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Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli.

Parisutham V, Lee SK - PLoS ONE (2015)

Bottom Line: Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism.While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability.This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea.

ABSTRACT
Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (β-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.

No MeSH data available.


Related in: MedlinePlus

(A) Comparison of growth of E. coli strains on cellobiose minimal medium. For these experiments, strains were grown overnight in LB, washed with M9 salts, and suspended in fresh M9-cellobiose minimal medium to a final OD of 0.05. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate. (B) Comparison of growth of E. coli strains on cellobiose minimal medium. These strains were pre-adapted on M9-cellobiose minimal medium to mid-log phase and diluted into fresh M9-cellobiose minimal medium. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate.
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pone.0131928.g001: (A) Comparison of growth of E. coli strains on cellobiose minimal medium. For these experiments, strains were grown overnight in LB, washed with M9 salts, and suspended in fresh M9-cellobiose minimal medium to a final OD of 0.05. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate. (B) Comparison of growth of E. coli strains on cellobiose minimal medium. These strains were pre-adapted on M9-cellobiose minimal medium to mid-log phase and diluted into fresh M9-cellobiose minimal medium. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate.

Mentions: ESS is one of the efficient cellobiose metabolizing strains of E. coli constructed by replacing the cryptic promoters of chb and asc operons with constitutive promoters and adaptive evolution on cellobiose [6]. Comparative whole genome re-sequencing revealed 12 and 16 mutations in strains OSS and ESS, respectively. Comparison of the mutations in ESS and OSS with the reference sequence of MG1655 indicates that at least 11 mutations differed between the reference strain of MG1655 and the laboratory strain. Conventional PCR-based re-sequencing helped to verify only two true-positive mutations in strain ESS, yet through this approach longer deletion and insertion could not be identified. The first mutation leads to the duplication of 10-nt (GAGGATGAAA) upstream of the ascB gene of the asc operon. The second mutation was a nonsense mutation on a previously uncharacterized transcription factor, yebK, resulting in the expression of a truncated protein (79 amino acids) containing only the DNA binding domain. We then characterized the role of independent mutations on cellobiose metabolism. Allelic replacement of each of the two mutations independently or in combination in strain OSS leads to enhancing the growth rate on cellobiose in the following order: OSS-yebK*ascB*>ascB*>yebK*>OSS, indicating that both these mutations are beneficial for growth on cellobiose (Fig 1A and 1B). The final strain, OSS-yebK*ascB*, had a specific growth rate similar to the strain ESS when grown on cellobiose minimal medium, indicating that key mutations related to cellobiose metabolism in strain ESS was deciphered.


Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli.

Parisutham V, Lee SK - PLoS ONE (2015)

(A) Comparison of growth of E. coli strains on cellobiose minimal medium. For these experiments, strains were grown overnight in LB, washed with M9 salts, and suspended in fresh M9-cellobiose minimal medium to a final OD of 0.05. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate. (B) Comparison of growth of E. coli strains on cellobiose minimal medium. These strains were pre-adapted on M9-cellobiose minimal medium to mid-log phase and diluted into fresh M9-cellobiose minimal medium. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4488073&req=5

pone.0131928.g001: (A) Comparison of growth of E. coli strains on cellobiose minimal medium. For these experiments, strains were grown overnight in LB, washed with M9 salts, and suspended in fresh M9-cellobiose minimal medium to a final OD of 0.05. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate. (B) Comparison of growth of E. coli strains on cellobiose minimal medium. These strains were pre-adapted on M9-cellobiose minimal medium to mid-log phase and diluted into fresh M9-cellobiose minimal medium. OSS–closed diamond; OSS-yebK*–closed triangle; OSS-ascB*–closed square; OSS-yebK*/ascB*–closed circle; ESS–open circle. Error bars indicate the standard deviation of experiments performed in triplicate.
Mentions: ESS is one of the efficient cellobiose metabolizing strains of E. coli constructed by replacing the cryptic promoters of chb and asc operons with constitutive promoters and adaptive evolution on cellobiose [6]. Comparative whole genome re-sequencing revealed 12 and 16 mutations in strains OSS and ESS, respectively. Comparison of the mutations in ESS and OSS with the reference sequence of MG1655 indicates that at least 11 mutations differed between the reference strain of MG1655 and the laboratory strain. Conventional PCR-based re-sequencing helped to verify only two true-positive mutations in strain ESS, yet through this approach longer deletion and insertion could not be identified. The first mutation leads to the duplication of 10-nt (GAGGATGAAA) upstream of the ascB gene of the asc operon. The second mutation was a nonsense mutation on a previously uncharacterized transcription factor, yebK, resulting in the expression of a truncated protein (79 amino acids) containing only the DNA binding domain. We then characterized the role of independent mutations on cellobiose metabolism. Allelic replacement of each of the two mutations independently or in combination in strain OSS leads to enhancing the growth rate on cellobiose in the following order: OSS-yebK*ascB*>ascB*>yebK*>OSS, indicating that both these mutations are beneficial for growth on cellobiose (Fig 1A and 1B). The final strain, OSS-yebK*ascB*, had a specific growth rate similar to the strain ESS when grown on cellobiose minimal medium, indicating that key mutations related to cellobiose metabolism in strain ESS was deciphered.

Bottom Line: Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism.While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability.This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea.

ABSTRACT
Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (β-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.

No MeSH data available.


Related in: MedlinePlus