Cloning and sequencing of two genes fromStaphylococcus carnosus coding for glucose-specific PTS and their expression inEscherichia coliK-12

Phosphoenolpyruvate (PEP)-dependent phosphorylation experiments have indicated that the grampositive bacteriumStaphylococcus carnosus possesses an EIICBA fusion protein specific for glucose. Here we report the cloning of a 7 kb genomic DNA fragment containing two genes,glcA andglcB, coding for the glucose-specific PTS transporters EII^Glc1 and EII^Glc2 which are 69% identical. The translation products derived from the nucleotide sequence consist of 675 and 692 amino acid residues and have calculated molecular weights of 73 025 and 75 256, respectively. Both genes can be stably maintained inEscherichia coli cells and restore the ability to ferment glucose toptsG deletion mutants ofE. coli. This demonstrates the ability of the PTS proteins HPr and/or EIIA^Glc of a gram-negative organism (E. coli) to phosphorylate an EIICBA^Glc from a gram-positive organism (S. carnosus).     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.     

Cloning and sequencing of two genes fromStaphylococcus carnosus coding for glucose-specific PTS and their expression inEscherichia coliK-12

Phosphoenolpyruvate (PEP)-dependent phosphorylation experiments have indicated that the grampositive bacteriumStaphylococcus carnosus possesses an EIICBA fusion protein specific for glucose. Here we report the cloning of a 7 kb genomic DNA fragment containing two genes,glcA andglcB, coding for the glucose-specific PTS transporters EII^Glc1 and EII^Glc2 which are 69% identical. The translation products derived from the nucleotide sequence consist of 675 and 692 amino acid residues and have calculated molecular weights of 73 025 and 75 256, respectively. Both genes can be stably maintained inEscherichia coli cells and restore the ability to ferment glucose toptsG deletion mutants ofE. coli. This demonstrates the ability of the PTS proteins HPr and/or EIIA^Glc of a gram-negative organism (E. coli) to phosphorylate an EIICBA^Glc from a gram-positive organism (S. carnosus).     

Complementation of a truncated membrane-bound Enzyme IINag from Klebsiella pneumoniae with a soluble Enzyme III in Escherichia coli K12

Summary Cloning and analysis of the gene nagE encoding Enzyme II^Nag (EII^Nag) from Klebsiella pneumoniae revealed strong similarities with the corresponding gene from Escherichia coli K12. Truncated EII^Nag proteins were generated by inserting a series of Tn1725 transposons into the structural gene; the positions of the insertions were mapped by restriction enzyme analysis, and the activity of the polypeptides determined by in vitro and in vivo tests. Insertions in the region encoding the amino-terminal half of the protein invariably abolished transport and phosphorylation activity, while truncated proteins lacking a C-terminal domain homologous to the soluble Enzyme III (crr gene) could be complemented by this molecule to nearly wild-type activity.     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.     

Comparison of the sequences of the nagE operons from Klebsiella pneumoniae and Escherichia coli K12: enhanced variability of the enzyme IIN-acetylglucosamine in regions connecting functional domains.

Summary The nagE operon, encoding the enzyme II specific for N-acetylglucosamine (EII^Nag), and adjacent DNA from the chromosome of Klebsiella pneumoniae were sequenced and compared with the corresponding sequence from Escherichia colt K12. The deduced EII^Nag sequences differ in 72 out of 651 amino acids, the K. pneumoniae sequence being three residues longer. The amino acid differences were distributed unevenly, and were most frequent in regions connecting the three functional domains of the protein. In the nagE-nagB intergenic region, two promoter, two operator, and one CAP consensus sequence with regulatory functions were highly conserved. The nag structural genes from both species were very similar (83% DNA similarity; 89% amino acid similarity) except for frequent AT to GC exchanges in the wobble base of codons in K. pneumoniae DNA relative to the E. coli DNA.     

TheEscherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and theEscherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.     

Structural characterization of the PTS IIA and IIB proteins associated with pneumococcal fucose utilization

Streptococcus pneumoniae harbors a significant number of transporters, including phosphotransferase (PTS) systems, allowing the bacterium to utilize a number of different carbohydrates for metabolic and other purposes. The genes encoding for one PTS transport system in particular (EII^fuc) are found within a fucose utilization operon in S. pneumoniae TIGR4. Here, we report the three‐dimensional structures of IIA^fuc and IIB^fuc providing evidence that this PTS system belongs to the EII^man family. Additionally, the predicted metabolic pathway for this distinctive fucose utilization system suggests that EII^fuc transports the H‐disaccharide blood group antigen, which would represent a novel PTS transporter specificity. Proteins 2017; 85:963–968. © 2016 Wiley Periodicals, Inc.     

