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| Status |
Public on Feb 03, 2023 |
| Title |
The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress [RNA-seq] |
| Organism |
Porphyromonas gingivalis |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
To understand the role of CdhR and its adjacent gene PG1236 in nitric oxide (NO) stress resistance, isogenic mutants P. gingivalis FLL457 (ΔPG1237::ermF), FLL458 (ΔPG1236::ermF) and FLL459 (ΔPG1236-37::ermF) were made by allelic exchange mutagenesis and their gene expression was studied under control and NO stress conditions. DNA microarray analysis of FLL457 showed that approximately 2% of the genes were up regulated and over 1% of the genes down regulated. Transcriptome analysis of FLL458 and FLL459 under NO stress showed similar modulation patterns in both mutants for a few genes. The PG1236-7 gene cluster seemed to be part of the same transcriptional unit that showed increased expression under NO stress. Recombinant CdhR showed binding activity to the predicted promoter regions of PG1459 and PG0495. Taken together, the data indicate that CdhR may play a role in NO stress resistance and be involved in a regulatory network in P. gingivalis.
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| Overall design |
Fresh cultures of P. gingivalis strains (mutants and wild type) were grown from overnight cell cultures under anaerobic conditions at 37ºC in BHI broth. P. gingivalis strains were grown to early exponential phase (OD600:~0.3) in BHI broth under anaerobic conditions at 37ºC. At that time point, the bacteria cultures were stressed with Diethylamine (DEA) NONOate (NO stress condition). Untreated wild type and mutant strains cell cultures grown under the same conditions were used as controls. Experiments were done in triplicates. After 15 minutes, stressed and control samples were taken from the fresh cultures. The library was generated using RiboMinus Transcriptome Isolation Kit (Life Technologies, CA) and NEXTflex RNA-Seq Kit (Bioo Scientific, TX) following the vendors’ protocols. Briefly, 2 μg total RNA (20 μl) was hybridized with 4 μl of RiboMinus probe in 100 μl hybridization buffer at 37 ºC for 5 min, which was then cooled on ice for at least 30 seconds. The sample was transferred into an Eppendorf tube containing RiboMinus Beads resuspended in 100 μl hybridization buffer, which was incubated at 37 ºC for 15 min to allow beads to bind with probes. The tube was placed on a magnetic separator for 1 min to pellet the rRNA-probe complex. The supernatant containing RiboMinus RNA was transferred into a new tube. The RiboMinus RNA was concentrated by ethanol precipitation and was resuspended in 14 μl nuclease free water. Five μl of NEXTflex RNA Fragmentation buffer (Bioo Scientific, TX) was mixed with the 14 μl RiboMinus RNA. The mixture was incubated at 95 ºC for 10 min and then chilled on ice immediately. The first and second strand cDNAs were synthesized following the Bioo Scientific protocol and the NEXTflex RNA-Seq barcode was ligated to double strand cDNA after the end repair and adenylation for multiplexing. The library was amplified by PCR. The quality of each RNA-Seq library was checked using Agilent Bioanaylzer and DNA 1000 Nano chip and the library was quantified using Qubit (Life Technology, CA). The libraries (20 samples) were loaded into a single lane of Illumina V3 flow cell for cluster generation and the sequencing was done on Illumina HiScanSQ with 100 x 100 BP of paired-end reads.
***Please note that raw data files have been lost and thus are not provided***
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| Contributor(s) |
Boutrin M, Mishra A, Wang C, Dou Y, Flether HM |
| Citation(s) |
37134265 |
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| Submission date |
Jan 30, 2023 |
| Last update date |
May 01, 2024 |
| Contact name |
Marie-Claire Boutrin |
| Organization name |
Oakwood University
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| Street address |
7000 Adventist Blvd NW
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| City |
Huntsville |
| ZIP/Postal code |
35896 |
| Country |
USA |
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| Platforms (1) |
| GPL33075 |
Illumina HiScanSQ (Porphyromonas gingivalis) |
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| Samples (12)
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| GSM7011225 |
Pophyromonas gingivalis PG1236-deficient mutant control [X1236_00_YY_A] |
| GSM7011226 |
Pophyromonas gingivalis PG1236-deficient mutant control [X1236_00_YY_B] |
| GSM7011228 |
Porphyromonas gingivalis PG1236-deficient mutant NO stress [X1236_00_NO_A] |
| GSM7011229 |
Porphyromonas gingivalis PG1236-deficient mutant NO stress [X1236_00_NO_B] |
| GSM7011230 |
Porphyromonas gingivalis PG1236-37-deficient mutant control [X1236-37_00_YY_A] |
| GSM7011232 |
Porphyromonas gingivalis PG1236-37-deficient mutant control [X1236-37_00_YY_B] |
| GSM7011233 |
Porhyromonas gingivalis PG1236-37-deficient mutant NO stress [X1236-37_00_NO_A] |
| GSM7011234 |
Porphyromonas gingivalis PG1236-37-deficient mutant NO stress [X1236-37_00_NO_B] |
| GSM7011236 |
Porphyromonas gingivalis W83 control [WT83_00_YY_A] |
| GSM7011237 |
Porphyromonas gingivalis W83 control [WT83_00_YY_B] |
| GSM7011238 |
Porphyromonas gingivalis W83 NO stress [WT83_00_NO_A] |
| GSM7011240 |
Porphyromonas gingivalis W83 NO stress [WT83_00_NO_B] |
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| This SubSeries is part of SuperSeries: |
| GSE224067 |
The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress |
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| Relations |
| BioProject |
PRJNA929508 |
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