From 2007.igem.org

The regulation of carbohydrate uptake and metabolism in E. coli - <a href="http://www.mun.ca/biochem/courses/4103/topics/sugar_transport.html">Bacterial sugar transport</a>

• The bacterial phosphoenolpyruvate-dependent sugar:phosphotransferase system (PEP-PTS; EC 2.7.1.69)
• The PTS is widespread in bacteria but absent in Archaea and eukaryotic organisms.
• In many bacteria, the PTS is responsible for the uptake and phosphorylation of various carbohydrates at a very high rate.
• <a href="http://igem.ym.edu.tw/private_2007/images/5/57/Eco02060.gif">The about 20 different phosphotransferase systems (PTSs) of the cell</a> fulfill besides the transport of various carbohydrates, 
  also the function of one signal processing system. 
• Extra- and intracellular signals are converted within the PTS protein chain to important regulatory signals affecting, e.g. carbon metabolism and chemotaxis.
• Due to its spatial organization, the functioning of the PTS depends on diffusion:
  • a phosphoryl group derived from cytoplasmic phosphoenolpyruvate (PEP) is transferred by cytoplasmic proteins to a membrane protein that imports and phosphorylates the carbohydrate.
• In E. coli K-12, as in many other gram-positive and gram-negative bacteria, the phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTSs) are the major transport and sensor systems for carbohydrates.
• The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) consists of
  • two general cytoplasmic proteins, enzyme I and histidine phosphocarrier protein HPr, and
  • some sugar-specific components collectively known as enzyme II.
• In E. coli K-12, D-glucose (Glc) is taken up and concomitantly phosphorylated either by
  • the glucose-specific enzyme II (EII) transporter (II^Glc) or
  • the mannose-specific EII transporter (II^Man) (genes manXYZ) of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). 
• As for most other PTS carbohydrates, the phosphoryl groups are sequentially transferred from PEP through two common intermediates,
  • enzyme I (EI; gene: ptsI) and
  • the phosphohistidine carrier protein (HPr; gene: ptsH), to sugar-specific EII (IICB^Glc) and to glucose.
• II^Glc consists of two subunits,
  • IIA^Glc (crr [catabolite repression resistance]) and
  • membrane-bound IICB^Glc (ptsG).
• The crr gene is part of the ptsHI crr operon separated from the ptsG gene, which maps at 25.0 min.
• IIA^Glc is a small hydrophilic protein which has, in addition to its transport function, a central regulatory role in carbon catabolite repression and inducer exclusion.
• The IICB^Glc subunit is composed of
  • an amino-terminal, hydrophobic IIC^Glc domain, which largely determines substrate specificity, and
  • a carboxy-terminal, hydrophilic IIB^Glc domain, which is phosphorylated at the Cys421 residue.
• The system normally recognizes glucose as well as methyl-alpha-D-glucoside (alphaMG), 5-thio-D-glucoside, L-sorbose and, with a low affinity, 2-deoxyglucose (2DG).
• For both E. coli and Salmonella enterica serovar Typhimurium, it was demonstrated that the activity of IICB^Glc is the rate-limiting step in glucose utilization.
• Both ptsG expression and manXYZ expression are
  • positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CrpA) complex and
  • negatively controlled by the DgsA (Mlc) protein.
• The dgsA locus (deoxyglucose sensitive) at 35.9 min on the E. coli chromosome was discovered as a suppressor mutation that enables ptsG-negative mutants to grow anaerobically on glucose via a constitutively expressed II^Man system and enhanced sensitivity to 2DG, a major substrate of this transport system.
• The DgsA protein was rediscovered recently and renamed Mlc (making large colonies). dgsA and mlc are the same gene. The DgsA protein represses its own synthesis as well as the expression of the ptsHI crr operon and the mal regulon. It may represent a novel global repressor and may counteract the global regulator cAMP-CrpA to ensure the expression of those genes, which are linked to glucose metabolism. The inducer for DgsA, however, has not been identified.
