Unknown,Transcriptomics,Genomics,Proteomics

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Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments

ABSTRACT: We performed RNA seq analysis on Escherichia coli MG1655 recovering from DNA damage induced by mitomycin C Escherichia coli was grown overnight in luria broth at 37°C with shaking at 200 rpm. The culture was back diluted to ~OD600 0.01 and allowed to grow to OD600 ~0.1. It was then treated with 1 μg/ml mitomycin C for 60 min. Cells were pelleted, washed with fresh media, resuspended to OD600 ~0.1 and then allowed to recover from damage treatment. Samples were collected before addition of damage as control. Samples were also collected every 20 min for 3 hrs during recovery and then every 1 hr for 3 hrs after that. Cultures were maintained in log phase (OD600 ~0.1 - 0.4) throughout the experiment. RNA was extracted using the Direct-zol™ RNA MiniPrep (Zymo, Cat no. R2052A81) and RNA Clean & Concentrator™-25 (Zymo, Cat no. R1018A82). RNA libraries were prepared using the TruSeq Stranded mRNA Library Preparation kit. Libraries were sequenced using Illumina MiSeq sequencing platform. Samples were collected from 2 independent experiments.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Escherichia coli O17 str. K12a

SUBMITTER: Suchitha Raghunathan

PROVIDER: E-MTAB-9696 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:6-Nonadecynoic acid (6-NDA), a plant-derived acetylenic acid, exhibits strong inhibitory activity against the human fungal pathogens Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae to investigate the mechanism of action of this compound. 6-NDA elicited a transcriptome response indicative of fatty acid stress, altering the expression of genes known to be affected when yeast cells are grown in the presence of oleate. Mutants of S. cerevisiae lacking transcription factors that regulate fatty acid ?-oxidation showed increased sensitivity to 6-NDA. Fatty acid profile analysis indicated that 6-NDA inhibited the formation of fatty acids longer than 14 carbons in length. In addition, the growth inhibitory effect of 6-NDA was rescued in the presence of exogenously supplied oleate. To investigate the response of a pathogenic fungal species to 6-NDA, transcriptional profiling and biochemical analyses were also conducted in C. albicans. The transcriptional response and fatty acid profile of C. albicans were comparable to those obtained in S. cerevisiae, and the rescue of growth inhibition with exogenous oleate was also observed in C. albicans. In addition, 6-NDA enhanced the potency of the antifungal drug fluconazole in a fluconazole-resistant clinical isolate of C. albicans. Collectively, our results indicate that the antifungal activity of 6-NDA is mediated by a disruption in fatty acid homeostasis, and that this compound has potential utility in combination therapy in the treatment of drug-resistant fungal infections. Cultures of S. cerevisiae strain S288C were started at OD600 of 0.1, allowed to grow to OD600 of 0.2, then treated with either DMSO (0.25%) or 6-NDA at the IC50 concentration (2.2 ug/ml). When OD600 of 0.5 was reached, cells were harvested and frozen. Two biological replicate cultures were grown for each treatment, and RNA from each replicate was hybridized to 2 independent arrays, resulting in 4 hybridizations for each treatment. Cultures of C. albicans strain SC5314 were started at OD600 of 0.1, allowed to grow to OD600 of 0.2, then treated with either DMSO (0.25%) or 6-NDA at the IC50 concentration (4 ug/ml). When OD600 of 0.5 was reached, cells were harvested and frozen. Three biological replicate cultures were grown for each treatment, and RNA from each replicate was hybridized as dye-swap pairs, resulting in 6 hybridizations for each treatment.

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Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments

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