nucleotides upstream of each TSS, which corresponds to the average distance of 6.4 nt described for Actinoplanes sp. SE50/110 by Schwientek et al. (2014).
Project description:In rainbow trout, type A spermatogonia can be split into SP cells and non-SP cells by the ability to exclude Hoechst 33342 dye (H33342). The H33342 fluorescence of SP cells are lower than that of non-SP cells, after H33342 staining. To investigate whether SP cells were transcriptomically distinct from non-SP cells, we compared the transcriptome of these cells. We used fluorescence-activated cell sorting (FACS) to isolate SP cells and non-SP cells from the type A spermatogonia in rainbow trout. To compensate unavailability of genetically uniform rainbow trout in independent sampling, SP cells and non-SP cells were collected at 3 times from 3 different parental fish groups. This experimental design allowed us to estimate effects specific to each parental fish genotype on mRNA expression in SP cells by a statistical modeling and to exclude the effects in subsequent analysis.
Project description:Small CAB-like proteins (SCPs) are single-helix light-harvesting-like proteins found in all organisms performing oxygenic photosynthesis. We investigated the function of these stress-induced proteins in the cyanobacterium Synechocystis sp. PCC 6803 by comparing a strain, which expresses SCPs constitutively, with a mutant lacking all five SCPs. In the absence of SCPs, cells were larger, and showed irregular thylakoid structure and cell-surfaces. Deletion of scp genes strongly affected the carbon-nitrogen balance, causing accumulation of carbohydrates and a decrease in N-rich compounds (proteins and chlorophyll a). Data from transcriptomic and metabolomic experiments demonstrated that SCPs mediated stabilization of chlorophyll a under stress conditions is crucial to maintain the C/N balance. SCPs diminished the formation of reactive oxygen species, preventing cellular damage by adjusting the de-novo production of chlorophyll a to demand levels. Lack of SCP expression under stress conditions had a large impact on the metabolism of the entire cell. Synechocystis 6803 PSI-less (Shen et al 1993) and PSI-less/ScpABCDE- (Xu et al 2004) mutants were cultivated at 30°C at a light intensity of 4–5 μmol photons m–2 s–1 in BG-11 growth medium (Rippka et al 1979) supplemented with 10 mM glucose. Cells (40–50 ml) from both control and the mutant cultures were collected at mid-log phase by rapid filtration (Pall Supor 800 Filter, 0.8 mm) and total RNA extracted after Pinto et al. (Pinto et al 2009). Single-stranded cDNA preparation, labelling, and hybridization onto the microarray slides were performed after Eisenhut et al. (2007). The microarray slides were scanned with an Agilent Microarray Scanner (model G2505B) with default settings.
Project description:We designed a group of probes for detection of known and some putative E. coli sRNAs and included them in a commercial microarray containing a standard set of probes developed by Agilent Technologies for hybridization with E. coli transcripts. To assess the potential of the re-designed microarray to detect E. coli sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells in log phase, grown in rich (Luria-Bertani) and minimall (M9/Glucose) media. The microarray data obtained indicate that many sRNAs could yield reasonably strong signals (i.e. considerably above the background). With few exceptions (such as SraA, IstR-1, OmrA, RseX and DicF), nearly all known trans-encoded sRNAs annotated in several databases including EcoCyc, RNAMap and BSRD were detected. The expression patterns of some transcripts were further validated by Northern blot analysis.
Project description:Aedes aegypti SP strain vs. SMK strain. Aedes aegypti is the major vector of yellow fever and dengue/dengue hemorrhagic fever. Starting with a population collected from Singapore, we established a pyrethroid-resistant A. aegypti strain (SP) and investigated three major possible mechanisms of insecticide resistance. After 10 generations of adult selection, an A. aegypti strain developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel genes (Vssc) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can contribute more to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than the susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over-expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2, and confirmed that they were capable of detoxifying permethrin to 4'-HO-permethrin. Over-expression of CYP9M6 was partially due to gene amplification. Association analysis demonstrated that CYP9M6 and CYP6BB2 complementarily conferred permethrin resistance. Two other P450s (CYP9J26 and CYP9J28), which are capable of metabolizing permethrin, were also over-expressed in the SP strain, indicating that at least four P450 isoforms are likely involved in resistance development. Our data show that it is unlikely that reduced cuticle penetration of permethrin contributes to resistance. One-color experiment with two strains (SP, SMK) and 3 developmental stages/genders (larvae, adult males, and adult females), 4 biological replicates each.
Project description:Azoarcus sp. BH72 is able to communicate via cell density-dependent gene regulation. Here, the impact of cell-free conditioned culture supernatants, obtained from stationary phase Azoarcus wild type cultures, on gene expression was investigated determining changes in transcript profiles when early exponential aerobic cultures were incubated with cell-free culture supernatants for one and four hours. Bacterial communication via quorum sensing (QS) is involved in the regulation of several cellular mechanisms such as metabolic processes, microbe-host interactions or biofilm formation. The nitrogen-fixing model endophyte of grasses Azoarcus sp. strain BH72 shows density-dependent gene regulation in the absence of common hydrophobic autoinducers for pilA encoding the structural protein of type IV pili that are essential for plant colonization. Here, we used a transcriptomic approach to identify target genes differentially regulated under QS conditions in conditioned supernatants in comparison to standard growth conditions. Analysis used RNA from the early exponential growth phase as control samples for comparison to the quorum-sensing condition samples taken at one hour and four hours after incubation with cell-free culture supernatants.
Project description:Commensal (symbiont) bacteria form communities in various regions of the bodies of vertebrates. Phylogenetic analysis of gut communities is advanced, but the relationships, especially at the trophic level, between commensals that share gut habitats of monogastric animals have not been investigated to any extent. Lactobacillus reuteri strain 100-23 and Lactobacillus johnsonii strain 100-33 cohabit in the forestomach of mice. According to the niche exclusion principle, this should not be possible because both strains utilise the two main fermentable carbohydrates present in the stomach digesta: glucose and maltose. We show, based on gene transcription analysis, in vitro physiological assays, and in vivo experiments that the two strains can co-exist in the forestomach habitat because L. reuteri 100-23 transports maltose into its cells more efficiently than does L. johnsonii 100-33. Conversely, strain 100-33 transports glucose more efficiently than 100-23. As a result, 100-23 shows a preference for growth using maltose, whereas 100-33 prefers glucose. Mutation of the maltose phosphorylase gene (malA) of strain 100-23 prevented its growth on maltose-containing culture medium, and resulted in the numerical dominance of 100-33 in the forestomach. The fundamental niche of L. reuteri 100-23 in the mouse forestomach can be defined in terms of glucose and maltose fermentation. Its realised niche when L. johnsonii 100-33 is present is maltose fermentation. Hence nutritional adaptations provided niche differentiation that enabled cohabitation by the two strains through resource partitioning in the mouse forestomach. This real life, trophic phenomenon conforms to a mathematical model. Analysis of the microarray data was obtained from two independent biological replicates. A dye swap was included in the analysis