Abstract
The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS− derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH−) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH− mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS− and rppH− mutations in the deep of a proteomic approach to understanding the relevance of rppH− phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH−, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH− were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS− strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS− derivatives during the ALE experiment. This result suggests that the evolving PTS− population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH− mutation in the PTS− background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.
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Acknowledgments
We thank Paul Gaytán, Jorge Yáñez, and Eugenio López for the synthesis of oligonucleotides and Sanger DNA sequencing. We thank Georgina Hernández and Luz María Martínez for technical assistance.
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This work was supported by CONACYT Ciencia Básica project 240519 and PAPIIT UNAM project IN209618.
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Aguilar, C., Martínez-Batallar, G., Flores, N. et al. Analysis of differentially upregulated proteins in ptsHIcrr− and rppH− mutants in Escherichia coli during an adaptive laboratory evolution experiment. Appl Microbiol Biotechnol 102, 10193–10208 (2018). https://doi.org/10.1007/s00253-018-9397-3
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DOI: https://doi.org/10.1007/s00253-018-9397-3