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Series GSE253516 Query DataSets for GSE253516
Status Public on Oct 09, 2024
Title The transcriptional program of Staphylococcus aureus phage K is affected by a host rpoC mutation that confers phage K resistance
Organism Staphylococcus aureus
Experiment type Expression profiling by high throughput sequencing
Summary To better understand host/phage interactions and the genetic bases of phage resistance in a model system relevant to potential phage therapy, we isolated several spontaneous mutants of the USA300 S. aureus clinical isolate NRS384 that were resistant to phage K. Six of these had a single missense mutation in the host rpoC gene, which encodes the RNA polymerase beta prime subunit. To examine the hypothesis that the mutations in the host RNA polymerase affect the transcription of phage genes, we performed RNA-seq analysis on total RNA samples collected from NRS384 wild-type (WT) and rpoC G17D mutant cultures infected with phage K, at different time points after infection. Infection of the WT host led to a steady increase of phage transcription relative to the host. Our analysis allowed us to define different early, middle, and late phage genes based on their temporal expression patterns and group them into transcriptional units. Predicted promoter sequences defined by conserved -35, -10, and in some cases extended -10 elements were found upstream of early and middle genes. However, sequences upstream of late genes did not contain clear, complete, canonical promoter sequences, suggesting that factors in addition to host RNA polymerase are required for their regulated expression. Infection of the rpoC G17D mutant host led to a transcriptional pattern that was similar to the WT at early time points. However, beginning at 20 minutes after infection, transcription of late genes (such as phage structural genes and host lysis genes) was severely reduced. Our data indicate that the rpoCG17D mutation prevents the expression of phage late genes, resulting in a failed infection cycle for phage K. In addition to illuminating the global transcriptional landscape of phage K throughout the infection cycle, these studies can inform our investigations into the bases of phage K’s control of its transcriptional program as well as mechanisms of phage resistance.
 
Overall design For transcriptomic analysis, four replicate 25 mL cultures of NRS384WT and rpoC G17D were grown to an OD600 of ~0.3, and phage K was added at an MOI of 5. 1 mL samples were collected at 2, 5, 10, 20, 30, 40 min after addition of phage, and total RNA was extracted from these samples using the QIAGEN RNEasy Mini Kit. The samples were further treated with Turbo DNAse to degrade phage/bacterial DNA, and the RNA quality was measured using an Agilent 2100 Bioanalyzer. Ribosomal RNA was depleted using the Illumina Ribozero plus kit, and Illumina RNA-seq libraries were prepared without any enrichment. A strand-specific RNA seq Novaseq S2 100-bp paired end run was performed on the samples using an Illumina Novaseq 6000 instrument
 
Contributor(s) Rohit Kongari R, Ray M, Lehman S, Plaut R, Hinton D, Stibitz E
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Submission date Jan 17, 2024
Last update date Oct 09, 2024
Contact name Ramchander Rohit Kongari
E-mail(s) ramchander.kongari@fda.hhs.gov
Phone 9794225701
Organization name United States Food and Drug Administration
Department Department of Bacterial, Parasitic and Allergenic products
Lab Laboratory of Mucosal Pathogens and Cellular Immunity
Street address 10903 New Hampshire Ave
City Silver Spring
State/province Maryland
ZIP/Postal code 20903
Country USA
 
Platforms (1)
GPL27158 Illumina NovaSeq 6000 (Staphylococcus aureus)
Samples (56)
GSM8022435 Wild type host infected with phage K - 0 min time point (replicate A)
GSM8022436 Wild type host infected with phage K- 0 min time point (replicate B)
GSM8022437 Wild type host infected with phage K- 0 min time point (replicate C)
Relations
BioProject PRJNA1065939

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Supplementary file Size Download File type/resource
GSE253516_GEO_SUB_RNASEQ_KONGARI_Processeddata_TPM.xlsx 123.6 Kb (ftp)(http) XLSX
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