Dataset Information

Effects of Campylobacter jejuni CJIE1 prophage on protein expression experiment 4

ABSTRACT: Temperate bacteriophages (prophages) have recently been demonstrated in Campylobacter jejuni. However, what they do there is largely unknown. In the series of studies that are the subject of these submissions we have investigated the relative expression levels of proteins in C. jejuni isolates that differ in the presence or absence of the CJIE1 prophage. At the time of the initial investigations whole genome sequence data were not available for the isolates used, though DNA microarray data indicated that the isolates were very closely related. The overall project was carried out through four separate experiments. Previous work in the scientific literature indicated that growth on medium lacking blood but containing sodium deoxycholate induced the expression of at least some proteins associated with virulence and provided data thought to be of relevance to the virulence of the bacterium. The second set of experiments (experiment 2) compared protein expression in 4-plex iTRAQ experiments using two isolates grown on medium with and without 0.1% sodium deoxycholate to further explore the effects of the CJIE1 prophage, if any, on protein expression. Results from that experiment indicated that there was an unexpectedly large bile response, and that there were differences in expression from the first experiment that may be attributable to the presence of blood in the culture medium in the first experiment, compared with the use of medium without blood in the second experiment. Experiment 3 was therefore designed to compare protein expression of the CJIE1-carrying isolate 00-2425 after growth on medium containing blood, without blood, and without blood but with bile salts. In experiment 4 we grew isolate 00-2426, which lacks prophage CJIE1, on the same four media. The goals were to determine if the differences in protein expression (detection) were similar to those in isolate 00-2425 and if there were any differences that could be attributed to the presence or absence of the CJIE1 prophage.

OTHER RELATED OMICS DATASETS IN: PRJNA213842PRJNA213841PRJNA211797PRJNA214465

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Campylobacter Jejuni Subsp. Jejuni

SUBMITTER: Clifford Clark

LAB HEAD: Garrett R. Westmacott

PROVIDER: PXD000801 | Pride | 2014-07-31

REPOSITORIES: Pride

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Publications

The CJIE1 prophage of Campylobacter jejuni affects protein expression in growth media with and without bile salts.

Clark Clifford G CG Chong Patrick M PM McCorrister Stuart J SJ Simon Philippe P Walker Matthew M Lee David M DM Nguy Kimberly K Cheng Keding K Gilmour Matthew W MW Westmacott Garrett R GR

BMC microbiology 20140319

<h4>Background</h4>The presence of Campylobacter jejuni temperate bacteriophages has increasingly been associated with specific biological effects. It has recently been demonstrated that the presence of the prophage CJIE1 is associated with increased adherence and invasion of C. jejuni isolates in cell culture assays.<h4>Results</h4>Quantitative comparative proteomics experiments were undertaken using three closely related isolates with CJIE1 and one isolate without CJIE1 to determine whether th ...[more]

PMID: 24641125

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Project description:Temperate bacteriophages (prophages) have recently been demonstrated in Campylobacter jejuni. However, what they do there is largely unknown. In the series of studies that are the subject of these submissions we have investigated the relative expression levels of proteins in C. jejuni isolates that differ in the presence or absence of the CJIE1 prophage. At the time of the initial investigations whole genome sequence data were not available for the isolates used, though DNA microarray data indicated that the isolates were very closely related. The overall project was carried out through four separate experiments. The first set of experiments (experiment 1) therefore compared protein expression in 4-plex iTRAQ experiments using three isolates carrying the (previously sequenced) prophage with a fourth isolate that was epidemiologically and genetically related to these isolates but which naturally lacked the CJIE1 prophage. Three replicate experiments were done. These isolates were grown on medium containing blood to obtain optimal growth and determine whether any differences were found. Three replicate experiments were done. However, since these growth conditions may not mimic in vivo growth of the organism the results may not be considered relevant or very interesting. Previous work in the scientific literature indicated that growth on medium lacking blood but containing sodium deoxycholate induced the expression of at least some proteins associated with virulence and provided data thought to be of relevance to the virulence of the bacterium. Therefore two isolates were chosen, one with and one without the CJIE1 prophage. Each of three replicates of a 4-plex iTRAQ experiment compared protein expression of the two isolates (00-2425 and 00-2426) after growth on Mueller-Hinton agar with and without 0.1% sodium deoxycholate. There was pretty good overlap between the proteins expressed in the first two experiments but, because all results were expressed as log2 change values, it was difficult to determine whether the expression of some proteins was completely absent in one or the other isolate. As well, protein expression overall showed quite extensive differences compared with the first experiment that we attributed to the lack of blood in the growth medium; we thought we might be seeing a response to limited iron as well. Finally, the response to the deoxycholate was extensive, and we wished to further explore this phenomenon as a means of determining or verifying what effects were due to the presence of the CJIE1 prophage and which were due to other differences. Further 4-plex iTRAQ experiments compared protein expression for each of isolate after growth on medium containing blood, without blood, without blood plus sodium deoxycholate, and without blood plus 2.5% Oxgall. Comparison of the results for isolate 00-2425 (with the CJIE1 prophage) and 00-2426 (without the prophage) identified a small number of proteins that could not be detected in one or the other isolate and allowed discrimination of the effects of the prophage from the responses to iron limitation and bile salt preparations. This dataset is associated with the first experiment, experiment 1, which compares C. jejuni isolates carrying the CJIE1 prophage (00-2425, 00-2538, 00-2544) with a very closely related isolate lacking the prophage (00-2426)

