Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling time series of Campylobacter jejuni reveals a stationary phase physiological switch

ABSTRACT: Growth curves of C. jejuni NCTC11168 were performed in BHI broth at 42C with agitation at 150rpm.o. At 16, 20, 40, 46 and 54 hours of growth, aliquots were removed from the culture, and the bacteria were mixed with RNA stabilisation agent. RNA was then extracted from the stabilised pellets, and treated with DNase I to remove any contaminating residual genomic DNA. The integrity and quantity of the RNA was then confirmed on RNA Nano 6000 lab chips using an Agilent 2100 Bioanalyzer. The total RNA was reverse transcribed to amino-allyl dUTP labelled cDNA, and chemically coupled to either Cy3 or Cy5-NHS ester dyes prior to hybridisation to microarrays. Hybridisations were performed between the 16h and 20h, 20h and 40h, 40h and 46h, 46h and 54h and 16h and 54h (loop) samples. Hybrididsations from three independent growth curves were performed, and two dye reversal repeats were performed for each comparison, giving a total of 30 microarray hybridisations.

INSTRUMENT(S): G2505A DNA microarray scanner [Agilent]

ORGANISM(S): Campylobacter jejuni

SUBMITTER: John Wright

PROVIDER: E-MEXP-1436 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It produces flavor compounds over the whole ripening period. During cheese ripening, P. freudenreichii is exposed to a temperature downshift, especially when cheeses are transferred from warm temperature (about 24°C) to cold temperature (about 4°C). The aim of this study was to investigate the adaptation of P. freudenreichii at cold temperature by means of the first global gene expression profile for this species. The temporal transcriptomic response of P. freudenreichii was analyzed at five times of growth, during growth at 30°C then for 9 days at 4°C, in the constant presence of lactate as the main carbon source. P. freudenreichii response was also investigated by RT-qPCR for 30 genes, by proteomics and metabolomics (main metabolites quantified in culture supernatant). Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from warm to cold temperature (P < 0.05 and |fold change| > 1). Most of the down-expressed genes were involved in cell machinery (cell division, protein turnover, translation, transcription and DNA replication). During incubation at cold temperature, P. freudenreichii accumulated carbon supplies by up-regulating genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis and in glycogen synthesis. Interestingly, some genes involved in the formation of important flavor compounds of cheese, coding for an extracellular lipolytic esterases and enzymes of the pathways of formation of branched-chain compounds, were not significantly affected by cold. In conclusion, P. freudenreichii is metabolically active at cold temperature and induces pathways to maintain its long-term viability, which could explain its contribution to cheese ripening even at low temperature. Gene expression was measured in the middle of exponential growth phase at 30°C (20 h, OD650 ≈ 0.5), at the end of exponential growth phase (40 h, OD650 ≈ 2), and after 3, 6 and 9 days of incubation at 4°C. Three independent biological experiments were performed at each time (20h, 40h, 3, 6 and 9 days) and labelled A, B, C. One technical repetition was performed using the RNA of the 20HA sample ; This technical repetition was labelled 20HAbis.

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Transcription profiling time series of Campylobacter jejuni reveals a stationary phase physiological switch

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