Sugar uptake and utilisation in Streptomyces coelicolor: a PTS view to the genome

Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation. We have surveyed the public available S. coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues (http://www.sanger.ac.uk/Projects/S_coelicolor/). Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIA^Crr (crr; IIA^Glc-homologue) and six genes encoding homologues of sugar-specific PTS components were identified. The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family. A model is presented, in which possible functions of the novel described PTS homologues are discussed.     

Regulation of expression of general components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) by the global regulator SugR in Corynebacterium glutamicum

The phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) catalyzes transport of carbohydrates by coupling carbohydrate translocation and phosphorylation. Enzyme I and HPr, encoded in ptsI and ptsH, respectively, are cytoplasmic proteins commonly used for transport of variety of PTS sugars. In this study, we investigated the role of SugR on the expression of the ptsI and ptsH which increases in the presence of PTS sugars in Corynebacterium glutamicum. Disruption of sugR resulted in the increased expression of ptsI and ptsH in the absence of PTS sugar. Introduction of a plasmid containing sugR gene complemented the effect of sugR disruption. SugR was purified and binding to the promoter regions of ptsI and ptsH was indicated by EMSA. DNase I footprinting analysis indicated the binding sites of SugR on the promoter region of divergently transcribed ptsI gene and fructose-pts operon. The binding sites contain a possible SugR binding motif which is conserved in the promoter regions of general and sugar-specific pts genes. Mutations in this motif resulted in the decrease of SugR binding to the ptsI promoter. These results suggest that SugR represses ptsI and ptsH in the absence of PTS sugar and derepression is the mechanism for the induction of the general components of PTS.     

Three-dimensional structures of the central regulatory proteins of the bacterial phosphotransferase system,HPr and enzyme IIAglc

Enzyme IIA and HPr are central regulatory proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase (PTS) system. Three-dimensional structures of the glucose enzyme IIA domain (IIA^glc) and HPr of Bacillus subtilis and Escherichia coli have been studied by both X-ray crystallography and Nuclear Magnetic Resonance (NMR) Spectroscopy. Phosphorylation of HPr of B. subtilis and IIA^glc of E. coli have also been characterized by NMR spectroscopy. In addition, the binding interfaces of B. subtilis HPr and IIA^glc have been identified from backbone chemical shift changes. This paper reviews these recent advances in the understanding of the three-dimensional structures of HPr and IIA^glc and their interaction with each other. © 1993 Wiley-Liss, Inc.     

Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB^Man, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB^Man, IIC^Man, IID^Man, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB^Man as well as membrane fragments containing IIC^Man and IID^Man. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB^Man.     

Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium

Summary We have studied the regulation of the synthesis and activity of a major galactose transport system, that of methyl -galactoside (MglP), in mutants of Salmonella typhimurium. Two classes of mutation that result in a (partially) defective phosphoenolpyruvate: sugar phosphotransferase system (PTS) interfere with MglP synthesis. pts mutations, which eliminate the general proteins of the PTS Enzyme I and/or HPr and crr mutations, which result in a defective glucose-specific factor III^Gle of the PTS, lead to a low MglP activity, as measured by methyl -galactoside transport. In both ptsH,I, and crr mutants the amount of galactose binding protein, one of the components of MglP, is only 5%–20% of that in wild-type cells, as measured with a specific antibody. We conclude that synthesis of MglP is inhibited in pts and crr mutants. Once the transport system is synthesized, its transport activity is not sensitive to PTS sugars (i.e., no inducer exclusion occurs). The defect in pts and crr mutants with respect to MglP synthesis can be relieved in two ways: by externally added cyclic adenosine 3, 5-monophosphate (cAMP) or by a mutation in the cAMP binding protein. The conclusion that MglP synthesis is dependent on cAMP is supported by the finding that its synthesis is also defective in mutants that lack adenylate cyclase. pts and crr mutations do not affect growth of S. typhimurium on galactose, however, since the synthesis and activity of the other major galactose transport system, the galactose permease (GalP), is not sensitive to these mutations. If the galactose permease is eliminated by mutation, growth of pts and crr mutants on low concentrations of galactose becomes very slow due to inhibited MglP synthesis. Residual growth observed at high galactose concentrations is the result of yet another transport system with low affinity for galactose.     

Isolation and Genetic Study of Erwinia Mutants Devoid of Common Components of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Mutants of bacteria belonging the genus Erwinia(Erwinia chrysanthemi andErwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI ⁺ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that theptsIand ptsH genes ofE. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas theptsH gene is located at 175 min. Sequencing of a portion of theE. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.     

