Genetic regulation of glycogen biosynthesis in Escherichia coli: in vitro effects of cyclic AMP and guanosine 5'-diphosphate 3'-diphosphate and analysis of in vivo transcripts

Abstract

Glycogen accumulation in Escherichia coli is inversely related to the growth rate and occurs most actively when cells enter the stationary phase. The levels of the three biosynthetic enzymes undergo corresponding changes under these conditions, suggesting that genetic control of enzyme biosynthesis may account for at least part of the regulation (J. Preiss, Annu. Rev. Microbiol. 38:419-458, 1984). We have begun to explore the molecular basis of this control by identifying factors which affect the expression of the glycogen genes and by determining the 5'-flanking regions required to mediate the regulatory effects. The in vitro coupled transcription-translation of two of the biosynthetic genes, glgC (ADPglucose pyrophosphorylase) and gigA (glycogen synthase), was enhanced up to 26-and 10-fold, respectively, by cyclic AMP (cAMP) and cAMP receptor protein (CRP). Guanosine 5'-diphosphate 3'-diphosphate stimulated the expression of these genes 3.6-and 1.8-fold, respectively. The expression of glgB (glycogen branching enzyme) was affected weakly or negligibly by the above-mentioned compounds. Assays which measured the in vitro formation of the first dipeptide of glgC showed that a restriction fragment which contained 0.5 kilobases of DNA upstream from the initiation codon supported cAMP-CRP-activated expression. Sequence-specific binding of cAMP-CRP to a 243-base-pair restriction fragment from the region upstream from glgC was observed by virtue of the altered electrophoretic mobility of the bound DNA. Sl nuclease protection analysis identffied 5' termini of four in vivo transcripts within 0.5 kilobases of the glgC coding region. The relative concentrations of transcripts were higher in the early stationary phase than in the exponential phase. Two mutants which overproduced the biosynthesis enzymes accumulated elevated levels 'of specific transcripts. The 5' termini of three of the transcripts were mapped to a high resolution. Their upstream sequences showed weak similarity to the E. coli consensus promoter. These results suggest complex transcriptional regulation of the glycogen biosynthesis genes involving multiple promoter sites and direct control of gene expression by at least two global regulatory systems.

Identification of a central regulator of stationary‐phase gene expression in Escherichia coli

“…Adenylate cyclase is stimulated by phosphate (Peterkofsky et al, 1989). A sudden lack of phosphate may therefore result in a decrease in the intracellular level of cAMP, thus allowing better expression of csi2 ::/acZ For the glycogen-synthetic genes (glgB, glgCA) , direct positive control by cAMP/CAP has been observed in in vitro transcription/translation assays (Romeo and Preiss, 1989). However, we observed only a minor reduction of glycogen synthesis in ~cya, ~crp or ~cya~crp mutants, compared to a dramatic reduction caused by disruptions in rpaS.…”

“…Information about transcripts is even more sparse. Interestingly, however, the xthA promoter used in viva lacks a -35 (u 7 Dj region (Saporito et al, 1988) and the start pOints of three different glgC transcripts are preceded by regions with poor homology to the -35 and -10 (u 7 Dj consensus sequences (Romeo and Preiss, 1989). This also strongly argues for the rpaS gene product as an alternative sigma factor (us).…”

“…2C). Transcription of the structural genes of glycogen-synthetic enzymes is regulated by cAMPI CAP and weakly by ppGpp in vitro (Romeo and Preiss, 1989) but induction during late exponential and early stationary phase has remained unexplained. We therefore tested whether glycogen synthesis would be affected by the presence of fusion csi2 :: lacZ, indicating that the latter disrupted a potential regulatory gene (Fig .…”

The RNA Molecule CsrB Binds to the Global Regulatory Protein CsrA and Antagonizes Its Activity in Escherichia coli

“…The enzymes for DNA manipulation were from the sources previously described (3,13). Sheep antirabbit horseradish peroxidase, prestained and unstained protein standards for SDS-PAGE, polyvinylidene difluoride membranes, and nitrocellulose membranes were purchased from Bio-Rad.…”

“…Molecular Biology, Nucleotide Sequencing, and Analysis-Standard procedures were used for isolation of plasmid DNA and restriction fragments, restriction mapping, transformation, and molecular cloning, as described previously (13) and for PCR amplification (17). Alternatively, plasmid DNA was purified using Qiagen plasmid mini-DNA cartridges.…”