| The phenomenon of carbon catabolite repression widely exists in microorganisms,which refers to a fact that when growing in a medium containing glucose and other utilizable carbon sources,microbes will prefer to consume glucose and meanwhile inhibit the synthesis of proteins that can breakdown or metabolize other carbon sources.In order to investigate genes regulated by carbon catabolite repression and characterize small molecules that can trigger carbon catabolite repression,we adopted wild-type strain NCM3722 as the object of our research,and continuously cultured NCM3722 in Chemostat at different growth rates by altering feeding rates to achieve stable cell growth(0.2 h-1,0.4 h-1,0.6 h-1 and 0.9 h-1)under nitrogen-limited and carbon-limited conditions.In each nutrient limited condtions,total four steady stages of bacterial cells were collected for study of transcriptome and metabolomics.We used RNA-seq to study the transcriptome changes of NCM3722 under various conditions indicated above.Transcriptomic data revealed that the expression of 31.9% and 40.3% of the whole genome 4522 genes was correlated to the growth rate under carbon-limited and nitrogen-limited conditions,respectively.In total,1008 differently expressed genes were fund in both carbon-limited and nitrogen-limited conditions,in which the expression of 30.2% genes were up-regulated with the increase of growth rate,and 64.8% genes were down-regulated with the increase of growth rate,and 5.0% genes were showed opposite expression trend with the increase of growth rates.A cluster analysis of genes changed with growth rates showed that these genes were mainly related to carbon source metabolism,amino acid metabolism,energy metabolism,nucleotide metabolism and signal transduction.We further analyzed the 51 genes which have opposite expression trend with the increase of growth rates under nitrogen-limited and carbon-limited conditions,and found 23 possible genes that have not been reported and were mainly regulated by carbon catabolite repression.Furthermore,in order to verify the accuracy of transcriptome data,we assayed the expression of glt B,ace B and ast C genes by real-time PCR using samples harvested at the lowest growth rate of 0.2 h-1 and the highest growth rate of 0.9 h-1 under both nitrogen-limited and carbon-limited conditions respectively.The result showed that the expression changes of selected genes obtained by real-time PCR were consistent well with our transcriptome data.We applied untargeted metabolomics by GC-MS to study the metabolite changes in both cell pellets and cell fluid under the lack of either carbon source or nitrogen source.In these two conditions,715 and 951 of physical peaks were detected,218 and 282 metabolites were identified in cell pellets,and 191 and 249 metabolites were identified in cell fluid.Through comparing the changes trend of metabolites following growth rates during nitrogen and carbon deficiency,we found 10 metabolites shown significant changes,including a-ketoglutaric acid.Therefore,our metabolome study provides the experimental proof that ?-ketoglutaric acid is one of the key catabolites that can trigger carbon catabolite repression.Moreover,we compared the expression changes of genes and fold changes of metabolites in the TCA cycle during both nitrogen and carbon limitation,we found the results of our transcriptome and metabolome study matched very well.In all,this study not only sets a foundation to the construction of a complete carbon catabolite repression regulatory network,but also provides a systemic way to the study of carbon catabolite repression. |
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