• According to a model (JOURNAL OF BACTERIOLOGY, Aug. 2000, p. 4443–4452), the phosphorylation state of IIB^Glc modulates IIC^Glc which, directly or indirectly, controls the repressor DgsA and hence ptsG expression. By the same mechanism, glucose uptake and phosphorylation also control the expression of the pts operon and probably of all operons controlled by the repressor DgsA. 
• Glucose-specific enzyme II of E. 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 membrane-bound protein, enzyme IICB^Glc (EIICB^Glc), encoded by the ptsG gene.
• During translocation of glucose, a phosphoryl group derived from phosphoenolpyruvate (PEP) 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 (PEP) to the general PTS proteins enzyme I and HPr and further to the carbohydrate-specific cytoplasmic EIIA^Glc, membrane-bound EIICB^Glc, and glucose.
• EIIA^Glc of the PTS also regulates the flux between respiration and fermentation pathways by sensing the available sugar species 
  via a phosphorylation state-dependent interaction with the fermentation/respiration switch protein FrsA.
• All of the PTSs except the mannose-specific PTS consist of five conserved functional domains, designated
  • enzyme I (EI) (gene, ptsI),
  • the histidine-containing phosphoryl carrier protein HPr (gene, ptsH),
  • enzyme IIA (EIIA),
  • enzyme IIB (EIIB), and
  • enzyme IIC (EIIC).
• Depending on the organism or system, these functional PTS domains exist as single or multidomain proteins.
• The two cytoplasmic proteins, EI and HPr, are the general components of all PTS, whereas the EII complexes are carbohydrate specific.
• The protein kinase EI uses phosphoenolpyruvate (PEP) in an autophosphorylation reaction, and 
  the phosphoryl group is subsequently transferred to HPr, EIIA, and EIIB. 
  Finally, the carbohydrate substrate, which is bound by the integral membrane domain of EIIC, is phosphorylated and concomitantly translocated across the membrane.
• The preferred carbon source of E. coli, D-glucose, is taken up by two different EIIs,
  • the high affinity glucose-specific molecule EII^Glc (Glc-PTS) and
  • the low-affinity mannose-specific molecule EII^Man (Man-PTS).
• The Glc-PTS consists of the cytoplasmic protein EIIA^Glc, encoded by the crr gene (part of the ptsHI crr operon), and the membrane protein EIICB^Glc (gene, ptsG). 
  In addition to its transport function, EIIA^Glc has a central regulatory role in
  • carbon catabolite repression involving activation of the adenylate cyclase in its phosphorylated state (in the absence of glucose) and
  • inducer exclusion by binding to several non-PTS carbohydrate systems in its unphosphorylated state (in the presence of glucose).
• Glucose uptake derepresses ptsG expression by inactivation of the glucose repressor Mlc (makes large colonies). 
  The mlc gene has been mapped at 35 min on the E. coli chromosome. There is good evidence that mlc is identical to the previously identified dgsA gene.
• The current model is that dephosphorylated EIICB^Glc generated during glucose uptake binds Mlc and sequesters the repressor away from its DNA-binding sites.
  Membrane localization, and not binding to EIICB^Glc, seems to be responsible for the inactivation of Mlc in this process. 
  Furthermore, it has been shown that Mlc is also involved in the glucose-dependent regulation of
  • the ptsHI crr operon,
  • the malT gene encoding the transcriptional activator of the maltose regulon, and
  • the manXYZ operon.
• Mlc is therefore considered to be a global transcription factor responsible for the induction of genes in the presence of glucose.
• The second major regulator of ptsG transcription is the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex, which activates transcription.
• The two global regulators work antagonistically, since cAMP levels should be low during growth on glucose. 
  This indicates that precise control of ptsG expression is necessary under various growth conditions, since EIICB^Glc activity has a major effect on the levels of phosphorylation of all other PTS proteins, especially EIIA^Glc. In this context it was not surprising that several more transcription factors which are also involved in ptsG gene regulation were