Project description:Pf-5 is an important biocontrol strain of Pseudomonas fluorsecens, that improves plant growth, mainly via the production of secondary metabolites and growth/colonisation competition. Copper is a broad-spectrum antimicrobial that is effective against bacteria, fungi and even viruses in soluble forms such as copper sulfate and to a lesser extent in solid form, such as copper surfaces. Copper sulfate is commonly sprayed on food crops to increase yield and is becoming more routinely used due to the increasing problem of resistance to standard pesticides. Thus, an obvious question that remains is: what effect is this introduced copper having on these important biocontrol strains? First, the phenotypic effects of copper on carbon utilisation and pH tolerance was tested using Biolog. Interestingly, the ability of Pf-5 to utilise amino acids as a sole carbon source was largely unaltered, but the presence of copper completely eliminated the utilisation of carbohydrates or fatty acids, and diminished the use of carboxylic acids and amines. This could be explained by copper-mediated disruptions of enzymes in metabolic pathways, such as the TCA cycle. The effect on gene expression was examined using reverse transcriptomics, which established molecular bases for the phenotypic results, and confirmed some known mechanisms for copper resistance, efflux and transporter proteins, and highlighted the delicate interplay with cellular control of iron, as well as uncovered the interesting relationship between integrated bacteriophage and copper as a molecular switch. The integrated phage, Prophage 01, was downregulated in the presence of copper and when this phage was activated with Mitomycin C, the copper appeared to stop the phage from going into lytic cycle and protected lysis of the cells. Prophage 01 is integrated between the housekeeping genes mutS and cinA and is conserved throughout many Pseudomonas. However, only Pf-5, out of the 10 strains tested seemed to be protected from cell lysis by copper, indicating that Prophage 01 in Pf-5 has unique features that interact with Cu. Two-condition experiment, where normal culture in rich medium versus cell treated with CuSO4. 3 biological replicates including 3 technical replicates for one of the biological replicates and 2 technical replicates for another of biological replicates. Swap-dye experiments were performed.

Project description:We combined heritability analysis of larval development rate with a global expression analysis of this phenotype to investigate genotype by environment interactions across three ecologically relevant temperatures in the Glanville fritillary butterfly (Melitaea cinxia). We focused upon the development of final instar caterpillars which is greatly affected by temperature, and during this stage the caterpillars build up most of the resources for adult life. Second generation, lab reared larvae, initially collected from the M-CM-^Eland metapopulation, were reared in standard lab condition until 6th larval instar. At the beginning of the final (7th) instar stage the larvae were separated into of three temperature conditions: Cold treatment (temperature profile: 8M-BM-0C 18:00-9:59, 14M-BM-0C 10:00-11:59, 20M-BM-0C 12:00-15:59 and 14M-BM-0C 16:00-17:59) Standard treatment (temperature profile: 15M-BM-0C 17:00-6:59, 18M-BM-0C 7:00-8:59, 22M-BM-0C 9:00-10:59 and 26M-BM-0C 11:00-16:59) Hot treatment (temperature profile: 8M-BM-0C 20:00-7:59, 15M-BM-0C 8:00-9:59, 35M-BM-0C 10:00-17:59 and 15M-BM-0C 18:00-19:59) The temperature profiles mimic the diurnal thermal variation of the natural habitat (M-CM-^Eland islands) of samples. Cold treatment mimics a cool and cloudy summer, Standard represents an average temperature profile in the M-CM-^Eland islands and Hot treatment mimics an exceptionally hot and sunny summer, with cold night-time temperatures. The experiment contained several larval families (full-sib) of which three were selected for gene expression analysis. Samples were snap-frozen in liquid nitrogen during mid-development (after 6, 5 and 4 days, for Cold, Standard and Hot respectively). Additional larvae from the same treatments were assayed for survival and growth. Gene expression was analyzed using a mixed model approach to identify genes with potential heritable expression variation (variation among families), genes with plastic expression responses (treatment induced changes) and genes with treatment dependent expression that varies among families (family by treatment interactions). Full-sib larvae from three families (N170, N74 and O171) were exposed to three temperature treatments (Cold, Standard and Hot) during final (7th) instar stage. A total of 35 samples were used to analyze family, treatment and family by treatment interactions in gene expression, using a mixed model approach. This included 3 biological replicates in Cold and Hot treatments from each family and 5-6 biological replicates in Standard (5 in O171) per family. Techinal replicates from each family and additional techical replicates in family O171 (including dye swap replicates) were used to assess robustness of the findings.