Dependency of sugar transport and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system on membranous phosphatidylethanolamine in Escherichia coli: studies with a pssA mutant lacking phosphatidylserine synthase

An isogenic pair of Escherichia coli strains lacking (pssA) and possessing (wild-type) the enzyme phosphatidylserine synthase was used to estimate the effects of the total lack of phosphatidylethanolamine (PE), the major phospholipid in E. coli membranes, on the activities of several sugar permeases (enzymes II) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The mutant exhibits greatly elevated levels of phosphatidylglycerol (PG), a lipid that has been reported to stimulate the in vitro activities of several PTS permeases. The activities, thermal stabilities, and detergent sensitivities of three PTS permeases, the glucose enzyme II (II^Glc), the mannose enzyme II (II^Man) and the mannitol enzyme II (II^Mtl), were characterized. Western blot analyses revealed that the protein levels of II^Glc were not appreciably altered by the loss of PE. In the pssA mutant, II^Glc and II^Man activities were depressed both in vivo and in vitro, with the in vivo transport activities being depressed much more than the in vitro phosphorylation activities. II^Mtl also exhibited depressed transport activity in vivo but showed normal phosphorylation activities in vitro. II^Man and II^Glc exhibited greater thermal lability in the pssA mutant membranes than in the wild-type membranes, but II^Mtl showed enhanced thermal stability. All three enzymes were activated by exposure to TritonX100 (0.4%) or deoxycholate (0.2%) and inhibited by SDS (0.1%), but II^Mtl was the least affected. II^Man and, to a lesser degree, II^Glc were more sensitive to detergent treatments in the pssA mutant membranes than in the wild-type membranes while II^Mtl showed no differential effect. The results suggest that all three PTS permeases exhibit strong phospholipid dependencies for transport activity in vivo but much weaker and differential dependencies for phosphorylation activities in vitro, with II^Man exhibiting the greatest and II^Mtl the least dependency. The effects of lipid composition on thermal sensitivities and detergent activation responses paralleled the effects on in vitro phosphorylation activities. These results together with those previously published suggest that, while the in vivo transport activities of all PTS enzymes II require an appropriate anionic to zwitterionic phospholipid balance, the in vitro phosphorylation activities of these same enzymes show much weaker and differential dependencies. Alteration of the phospholipid composition of the membrane thus allows functional dissection of transport from the phosphorylation activities of PTS enzyme complexes.     

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme II^Glc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme II^Glc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme II^Glc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y _Glc values. Assuming equal Y _ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme II^Glc.Abbreviations PTS phosphoenolpyruvate: carbohydrate phosphotransferase system - HPr histidine-containing phosphocarrier protein - GalP galactose permease     

Cloning and molecular analysis of a mannitol operon of phosphoenolpyruvate-dependent phosphotransferase (PTS) type from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EII^Mtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [³H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EII^Mtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICB(Glc) (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc). We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside. Binding of Mlc to Enzyme IICB(Glc) in vitro required the IIB domain and the IIC-B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross-dephosphorylation of IIB(Glc) during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.     

Analysis of the nag regulon from Escherichia coli K12 and Klebsiella pneumoniae and of its regulation

Summary Four genes, nagR, A, B and E, clustered in the nag locus of Escherichia coli K12 and Klebsiella pneumoniae, were cloned and physically mapped, and the corresponding gene products involved in amino sugar metabolism identified. Expression of the nag genes was also analysed using a series of lacZ fusions. In both bacteria, the genes are arranged in two divergent operons and controlled by a common NagR repressor. The corresponding gene nagR was found to map in the first operon together with the promoter proximal gene nagB, encoding the enzyme d-glucosamine isomerase (deaminase) (NagB) and the middle gene nagA, coding for N-acetyl-glucosamine deacetylase (NagA). Polar mutations in nagB and nagA prevent the efficient expression of nagR and cause constitutive expression of all nag genes. This includes the gene nagE encoding Enzyme II^Nag of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS), encoded in the second divergently transcribed operon. No further gene is found in this operon which in both organisms is directly adjacent to the gene glnS. It is interesting that the NagR repressor also affects the mannose PTS (genes manX, Y, Z), the second transport system involved in amino sugar uptake and phosphorylation.