  identified recently. Among these factors are
  • ArcA, a major transcription factor for the switch between aerobic growth and anaerobic growth in E. coli,
  • two alternative sigma factors, sigma32 for the heat shock response (40) and sigmaS for expression of genes in the stationary growth phase, and
  • the small DNA-binding protein Fis.
• In addition to these regulation mechanisms at the transcriptional level, ptsG expression is posttranscriptionally regulated by modulation of ptsG mRNA stability
  in response to the glycolytic flux in the cells.
• YeeI is an Mlc-binding and -inactivating protein and is therefore involved in the regulation of expression of the ptsG gene encoding the major glucose transporter EIICB^Glc. 
• As shown previously, the EIIB^Glc domain of the glucose transporter is responsible for the interaction with Mlc. 
  Soluble unphosphorylated EIIB^Glc can bind to Mlc, as demonstrated in SPR experiments, but is not capable of preventing Mlc from binding to its cognate operator sites.
  Consequently, it was demonstrated that EIIB^Glc could repress Mlc activity only if it was attached either to the membrane by the EIIC^Glc domain 
  or to a heterologous membrane anchor (e.g., the Gp8 protein, the bacteriophage M13 major coat protein, or the lactose permease LacY). 
  These results suggest that under physiological conditions membrane sequestration of Mlc by unphosphorylated EIICB^Glc, 
  which builds up during the transport of glucose into the cell, is the key process in the inactivation of Mlc and leads to physical separation of Mlc from its DNA target sites.
• Furthermore, it was suggested that in addition to the physical separation mechanism, 
  a membrane environment-induced conformational change of Mlc might be necessary to prevent the repressor from binding to DNA. 
  In contrast to these results, we (J Bacteriol. 2006 August; 188(15): 5439–5449) obtained strong evidence that 
  YeeI is not an integral membrane protein but is nevertheless capable of inactivating Mlc directly in the cytosol, even in the absence of EIICB^Glc. 
  Therefore, there must be a fundamental difference in the mode of action between YeeI and EIICB^Glc during inactivation of Mlc. 
• Native Mlc exists as a tetramer. Deletion of nine carboxy-terminal amino acids did not have a severe effect on the activity, 
  whereas deletion of 18 carboxy-terminal amino acids removed an amphipathic helix that is necessary for tetramerization. 
  In addition, as revealed by the crystal structure of Mlc, this carboxy-terminal amphipathic helix stabilizes the amino-terminal helix-turn-helix domain, which is responsible for the DNA binding of Mlc. Thus, delete18C-Mlc can only form dimers that cannot repress ptsG expression.
• Interestingly, the carboxy-terminal part of Mlc seems to be the target site for YeeI binding, 
  since fully active delete9C-Mlc had only a 20% residual binding response and delete18C-Mlc did not interact at all with YeeI in our SPR experiments.
  It is tempting to speculate that tetramerization of the repressor is inhibited by binding of YeeI to the carboxy-terminal region of Mlc 
  and repression of ptsG expression cannot take place. In this case membrane sequestration might not be necessary for inactivation of Mlc by YeeI.
• During the last few years a remarkable number of regulators of ptsG expression have been found, and these regulators can be divided into different classes.
  • The first class consists of Mlc and cAMP-CRP.
    • Whereas Mlc is responsible for induction of ptsG (and several other genes) in the presence of glucose, 
      the catabolite activator complex cAMP-CRP is also absolutely necessary for ptsG expression. 
    • Since cAMP levels are low during growth on glucose, the two regulatory systems work antagonistically. 
      Indeed, addition of cAMP to cells growing on glucose resulted in a significant increase in ptsG expression. 
      These two systems seem to be responsible for sophisticated fine-tuning of ptsG expression under various growth conditions.