Project description:The goal of the experiment was to perform a large scale study of circadian regulation of gene expression in maize. To identify maize genes with expression regulated by the circadian clock, transcript levels in the aerial tissues of young maize seedlings were determined by transcriptional profiling with the Affymetrix GeneChip Maize Genome Array. Maize inbred B73 seedlings were grown inside Conviron growth chamber. B73 seedlings were grown for 7 days under 12 h light:12 h dark (LD) photocycles, 26° C temperature and 70% humidity. At the 8th day, seedlings were transferred to continuous light (LL) and were allowed to entrain completely for 24 h prior to tissue harvest following which tissue was harvested every 4 hours under LL conditions for a period of 48h. Therefore, for the circadian LL time course 12 time points were collected as follows (also defined as factors in the treatment section): ZT0 - 8:00 am/ subjective dawn/ Day1 ZT4 - 12:00 pm/ subjective mid-day/ Day1 ZT8 - 4:00 pm/ subjective late-day/ Day1 ZT12 - 8:00 pm/ subjective dusk/ Day1 ZT16 - 12:00 am/ subjective mid-night/ Day1 ZT20 - 4:00 am/ subjective pre-dawn/ Day1 ZT24- 8:00 am/ subjective dawn/ Day2 ZT28 - 12:00 pm/ subjective mid-day/ Day2 ZT32 - 4:00 pm/ subjective late-day/ Day2 ZT36 - 8:00 pm/ subjective dusk/ Day2 ZT40 - 12:00 am/ subjective mid-night/ Day2 ZT44 - 4:00 am/ subjective pre-dawn/ Day2 Tissue comprised of aerial portion of the seedlings (corresponding to tissue from the prop roots and up) for RNA isolation. Total RNA was isolated from the entire aerial portion of 7 day-old seedlings (corresponding to tissue from the prop roots and up) by Trizol extraction followed by Qiagen RNeasy columns and treatment with RNase-free DNase I (Qiagen; qiagen.com). RNA was isolated from 3 independent biological replicates was pooled. cRNA was generated from pooled total RNA from 3 biological replicates with the GeneChip One-Cycle Target Labeling kit according to the manufacturer’s recommendations (Affymetrix, affymetrix.com). The University of California, Berkeley Functional Genomics Laboratory hybridized samples to Affymetrix GeneChip Maize Genome Arrays and scanned the washed arrays as suggested by manufacturer. Probe sets called “Not Present” or “Marginal” on one or more microarrays were removed from the downstream analysis, as is common practice with circadian studies. Raw hybridization intensities were normalized across all twelve arrays using RMA express in Perfect Match mode. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Frank G. Harmon. The equivalent experiment is ZM28 at PLEXdb.]

Project description:Because iron toxicity and deficiency are equally life threatening, maintaining intracellular iron levels within a narrow optimal range is critical for nearly all known organisms. However, regulatory mechanisms that establish homeostasis are not well understood in organisms that dwell in environments at the extremes of pH, temperature, and salinity. Under conditions of limited iron, the extremophile Halobacterium salinarum, a salt-loving archaeon, mounts a specific response to scavenge iron for growth. We have identified and characterized the role of two transcription factors (TFs), Idr1 and Idr2, in regulating this important response. An integrated systems analysis of TF knockout gene expression profiles and genome-wide binding locations in the presence and absence of iron has revealed that these TFs operate collaboratively to maintain iron homeostasis. In the presence of iron, Idr1 and Idr2 bind near each other at 24 loci in the genome, where they are both required to repress some genes. In contrast, Idr1 and Idr2 are both necessary to activate other genes in a putative a feed forward loop. Even at loci bound independently, the two TFs target different genes with similar functions in iron homeostasis. We discuss conserved and unique features of the Idr1-Idr2 system in the context of similar systems in organisms from other domains of life. Data in this GEO archive are linked to the publication: Schmid AK, Pan M, Sharma K, Baliga NS.2011. Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon.Nucleic Acids Res.39(7):2519-33. Cultures containing either the gene encoding the Idr1 or Idr2 transcription factors with c-terminal fusions to the myc epitope were grown to mid-logarithmic phase in the presence or absence of 100 uM FeSO4. Cultures were subjected to ChIP-chip as described in Facciotti, MT, Reiss, DJ, Pan, M, Kaur, A, Vuthoori, M, Bonneau, R, Shannon, P, Srivastava, A, Donohoe, SM, Hood, LE and Baliga, NS. General transcription factor specified global gene regulation in archaea. Proc Natl Acad Sci U S A. 2007;104: 4630-4635. Each Sample is based on two arrrays (one with dye-swap).

Dataset Information

Effects of Campylobacter jejuni CJIE1 prophage on protein expression experiment 4

Publications

The CJIE1 prophage of Campylobacter jejuni affects protein expression in growth media with and without bile salts.

Similar Datasets

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