Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

Abstract

Because the phosphoenolpyruvate:sugar phosphotransferase system plays multiple regulatory roles in addition to the phosphorylation-coupled transport of many sugars in bacteria, synthesis of its protein components is regulated in a highly sophisticated way. Thus far, the cAMP receptor protein (CRP) complex and Mlc are known to be the major regulators of ptsHIcrr and ptsG expression in response to the availability of carbon sources. In this report, we performed ligand fishing experiments by using the promoters of ptsHIcrr and ptsG as bait to find out new factors involved in the transcriptional regulation of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, and we found that the anaerobic regulator ArcA specifically binds to the promoters. Deletion of the arcA gene caused about a 2-fold increase in the ptsG expression, and overexpression of ArcA significantly decreased glucose consumption. In vitro transcription assays showed that phospho-ArcA (ArcA-P) represses ptsG P1 transcription. DNase I footprinting experiments revealed that ArcA-P binds to three sites upstream of the ptsG P1 promoter, two of which overlap the CRP-binding sites, and the ArcA-P binding decreases the CRP binding that is essential for the ptsG P1 transcription. These results suggest that the response regulator ArcA regulates expression of enzyme IICB Glc mediating the first step of glucose metabolism in response to the redox conditions of growth in E. coli.The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) 1 consists of two general cytoplasmic proteins, enzyme I and histidine phosphocarrier protein HPr, and some sugar-specific components collectively known as enzyme II (1, 2). Glucose-specific enzyme II of Escherichia coli consists of two subunits, soluble enzyme IIA Glc (EIIA Glc ) and membrane-bound enzyme IICB Glc . Thus, glucose transport in E. coli involves three soluble PTS components (enzyme I, HPr, and EIIA Glc , encoded by the ptsHIcrr operon) and one membranebound protein, enzyme IICB Glc (EIICB Glc ), encoded by the ptsG gene. During translocation of glucose, a phosphoryl group derived from phosphoenolpyruvate is transferred sequentially along a series of proteins to the transported glucose molecule, eventually converting it into glucose 6-phosphate. The sequence of phosphotransfer is from phosphoenolpyruvate to the general PTS proteins enzyme I and HPr and further to the carbohydrate-specific cytoplasmic EIIA Glc , membrane-bound EIICB Glc , and glucose.The PTS takes an important part in metabolic adaptation to environmental changes to compete effectively with ambient organisms by sensing the availability of nutrients in the environment. In addition to sugar transport, multiple roles are exerted by the PTS and these include chemoreception (3), catabolite repression (4), carbohydrate transport and metabolism (1, 5, 6), carbon storage (7,8), and the coordination of carbon and nitrogen metabolism (9). More recently, we found that EIIA Glc of the PTS also regulates the flux between respirat...

A two-component phosphotransfer network involving ArcB, ArcA, and RssB coordinates synthesis and proteolysis of σS (RpoS) in E. coli

“…As shown by gel retardation, ArcA binds, but a smaller complex is formed (Fig. 3A), consistent with nonphosphorylated ArcA forming a dimer (Jeon et al 2001) and previous observations of similar ArcA band-shift patterns at other promoters (Lynch and Lin 1996b;Jeon et al 2001;Jeong et al 2004). In the footprinting assay, nonphosphorylated ArcA produced some very minor alterations but no clear protection in the rpoSp1 region (Fig.…”

“…Due to the overlap with the activating cAMP-CRP-binding site, phosphorylated ArcA would not only act as a "classical" repressor, but also as an antiactivator for cAMP-CRP. A similar arrangement, in which cAMP-CRP and ArcA compete, has also been observed in the ptsG promoter (Jeong et al 2004). With nonphosphorylated ArcA, the binding pattern in the rpoSp1 region is different.…”

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…In addition, under conditions of nutrient limitation, ptsG expression is repressed by E s (RpoS), and this repression seems to act in synergy with that of Mlc (792). Furthermore, using ligand fishing with the promoter region of E. coli ptsG, it was found that the two-component response regulator ArcA, when phosphorylated, binds the promoter region at three positions: two between P 2 and P 1 overlapping the Crp/cAMP binding sites and one next to P 1 overlapping the Mlc binding site (373). PϳArcA also binds to the promoter region of the ptsHI-crr operon but with lower affinity.…”

“…Crp/cAMP-dependent expression from P 1 prevails, as P 2 transcripts amount to only 10% of the total ptsG mRNA (664). crp or cyaA null mutants do not express ptsG (408,409,727), while ptsG is up to 18-fold overexpressed in mlc null mutants (depending on the growth conditions) (373,664). In wild-type cells, growth on glucose causes an eightfold induction of ptsG expression compared to growth on non-PTS carbon sources (215,664).…”

“…70,2006 PHOSPHOTRANSFERASE SYSTEM-RELATED PHOSPHORYLATION (271). PϳArcA then binds to the ptsG promoter region and represses the transcription of ptsG (373), thus reducing the uptake of glucose and the glycolytic flux. A different mode of regulation of the EIICB Glc concentration is related to the stability of the RNA message.…”