  • The second class consists of several other factors that have minor effects on ptsG expression levels.
    • The members of this group include
      • ArcA, a major regulator of the switch between aerobic growth and anaerobic growth in E. coli,
      • two sigma factors, sigma32 for the heat shock response and sigmaS for expression of genes in the stationary growth phase, and
      • the small DNA-binding protein Fis.
  • There is a third class of regulatory effects, which occur at the level of ptsG mRNA stability. 
    • Several workers have provided evidence that intracellular accumulation of glucose-6-phosphate or fructose-6-phosphate 
      leads to specific degradation of ptsG mRNA in an RNase E-dependent manner.
    • Vanderpool and Gottesman identified a transcriptional activator called SgrR, which is activated under these conditions and causes enhanced transcription of a small RNA designated SgrS.
    • SgrS is complementary to the 5' end of ptsG and is capable of forming Hfq-dependent RNA-RNA hybrids. This is the first step in ptsG mRNA degradation.
• Several of these regulatory systems, including YeeI, cause rather small changes in the amounts of EIICB^Glc under various growth conditions. 
  However, experiments to determine the enzyme flux control coefficients of all phosphotransferase reactions of the glucose-PTS revealed that 
  only the EIICB^Glc activity controls the flux through the glucose phosphotransferase system with wild-type levels of expression of the proteins involved, EI, HPr, EIIA^Glc, and EIICB^Glc. Furthermore, simulation experiments showed that if the EIICB^Glc activity is less than 40% of the maximum induction level, 
  the amount of unphosphorylated EIIA^Glc starts to increase even under glucose saturating conditions. 
• The multicomponent PEP-PTS includes membrane-localized, sugar-specific transporters (enzymes IICB) that are fused or associated with a third domain (IIA) and 
  two general cytoplasmic proteins (enzyme I and HPr). Collectively, these interactive components constitute a phospho-relay that (in five sequential stages) transfers the high energy phosphoryl moiety from PEP to catalyze the simultaneous phosphorylation and translocation of sugars through the cytoplasmic membrane.
• Completion of the chromosomal DNA sequence of E. coli K12 strain MG-1655 in 1997, confirmed that
  • ptsG encodes one (IICB^Glc/PtsG) of two glucose-specific PTS transporters, whereas
  • the genes for the general proteins and IIA^Glc reside within a separate ptsHIcrr operon.
• The glucose-PTS of E. coli consists of four proteins (Biophys J. 2003 Jul;85(1):612-622):
  • the general PTS-proteins enzyme I (EI) and HPr and the carbohydrate-specific proteins IIA^Glc and IICB^Glc.
  • The former three proteins are located in the cytoplasm and relay a phosphoryl-group derived from phosphoenolpyruvate (PEP) in a consecutive manner to the latter membranebound protein, which in turn imports glucose and concomitantly phosphorylates it.
  • The IICB^Glc subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate. 
    • It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus.
  • In E. coli K-12, the major glucose transporter with a central role in carbon catabolite repression and in inducer exclusion is the phosphoenolpyruvate-dependent glucose:phosphotransferase system (PTS).
    • Its membrane-bound subunit, IICB^Glc, is encoded by the gene ptsG;
    • its soluble domain, IIA^Glc, is encoded by crr, which is a member of the pts operon.
  • The system is inducible by D-glucose and, to a lesser degree, by L-sorbose.
  • Enzyme IIA^Glc and IIA^Glc-P are considered sensors for glucose. We mimicked the absence of glucose by lowering its concentration from 500 to 0.5 uM.
  • IIA^Glc ‘‘senses’’ the concentration of PEP via two intermediate proteins, i.e., EI and HPr.
  • Enzyme IIA^Glc of E. coli mediates another signal transduction route also effecting glucose repression. 
    At low glucose (and high PEP), many catabolic operons are activated by the action of cAMP. 
    The cAMP is produced by adenylate cyclase, and that enzyme is activated by phosphorylated IIA^Glc (Saier et al., 1996).

