A widely accepted model for catabolite repression posits that phospho-IIA Glc of the bacterial phosphotransferase system activates adenylyl cyclase (AC) activity. For many years, attempts to observe such regulatory properties of AC in vitro have been unsuccessful. To further study the regulation, AC was produced fused to the transmembrane segments of the serine chemoreceptor Tsr. Cells harboring Tsr-AC and normal AC, expressed from the cya promoter on a low copy number vector, exhibit similar behavior with respect to elevation of cAMP levels resulting from deletion of crp, expressing the catabolite regulatory protein. Membrane-bound Tsr-AC exhibits activity comparable with the native form of AC. Tsr-AC binds IIA Glc specifically, regardless of its phosphorylation state, but not the two general phosphotransferase system proteins, enzyme I and HPr; IIA Glc binding is localized to the C-terminal region of AC. Binding to membranes of either dephospho-or phospho-IIA Glc has no effect on AC activity. However, in the presence of an Escherichia coli extract, P-IIA Glc , but not IIA Glc , stimulates AC activity. Based on these findings of a direct interaction of IIA Glc with AC, but activity regulation only in the presence of E. coli extract, a revised model for AC activity regulation is proposed.In Escherichia coli, cAMP, produced by adenylyl cyclase (AC), 5 is an important regulatory molecule, essential for controlling the expression of numerous operons. The cellular levels of cAMP are regulated mainly via effects on AC activity. It has been firmly established that the phosphoenolpyruvate:sugar phosphotransferase system (PTS) plays an important role in the regulation mechanism. Thus, in wild-type but not in PTS mutant cells, exposure to glucose results in decreased cellular cAMP levels; this decrease accounts for the phenomenon of catabolite repression (1).A popular, but never proven, model for the regulation of AC activity is that the phosphorylated form of IIA Glc of the PTS stimulates AC activity; thus, glucose transport is presumed to lead to dephosphorylation of IIA Glc resulting in a de-activation of AC. It has also been observed that strains of E. coli deficient in the cAMP-binding protein, CRP, produce extraordinarily large amounts of cAMP (2). This CRP-dependent regulation of cAMP levels depends on the presence of IIA Glc (3). It has been proposed that the CRP-cAMP complex promotes expression of a phosphatase that converts P-IIA Glc to dephospho-IIA Glc (4). Consequently, in the absence of CRP, a greater proportion of the pool of IIA Glc is in the phospho-form and the AC is more fully activated.One approach to allow a further understanding of the mechanism by which AC is regulated has involved the use of permeable cells. In this case, exposure of the permeable cells to glucose results in inhibition of AC activity (5). In this system, it was discovered that P i is essential for high activity of AC as well as for the capability of the cells to be inhibited by glucose. Because P i was also shown to stimulate PTS ...

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA Glc (EIIA Glc ) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA Glc in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ 70 in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ 70. Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ 70-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ 70 and σ S for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ 70 activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.glucose signaling | sigma factor competition | transcriptional regulation B y monitoring their environment, bacteria ensure the most appropriate response for each environment. One of the sensory systems for monitoring changes in nutrient availability is the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS). The PTS is a multicomponent system that catalyzes the concomitant phosphorylation and translocation of numerous sugar substrates across the cytoplasmic membrane. This system consists of two general components, enzyme I (EI) and the histidine-containing phosphocarrier protein HPr, which are common to all PTS sugars, along with many sugar-specific components collectively known as enzyme IIs (EIIs) (1, 2).Each EII complex generally consists of three domains: one integral membrane domain forming the sugar translocation channel (EIIC) and two cytosolic domains (EIIA and EIIB). EI transfers a phosphoryl group from PEP to HPr, and HPr then transfers the phosphoryl group to the different EIIs. Each EII complex forms a cascade of phosphorylated intermediates, and in the presence of a PTS sugar, the EIIA and EIIB domains sequentially transfer the phosphate group from HPr to the incoming sugar. Thus, the phosphorylation states of the PTS components change depending on the availability of a PTS sugar substrate (3, 4).In addition to sugar uptake, the PTS plays an important role as a sensory transduction system to monitor nutritional changes, and its components are involved in the regula...

Summary In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTSNtr). The first proteins in both pathways are enzymes (enzyme Isugar and enzyme INtr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme Isugar and enzyme INtr is the presence of a GAF domain at the N-terminus of enzyme INtr. Since the PTSNtr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTSNtr components. However, there has been little evidence that any component of this so-called PTSNtr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTSNtr had not been uncovered. Here, we demonstrate that glutamine and α-ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTSNtr by direct effects on enzyme INtr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme INtr activity by the two signal molecules. Taken together, our results suggest that the PTSNtr senses nitrogen availability.

SignificanceMost bacteria accumulate the molecular alarmone (p)ppGpp to divert resources away from growth and division toward biosynthesis under various nutrient limitations. Despite its crucial role, uncontrolled accumulation of this alarmone causes severe growth inhibition and cell death. Thus, fine-tuning the cellular (p)ppGpp level is required to ensure survival and adaptation under harsh nutritional conditions. Here, we identify Rsd as a stimulator of the (p)ppGpp-degrading activity of SpoT during carbon source downshift in Escherichia coli, and this regulation is controlled by the phosphorylation state of HPr, a general component of the PEP-dependent sugar transport system. This study establishes a direct link between sugar signaling and the bacterial stringent response.

