Unknown,Transcriptomics,Genomics,Proteomics

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Next generation sequencing of Pseudomonas aruginosa PAO1 in the presence of Free Nitrous Acid stress

ABSTRACT: Purpose: Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent to a range of microorganisms. However, its antimicrobial mechanism is largely unknown. For this study Pseudomonas aeruginosa PAO1 was selected to gain a systematic understanding of the antimicrobial mechanism of FNA. Methods: PAO1 was grown under anaerobic denitrifying conditions and when FNA was added (0.1 mg-N/L) growth temporarily stopped. From cultures with and without added FNA, RNA was extracted and the abundance of gene transcripts was detected by sequencing. Transcripts exhibiting â¥2.5 or â¤-2.5 fold abundance change in the presence of FNA were designated as differentially expressed. Results:In response to FNA, 167 genes showed increased transcription while 424 exhibited deceased transcript levels. Respiration was likely inhibited as denitrification activity was severely decreased. Genes coding for enzymes of the tricarboxylic acid cycle were down regulated, while highly increased transcripts were detected for pyruvate fermentation, indicating a possible survival response to FNA. Moreover, genes coding ribosomal proteins had significantly decreased transcript levels, implicating that protein biosynthesis was severely inhibited by FNA. Whereas, genes encoding proteins functioning to preserve ribosome units under stressful conditions, were up-regulated during FNA exposure. Conclusion: In this study, we performed genome-wide high throughput RNA sequencing (RNA-Seq) analysis on PAO1 in the absence and presence of a bacteriostatic-level of FNA (0.1 mg N/L). By combining the global activated/suppressed gene profiles and detected physiological responses, we detected the microbial response to FNA exposure and revealed potential antimicrobial mechanisms of FNA on this prevalent opportunistic pathogen and environmentally relevant denitrifier. mRNA profiles of P. aeruginosa PAO1 towards FNA stress were generated by next generation sequencing in triplicate via Illumina 2000.

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Shuhong Gao

PROVIDER: E-GEOD-73323 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent to a range of microorganisms. However, its antimicrobial mechanism is largely unknown. For this study Desulfovibrio vulgaris Hildenborough was selected to gain a systematic understanding of the antimicrobial mechanism of FNA. Methods: D. vulgaris was grown under anaerobic denitrifying conditions and when FNA was added (4 Î¼g-N/L) growth temporarily stopped. From cultures with and without added FNA, RNA was extracted and the abundance of gene transcripts was detected by sequencing. Transcripts exhibiting â¥1 or â¤-1 fold abundance change in the presence of FNA were designated as differentially expressed. Results:The results imply that FNA exerted multiple antimicrobial effects. Growth of the culture was inhibited by FNA and significant proportions of the cells were killed during the exposure. Respiration by sulfate reduction and lactate oxidation was shut down along with ATP production when exposed to FNA. Correspondingly, decreased transcript levels of genes involved in these metabolic pathways were detected. Detoxification of FNA was evident by nitrite reduction and the genes coding both nitrite reductase (NrfA) and hydroxylamine reductase were highly up regulated. During the FNA induced energy depleted state, there was evidence that D. vulgaris lowered both ribosome activity and protein production. Conditions within the cells were more oxidizing and genes coding for proteins providing resistance to oxidative stress were up regulated. Culture thiol levels increased during the exposure, suggesting that FNA had caused protein structures to alter. Furthermore, genes for motility were up regulated possibly as an attempt to avoid FNA stress. These findings provide new insight for understanding the response of D. vulgaris to FNA exposure. Conclusion: In this study, we performed genome-wide high throughput RNA sequencing (RNA-Seq) analysis on D. vulgaris in the absence and presence of a bacteriostatic-level of FNA (4 Î¼g-N/L). By combining the global activated/suppressed gene profiles and detected physiological responses, we detected the microbial response to FNA exposure and revealed potential antimicrobial mechanisms of FNA on this prevalent model sulfate reducing bacteria. mRNA profiles of D. vulgaris Hildenborough towards FNA stress were generated by next generation sequencing in triplicate via Illumina 2000.

Project description:Purpose: Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent to a range of microorganisms. However, its antimicrobial mechanism is largely unknown. For this study Desulfovibrio vulgaris Hildenborough was selected to gain a systematic understanding of the antimicrobial mechanism of FNA. Methods: D. vulgaris was grown under anaerobic denitrifying conditions and when FNA was added (4 μg-N/L) growth temporarily stopped. From cultures with and without added FNA, RNA was extracted and the abundance of gene transcripts was detected by sequencing. Transcripts exhibiting ≥1 or ≤-1 fold abundance change in the presence of FNA were designated as differentially expressed. Results:The results imply that FNA exerted multiple antimicrobial effects. Growth of the culture was inhibited by FNA and significant proportions of the cells were killed during the exposure. Respiration by sulfate reduction and lactate oxidation was shut down along with ATP production when exposed to FNA. Correspondingly, decreased transcript levels of genes involved in these metabolic pathways were detected. Detoxification of FNA was evident by nitrite reduction and the genes coding both nitrite reductase (NrfA) and hydroxylamine reductase were highly up regulated. During the FNA induced energy depleted state, there was evidence that D. vulgaris lowered both ribosome activity and protein production. Conditions within the cells were more oxidizing and genes coding for proteins providing resistance to oxidative stress were up regulated. Culture thiol levels increased during the exposure, suggesting that FNA had caused protein structures to alter. Furthermore, genes for motility were up regulated possibly as an attempt to avoid FNA stress. These findings provide new insight for understanding the response of D. vulgaris to FNA exposure. Conclusion: In this study, we performed genome-wide high throughput RNA sequencing (RNA-Seq) analysis on D. vulgaris in the absence and presence of a bacteriostatic-level of FNA (4 μg-N/L). By combining the global activated/suppressed gene profiles and detected physiological responses, we detected the microbial response to FNA exposure and revealed potential antimicrobial mechanisms of FNA on this prevalent model sulfate reducing bacteria.

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Next generation sequencing of Pseudomonas aruginosa PAO1 in the presence of Free Nitrous Acid stress

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