Genome-wide screening of genes whose enhanced expression affects glycogen accumulation in Escherichia coli

Abstract

Using a systematic and comprehensive gene expression library (the ASKA library), we have carried out a genome-wide screening of the genes whose increased plasmid-directed expression affected glycogen metabolism in Escherichia coli. Of the 4123 clones of the collection, 28 displayed a glycogen-excess phenotype, whereas 58 displayed a glycogen-deficient phenotype. The genes whose enhanced expression affected glycogen accumulation were classified into various functional categories including carbon sensing, transport and metabolism, general stress and stringent responses, factors determining intercellular communication, aggregative and social behaviour, nitrogen metabolism and energy status. Noteworthy, one-third of them were genes about which little or nothing is known. We propose an integrated metabolic model wherein E. coli glycogen metabolism is highly interconnected with a wide variety of cellular processes and is tightly adjusted to the nutritional and energetic status of the cell. Furthermore, we provide clues about possible biological roles of genes of still unknown functions.

Figure 1

Glycogen content (referred as percentage of glycogen accumulated by WT cells) of glycogen-excess and glycogen-deficient clones of the ASKA library. Averaged glycogen content in WT cells was 45 nmol glucose mg protein⁻¹. Cells were grown at 37°C with rapid gyratory shaking in liquid Kornberg medium (1.1% K₂HPO₄, 0.85% KH₂PO₄, 0.6% yeast extract) supplemented with 50 mM glucose. Because some yeast extracts are deficient in Mg²⁺, and because Mg²⁺ is a major determinant of cell metabolic and energetic status and of expression of genes affecting glycogen metabolism,^, the Kornberg medium was also supplemented with 1 mM MgCl₂.

Figure 2

AG1 is not a relA1 mutant. In (A) and (B), relA over-expressing cells of the ASKA library accumulate WT glycogen content. In (C) and (D), relA expression vector of the ASKA library complements the glycogen-deficient phenotype of ΔrelA cells of the Keio collection. ΔglgA cells of the Keio collection are used as negative control for glycogen accumulation.

Figure 3

Suggested model of glycogen metabolism in E. coli wherein major determinants of glycogen accumulation include availability of ATP for ADPG synthesis, levels of (p)ppGpp (which accumulates in a RelA- SpoT- and/or Gpp-dependent manner under conditions of limited provision of nutrients such as amino acids, sulphur, Mg²⁺, iron, etc.), factors determining intercellular communication, aggregative and social behaviour modes (which in turn determine the nutritional status of the cell), expression levels of the general stress regulator RpoS and of the global regulator CsrA, availability of a carbon source, redox status of the cell and less well-defined systems sensing the cell energy status through the activity of the electron transport chain. CsrB and CsrC small non-coding RNAs, represented by stem-loops, are likely involved in the regulation of functions strongly affecting glycogen accumulation through interaction with CsrA (Adk, adenylate kinase; c-di-GMP, cyclic diguanylate; ETC, electron transport chain).