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Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase. One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen. To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-'lacZ fusion. The glycogen excess mutation TR1-5 was found to be pleiotropic. It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties. The mutated gene was designated csrA for carbon storage regulator. Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators ofglgC expression. A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth. Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis. Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.A large body of evidence shows that during the transition into stationary phase, bacteria acquire numerous new physiological properties which enhance their ability to compete and survive under suboptimal conditions (for reviews, see references 16, 25, 26, and 44). While it has become clear that in Escherichia coli the induction of several genes and operons in the stationary phase requires a putative sigma factor, katF or rpoS (2,20,25,41), the expression of stationaryphase genes such as mcbA for microcin production (2) and glgCA for glycogen synthesis do not require katF (13, 38a), suggesting that additional regulators exist. We have initiated studies to identify and characterize factors that control the glycogen biosynthesis genes in E. coli. Our previous experiments showed that cyclic AMP (cAMP), cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp) stimulate the expression of the genes for the essen-

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The csrA gene encodes a global regulatory protein which facilitates glgC mRNA decay in vivo. A purified recombinant CsrA protein was found to inhibit in vitro glg (glycogen biosynthesis) gene expression posttranscriptionally and bind specifically to a glgC runoff transcript without causing its decay. Our results provide further insight into the mechanism by which CsrA functions as an mRNA decay factor.

The carbon storage regulator gene, csrA, encodes a factor which negatively modulates the expression of the glycogen biosynthetic gene glgC by enhancing the decay of its mRNA (M. Y. Liu, H. Yang, and T. Romeo, J. Bacteriol. 177:2663-2672, 1995). When endogenous glycogen levels in isogenic csrA ؉ and csrA::kanR strains were quantified during the growth curve, both the rate of glycogen accumulation during late exponential or early stationary phase and its subsequent rate of degradation were found to be greatly accelerated by the csrA::kanR mutation. The expression of the biosynthetic genes glgA (glycogen synthase) and glgS was observed to be negatively modulated via csrA. Thus, csrA is now known to control all of the known glycogen biosynthetic genes (glg), which are located in three different operons. Similarly, the expression of the degradative enzyme glycogen phosphorylase, which is encoded by glgY, was found to be negatively regulated via csrA in vivo. The in vitro transcription-translation of glgY was also specifically inhibited by the purified CsrA gene product. These results demonstrate that localization of glycogen biosynthetic and degradative genes within the Escherichia coli glgCAY operon facilitates their coordinate genetic regulation, as previously hypothesized (T. Romeo, A. Kumar, and J. Preiss, Gene 70:363-376, 1988). The csrA gene did not affect glycogen debranching enzyme, which is now shown to be encoded by the gene glgX.One feature of the complex adaptive response which occurs when Escherichia coli or various other bacteria enter stationary phase is the increased expression of genes involved in the biosynthesis of glycogen (reviewed in references 12 and 20-22). A shortage of a nutrient such as nitrogen in the presence of excess carbon is particularly effective in causing the intracellular accumulation of glycogen, which can be metabolized as an endogenous carbon and energy source. The regulatory mechanisms for glycogen synthesis in E. coli include (i) allosteric control via the metabolites AMP and fructose-1,6-bisphosphate of the committed step of the pathway, catalyzed by ADPglucose pyrophosphorylase (EC 2.7.7.27); (ii) stimulation of the genetic expression of glgC (which encodes ADPglucose pyrophosphorylase [2]) and glgA (which encodes glycogen synthase [EC 2.4.1.21] [13]) by guanosine 3Ј-bisphosphate 5Ј-bisphosphate and cyclic AMP (cAMP); and (iii) the transcriptional control of glgS, a monocistronic gene which stimulates glycogen synthesis in vivo, by cAMP and s (9, 20-22, 24, 28). We recently identified and characterized the csrA gene, which exerts a very strong negative effect on glycogen synthesis in E. coli (25,26). This gene encodes a 61-amino-acid protein, CsrA, which was shown to regulate the expression of glgB (encoding glycogen branching enzyme [EC 2.4.1.18]) and glgC (26). CsrA has been shown to be a factor which enhances the rate of decay of glgC mRNA via a novel though unresolved mechanism (16). The deduced amino acid sequence of CsrA is homologous to a diverse subset of RNA-bindin...