<a href="http://minst.org/ecoli_cyclase.htm">Escherichia coli adenylate cyclase homepage</a>

<a href="http://minst.org/ecoli_cyclase_mutants.htm">E. coli K12 adenylate cyclase</a>

• <a href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=2178176&dopt=AbstractPlus">Three amino-acids of E. coli K12 adenylate cyclase</a> - arginine 188, aspartic acid 414 and glycine 463 - were identified as essential residues for the activation process by Enzyme IIA-glc.

The phosphoenolpyruvate-dependent sugar transport system (PTS) is present in a large variety of bacteria. 
It catalyzes transport and phosphorylation of hexoses and hexitols at the expense of phosphoenolpyruvate (PEP). 
Only three of four enzymes are required for this entire sequence.

• The initial step in the energy conversion process is the EI catalyzed conversion of phosphoenolpyruvate (PEP) to pyruvate and P-HPr.
• EI is a metal requiring hydrophobic enzyme which is active only as a dimer. 
  Kinetic and gel filtration data confirm that it forms functional ternary complexes with HPr or P-Hpr and phosphoenolpyruvate or pyruvate 
  which influence both the degree of dimerization and the specific activity of the dimer. 
• The ptsH, ptsI, and crr genes, coding for three of the proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) 
  (HPr, enzyme I, and enzyme III^Glc, respectively).
• The three genes constitute an operon, but analysis of the ptsH, ptsI, and crr transcripts by Northern (RNA) blotting revealed the existence of three major mRNA species. 
  • One encompassed the three cistrons,
  • a second one the ptsH gene and part of the ptsI gene, and
  • the third one only the distal gene crr. The short crr transcripts were initiated inside the ptsI open reading frame at points which were identified by S1 mapping.
• Previous study (J Bacteriol. 1988 September; 170(9): 3827–3837) showed that
  • the ptsH, ptsI, and crr genes exhibited high basal expression,
  • transcription of the ptsH and ptsI genes was stimulated threefold by the cyclic AMP-cyclic AMP receptor protein complex and also by growth on glucose, 
    but only in the presence of an active enzyme II^Glc,
  • crr-specific expression was not sensitive to the complex or to growth on glucose, and
  • under the growth conditions tested, the major part of crr transcription was initiated from internal promoters.
• Growth of E. coli MG-1655 and its transformants on selected sugars
• Accumulation of phosphosugars such as glucose-6-phosphate causes a rapid degradation of ptsG mRNA encoding the major glucose transporter IICB^Glc 
  in an RNase E/degradosome-dependent manner. The destabilization of ptsG mRNA is caused by a small antisense RNA (SgrS) that is induced by phosphosugar stress.
  mRNA localization to the inner membrane coupled with the membrane insertion of nascent peptide mediates the Hfq/SgrS-dependent ptsG mRNA destabilization 
  presumably by reducing second rounds of translation.
• When glucose is present in the medium and it is being transported by the PTS, the IIA^Glc protein is non-phosphorylated, and in this state, it binds to various non-PTS permeases inhibiting uptake of other carbon sources. This form of IIA^Glc also binds to the enzyme glycerol kinase (GK), inhibiting its activity.
  • Glycerol kinase (GK) is the enzyme responsible for the transfer of the gamma-phosphoryl group from ATP to glycerol to produce glycerol-3-phosphate 
    and is the rate-limiting step in glycerol metabolism. 
• When glucose is absent from the culture medium, IIA^Glc is mainly in its phosphorylated state. In this condition, IIA^Glc~P binds to the enzyme adenylate cyclase (AC), activating its cyclic AMP (cAMP) biosynthetic capacity. Therefore, cAMP concentrations increase in the cell. Then cAMP binds to the cAMP receptor protein (CRP) and promotes the induction of catabolite-repressed genes.