The Doubly Phosphorylated Form of HPr, HPr(Ser-P)(His～P), Is Abundant in Exponentially Growing Cells of Streptococcus thermophilus and Phosphorylates the Lactose Transporter LacS as Efficiently as HPr(His～P)

In Streptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIA^LacS) possesses a histidine residue that can be phosphorylated by HPr(His~P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His~P), HPr(Ser-P), and HPr(Ser-P)(His~P), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr in S. thermophilus ATCC 19258 and S. thermophilus SMQ-301, respectively. Experiments conducted with [³²P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIA^LacS) showed that IIA^LacS was reversibly phosphorylated by HPr(Ser-P)(His~P) at a rate similar to that measured with HPr(His~P). Sequence analysis of the IIA^LacS protein domains from several S. thermophilus strains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIA^LacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIA^LacS from group II could also be phosphorylated by HPr(His~P) and HPr(Ser-P)(His~P), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIA^LacS very similar to group II S. thermophilus IIA^LacS. The results indicated that S. salivarius IIA^LacS was phosphorylated by HPr(Ser-P)(His~P) at a higher rate than that observed with HPr(His~P). Our results suggest that the reversible phosphorylation of IIA^LacS in S. thermophilus is accomplished by HPr(Ser-P)(His~P) as well as by HPr(His~P).     

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.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to adenylate cyclase.     

Positive selection for resistance to 2-deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsl missense mutants

We have used the toxic non-metabolizabie glucose/ mannose analogue 2-deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2-deoxyglucose and various metabolizable sugars, either lactose, meli-biose, galactose or fructose. We found that the frequency at which 2-deoxygiucose-resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2-deoxygiucose and was 15-fold higher than the rate observed with the mixture melibiose and 2-deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol analysis of III^Man, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of III^Man_L, a low-molecular-weight form of III^Man_L (35200), class 2 was composed of strains exhibiting a reduced level of III^Man_L, class 3 was composed of strains devoid of both forms of III^Man (III^Man_L as well as III^Man_H, the high-molecular-weight form of III^Man (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2-deoxygiucose-resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of III^Man but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.     

Relationship between pseudo-HPr and the PEP: fructose phosphotransferase system in Salmonella typhimurium and Escherichia coli

Summary We have studied in Salmonella typhimurium and Escherichia coli the properties of pseudo-HPr suppressor mutations. These mutations suppressed the defects in a ptsH mutant which lacks HPr, one of the enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system. The suppressor mutation was mapped in S. typhimurium at 3 min, closely linked to leu. The corresponding chromosomal fragment of 1.7 kb from S. typhimurium and E. coli (extending clockwise from ilvH) was cloned. In a maxicell system a protein with an approximate molecular weight of 36,000 was synthesized. Pseudo-HPr suppressor mutations (fruR) and a deletion extending clockwise from leu resulted in the constitutive expression of the fru operon containing the genes for II^Fru (fruA), III^Fru (fruB), fructose 1-phosphate kinase (fruK) and pseudo-HPr (fruF). fruR probably codes for a repressor of the fru operon. Tn10 mutagenesis revealed the following order of genes in the fru operon: fruB-(fruK, fruF)-fruA. Pseudo-HPr activity could replace HPr in PEP-dependent phosphorylation of PTS carbohydrates. III^Fru could be phosphorylated both via HPr and pseudo-HPr, since mutants lacking pseudo-HPr activity were still able to phosphorylate fructose in the presence of added HPr. Both the pseudo-HPr suppressor mutations at 3 min and the deletion extending from leu had an additional phenotype. Introduction of these mutations or deletions was always accompanied by disappearance of PEP synthase activity. Complementation of such a mutant with the cloned fragments reversed both phenotypes at the same time. Possibly, the fruR gene product acts as an activator of the gene coding for PEP synthase.     

Suppression of the ptsH Mutation in Escherichia coli and Salmonella typhimurium by a DNA Fragment from Lactobacillus casei

A DNA fragment from Lactobacillus casei that restores growth to Escherichia coli and Salmonella typhimurium ptsH mutants on glucose and other substrates of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been isolated. These mutants lack the HPr protein, a general component of the PTS. Sequencing of the cloned fragment revealed the absence of ptsH homologues. Instead, the complementation ability was located in a 120-bp fragment that contained a sequence homologue to the binding site of the Cra regulator from enteric bacteria. Experiments indicated that the reversion of the ptsH phenotype was due to a titration of the Cra protein, which allowed the constitutive expression of the fructose operon.     

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.     

The role of phosphorylation of HPr,a phosphocarrier protein of the phosphotransferase system,in the regulation of carbon metabolism in gram-positive bacteria

HPr of the Gram-positive bacterial phosphotransferase system (PTS) can be phosphorylated by an ATP-dependent protein kinase on a serine residue or by PEP-dependent Enzyme I on a histidyl residue. Both phosphorylation events appear to influence the metabolism of non-PTS carbon sources. Catabolite repression of the gluconate (gnt) operon of B. subtilis appears to be regulated by the former phosphorylation event, while glycerol kinase appears to be regulated by the latter phosphorylation reaction. The extent of our understanding of these processes will be described. © 1993 Wiley-Liss, Inc.