To survive in a continuously changing environment, bacteria sense concentration gradients of attractants or repellents, and purposefully migrate until a more favourable habitat is encountered. While glucose is known as the most effective attractant, the flagellar biosynthesis and hence chemotactic motility has been known to be repressed by glucose in some bacteria. To date, the only known regulatory mechanism of the repression of flagellar synthesis by glucose is via downregulation of the cAMP level, as shown in a few members of the family Enterobacteriaceae. Here we show that, in Vibrio vulnificus, the glucose-mediated inhibition of flagellar motility operates by a completely different mechanism. In the presence of glucose, EIIA(Glc) is dephosphorylated and inhibits the polar localization of FapA (flagellar assembly protein A) by sequestering it from the flagellated pole. A loss or delocalization of FapA results in a complete failure of the flagellar biosynthesis and motility. However, when glucose is depleted, EIIA(Glc) is phosphorylated and releases FapA such that free FapA can be localized back to the pole and trigger flagellation. Together, these data provide new insight into a bacterial strategy to reach and stay in the glucose-rich area.

Preferential sugar utilization is a widespread phenomenon in biological systems. Glucose is usually the most preferred carbon source in various organisms, especially in bacteria where it is taken up via the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The currently proposed model for glucose preference over non-PTS sugars in enteric bacteria including E. coli is strictly dependent on the phosphorylation state of the glucose-specific PTS component, enzyme IIAGlc (EIIAGlc). However, the mechanism of the preference among PTS sugars is largely unknown in Gram-negative bacteria. Here, we show that glucose preference over another PTS sugar, mannitol, is absolutely dependent on the general PTS component HPr, but not on EIIAGlc, in E. coli. Dephosphorylated HPr accumulates during the transport of glucose and interacts with the mannitol operon regulator, MtlR, to augment its repressor activity. This interaction blocks the inductive effect of mannitol on the mannitol operon expression and results in the inhibition of mannitol utilization.

Similar to decapping of eukaryotic mRNAs, the RppH-catalyzed conversion of 5′-terminal triphosphate to monophosphate has recently been identified as the rate-limiting step for the degradation of a subset of mRNAs in Escherichia coli. However, the regulation of RppH pyrophosphohydrolase activity is not well understood. Because the overexpression of RppH alone does not affect the decay rate of most target mRNAs, the existence of a mechanism regulating its activity has been suggested. In this study, we identified DapF, a diaminopimelate (DAP) epimerase catalyzing the stereoinversion of L,L-DAP to meso-DAP, as a regulator of RppH. DapF showed a high affinity interaction with RppH and increased its RNA pyrophosphohydrolase activity. The simultaneous overexpression of both DapF and RppH increased the decay rates of RppH target RNAs by about a factor of two. Together, our data suggest that the cellular level of DapF is a critical factor regulating the RppH-catalyzed pyrophosphate removal and the subsequent degradation of target mRNAs.

In Escherichia coli, glucose-dependent transcriptional induction of genes encoding a variety of sugar-metabolizing enzymes and transport systems is mediated by the phosphorylation state-dependent interaction of membrane-bound enzyme IICB Glc (EIICB Glc ) with the global repressor Mlc. Here we report the crystal structure of a tetrameric Mlc in a complex with four molecules of enzyme IIB Glc (EIIB), the cytoplasmic domain of EIICB Glc . Each monomer of Mlc has one bound EIIB molecule, indicating the 1:1 stoichiometry. The detailed view of the interface, along with the high-resolution structure of EIIB containing a sulfate ion at the phosphorylation site, suggests that the phosphorylation-induced steric hindrance and disturbance of polar intermolecular interactions impede complex formation. Furthermore, we reveal that Mlc possesses a built-in flexibility for the structural adaptation to its target DNA and that interaction of Mlc with EIIB fused only to dimeric proteins resulted in the loss of its DNA binding ability, suggesting that flexibility of the Mlc structure is indispensable for its DNA binding.enzyme IICB Glc ͉ glucose signaling ͉ protein-protein interaction ͉ transcription regulation B acteria sense continuous changes in their environment and adapt metabolically to compete effectively with other organisms for limiting nutrients. One sensory transduction system monitoring availability of a certain group of carbon sources is the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (1). In addition to concomitant transport and phosphorylation of sugars, PTSs take part in a variety of physiological processes through direct interactions with their target proteins (2-5).It is a normal occurrence that cells make more proteins necessary to transport and metabolize PTS sugars when they encounter an environment where PTS sugars are available. Glucose, a representative PTS sugar, mediates transcriptional activation of several genes for PTS-related sugar transporters and some enzymes involved in glycolysis (1, 6). Mlc, a tetrameric protein (7), is a signaling mediator for glucose induction of several PTS operons and related genes (8)(9)(10)(11)(12)(13)(14).A unique feature of induction of the Mlc regulon by glucose is that the effector molecule modulating Mlc activity is a membranebound protein, EIICB Glc (7,15,16), in which cytosolic EIIB protein is attached to membrane-embedded EIIC protein through a linker of Ͼ20 aa. In the absence of glucose, EIICB Glc mainly exists in the phosphorylated form (pEIICB Glc ). Because Mlc cannot interact with pEIICB Glc , Mlc dissociates from pEIICB Glc to bind to target promoters and repress their transcription. When glucose is available, transported glucose takes the phospho-groups away from pEIICB Glc and the dephosphorylated EIICB Glc recruits Mlc to sequester it from its target promoters. This leads to increased synthesis of EIICB Glc and other PTS proteins necessary to take up and metabolize glucose more efficiently.The physiological importance of the glucose-specific enz...