BMC Microbiology 2007, 7:53

• <a href="http://igem.ym.edu.tw/private_2007/images/c/ce/Glucose_response.jpg">Transcriptome data</a> from isogenic wild type and crp- strains grown in Luria-Bertani medium (LB) or LB + 4 g/L glucose (LB+G) were analyzed to identify 
  differentially transcribed genes. We detected 180 and 200 genes displaying increased and reduced relative transcript levels in the presence of glucose, respectively.
• Glucose-dependent induction was also detected for genes encoding proteins involved in the import of polyamines, inorganic phosphate and magnesium
  ions, thus suggesting that these nutrients are required to sustain the higher growth rate observed in the LB+G medium.
• In contrast, glucose had a repressive effect on genes encoding transporters and periplasmic receptor proteins related to the import of alternative carbon and
  carbon-nitrogen sources. These included: amino acids, carbohydrates, lactate, glycerol, peptides, dipeptides and nucleosides. Furthermore, a reduction in transcript levels
  was observed for genes encoding proteins involved in the catabolism of several sugars and amino acids. This transcriptome pattern is the expected result of carbon catabolite
  repression exerted by glucose.
• cAMP receptor protein (CRP) is a global dual regulator that governs the expression of at least 140 genes and corregulates gene expression with 75 other TFs. 
  In E. coli, carbon catabolite repression (CCR) is mainly mediated by the PTS. When glucose is present in the culture medium, protein IIA^Glc lacks the capacity to activate adenylate cyclase; therefore, cAMP is present at relatively low levels. Lacking cAMP, the CRP protein cannot bind DNA and activate catabolite-repressed genes. 
  Therefore, in the presence of glucose, CRP is unable to exert its usually positive effect on its regulated genes.
• The genes osmE and ompF displayed a significant change in their levels of expression being induced in the crp- mutant and repressed in the presence of glucose. 
  It has not been reported that CRP directly regulates these well characterized genes. Instead, <a href="http://igem.ym.edu.tw/private_2007/index.php/Image:Glucose_sensing_tfs.jpg">CRP directly controls the expression of the ompR gene</a>, 
  whose product controls the expression of ompF. Our result is consistent with a report showing an increment in the expression level of ompF under glucose limitation. 
  The effect is caused by the absence of cAMP that increases the levels of phosphorylated OmpR, which repress expression of ompF.
• The global transcription factor Fis assists both Mlc repression and CRP-cAMP activation of ptsG through the formation of Fis-CRP-Mlc or Fis-CRP nucleoprotein complexes
  at the ptsG promoter depending on the glucose availability in the growth medium.

J Mol Microbiol Biotechnol. 2003;5(4):206-15.

• Phosphorylation and dephosphorylation at Ser-46 in HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is controlled by the bifunctional HPr kinase/phosphorylase (HprK/P).
• In Gram-positive bacteria, P-Ser-HPr controls
  • (1) sugar uptake via the PTS;
  • (2) catabolite control protein A (CcpA)-mediated carbon catabolite repression, and
  • (3) inducer exclusion.
• Genome sequencing revealed that HprK/P is absent from Gram-negative enteric bacteria, but present in many other proteobacteria. These organisms also possess
  • (1) HPr, the substrate for HprK/P;
  • (2) enzyme I, which phosphorylates HPr at His-15, and
  • (3) one or several enzymes IIA, which receive the phosphoryl group from P approximately His-HPr.
• The genes encoding the PTS proteins are often organized in an operon with HPRK. However, most of these organisms miss CcpA and a functional PTS, as enzymes IIB and membrane-integrated enzymes IIC seem to be absent.
• HprK/P and the rudimentary PTS phosphorylation cascade in Gram-negative bacteria must therefore carry out functions different from those in Gram-positive organisms.
• The gene organization in many HprK/P-containing Gram-negative bacteria as well as some preliminary experiments suggest that HprK/P might control transcription regulators implicated in cell adhesion and virulence.
• In alpha-proteobacteria, HPRK is located downstream of genes encoding a two-component system of the EnvZ/OmpR family.
• In several other proteobacteria, HPRK is organized in an operon together with genes from the RPON region of ESCHERICHIA COLI (RPON encodes a sigma54).
• We propose that HprK/P might control the phosphorylation state of HPr and EIIAs, which in turn could control the transcription regulators.