Browsing by Author "Chen, Yen-Hua"

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CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication
(2014-06-01)
Yang, Chi-Dung
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Chen, Yen-Hua
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Huang, Hsi-Yuan
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Huang, Hsien-Da
;
Tseng, Ching-Ping
The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.
Large-Scale Functional Analysis of CRP-Mediated Feed-Forward Loops
(2018-08-01)
Yang, Chi-Dung
;
Huang, Hsi-Yuan
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Shrestha, Sirjana
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Chen, Yen-Hua
;
Huang, Hsien-Da
Feed-forward loops (FFLs) represent an important and basic network motif to understand specific biological functions. Cyclic-AMP (cAMP) receptor protein (CRP), a transcription factor (TF), mediates catabolite repression and regulates more than 400 genes in response to changes in intracellular concentrations of cAMP in Escherichia coli. CRP participates in some FFLs, such as araBAD and araFGH operons and adapts to fluctuating environmental nutrients, thereby enhancing the survivability of E. coli. Although computational simulations have been conducted to explore the potential functionality of FFLs, a comprehensive study on the functions of all structural types on the basis of in vivo data is lacking. Moreover, the regulatory role of CRP-mediated FFLs (CRP-FFLs) remains obscure. We identified 393 CRP-FFLs in E. coli using EcoCyc and RegulonDB. Dose-response genomic microarray of E. coli revealed dynamic gene expression of each target gene of CRP-FFLs in response to a range of cAMP dosages. All eight types of FFLs were present in CRP regulon with various expression patterns of each CRP-FFL, which were further divided into five functional groups. The microarray and reported regulatory relationships identified 202 CRP-FFLs that were directly regulated by CRP in these eight types of FFLs. Interestingly, 34% (147/432) of genes were directly regulated by CRP and CRP-regulated TFs, which indicates that these CRP-regulated genes were also regulated by other CRP-regulated TFs responding to environmental signals through CRP-FFLs. Furthermore, we applied gene ontology annotation to reveal the biological functions of CRP-FFLs.