Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes

Specialized lambda-transducing phages that carry the Escherichia coli genes ptsH, ptsI, crr, cysM, and cysA have been isolated, and the genes were subcloned in plasmid pBR322. Subcloning and restriction mapping data gave the following clockwise order of genes located at about 52 min on the E. coli genetic map: lig, cysK, ptsH, ptsI, crr, cysM, cysA. The nucleotide sequences of ptsH, ptsI, and crr and the corresponding flanking regions have been determined. These genes encode three cytoplasmic proteins of the phosphoenol-pyruvate:glycose phosphotransferase system: HPr, Enzyme I, and IIIGlc, respectively. The deduced amino acid sequences are consistent with amino acid composition and Edman degradation analyses obtained with the purified proteins. The calculated subunit molecular weight values (9,109 for HPr, 63,489 for Enzyme I, and 18,099 for IIIGlc) also agree well with values obtained with the proteins. Results of gamma delta-transposon insertional studies provided definitive evidence that IIIGlc is the gene product of crr, and therefore that IIIGlc plays a critical role in regulating the metabolism and uptake of certain non-PTS sugars (see accompanying papers: Mitchell, W.J., Saffen, D.W., and Roseman, S. (1987) J. Biol. Chem. 16254-16260; Misko, T.P., Mitchell, W.J., Meadow, N.D., and Roseman, S. (1987) J. Biol. Chem. 16261-16266). The gamma delta transposon studies also suggest that crr is transcribed from an independent promoter located within the ptsI gene. Putative regulatory sequence features include a catabolite gene activator protein-cAMP-binding site and two regions of 2-fold rotational symmetry adjacent to the potential promoter upstream from the HPr structural gene, several ribosome-binding sites, and a rho-independent RNA polymerase termination site downstream from crr. In addition, the ptsI gene contains two highly conserved direct repeats. The significance of these sequence features is discussed with respect to possible multiple forms of pts regulation.     

Deletion mapping of the genes coding for HPr and enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium

Sugars transported by a bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) require two soluble proteins: HPr, a low-molecular-weight phosphate-carrier protein, and enzyme I. The structural genes coding for HPr (ptsH) and Enzyme I (ptsI) are shown to be cotransducible in Salmonella typhimurium. The gene order of this region of the Salmonella chromosome is cysA-trzA-ptsH-ptsI...(crr). A method for the isolation of trzA-pts deletion is described. One class of pts deletions extends through ptsH and into ptsI; a second class includes both ptsH and ptsI and extends into or through the crr gene. The crr gene either codes for or regulates the synthesis of a third PTS protein (factor III) which is sugar-specific. A hypothesis is presented for a mechanism of deletion formation.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene

The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.     

Dominant ptsH mutations of the gene coding for synthesis of the HPr protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli K-12 merodiploids

Abstract The Escherichia coli ptsI and ptsH genes code for the synthesis of two proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), namely enzyme I and protein HPr. A number of ptsI ⁺ ptsH ⁺/F' ptsI ⁺ ptsH merodiploids was obtained. It was shown in experiments in vivo that ptsH mutations in the transposition are dominant. Bacterial extracts from these merodiploids supported [¹⁴C]methyl glucoside (MG) phosphorylation at the expense of phosphoenolpyruvate only half as much as extracts from the pts ⁺ cells. ptsI ⁺ ptsH /F' ptsI ⁺ ptsH ⁺ merodiploids appeared to be non-viable; the reason for this lack of viability is discussed.     

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 and Erwinia 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 the ptsI and ptsH genes of E. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas the ptsH gene is located at 175 min. Sequencing of a portion of the E. 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.     

Promoter-like mutation affecting HPr and enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium

A promoter-like mutation, ptsP160, has been identified which drastically reduces expression of the genes specifying two proteins, HPr and enzyme I, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Salmonella typhimurium. This mutation lies between trzA, a gene specifying susceptibility to 1,2,4-triazole, and ptsH, the structural gene for HPr. It leads to a loss of active transport of those sugars that require the PTS for entry into the cell. Pseudorevertants of strains carrying this promoter-like mutation have additional lesions very closely linked to ptsP160 by transduction analysis and are noninducible for HPr and enzyme I above a basal level. Presumably, strains carrying ptsP160 are defective in the normal induction mechanism for HPr and enzyme I, and the pseudorevertants derived from them result from second-site initiation signals within or near this promoter-like element. The induction of HPr and enzyme I above their noninduced levels apparently is not required for transport of at least one PTS sugar, methyl alpha-d-glucopyranoside, since this sugar is taken up by the pseudorevertants at the same rate as by the wild type. The existence of a promoter-like element governing the coordinate inducibility of both HPr and enzyme I suggests that ptsH and ptsI constitute an operon. Wild-type levels of a sugar-specific PTS protein, factor III, are synthesized in response to the crr(+) gene in both a ptsP160 strain and its pseudorevertants; this suggests that the crr(+) gene has its own promoter distinct from ptsP.     

Molecular cloning, sequencing, and expression of the crr gene: the structural gene for IIIGlc of the bacterial PEP:glucose phosphotransferase system.

The phosphoenolpyruvate:glucose phosphotransferase system (PTS) of Salmonella typhimurium is involved both in glucose transport and in the regulation and synthesis of adenylate cyclase and several transport systems. The crr gene has been implicated in this regulating mechanism. A 9.6-kb segment of the S. typhimurium chromosome containing the crr gene was cloned in pAT153. The cloned fragment also complemented cysA mutations but did not contain a functional pts operon which is closely linked to the crr gene and codes for two enzymes of the PTS. Although cysA and crr have been reported to be located on opposite sides of ptsHI, our results suggest that the correct gene order is cysK-ptsHI-crr-cysA. Expression of crr plasmids in a maxicell system yielded two proteins which reacted with specific anti-serum against IIIGlc. The apparent mol. wts. in SDS-polyacrylamide gels were 20 000 and 21 000, the former corresponding to the major band of purified IIIGlc. Both forms were also observed in bacterial extracts and purified IIIGlc. The crr gene was localized on a 1-kb EcoRI-EcoRV fragment of the 9.6-kb insert and sequenced. It codes for a single protein (18 556 D) containing 169 amino acid residues and identified as IIIGlc.     

Partial nucleotide sequence of the pts operon in Salmonella typhimurium: comparative analyses in five bacterial genera

The nucleotide sequence of a Salmonella typhimurium DNA segment of 549 base pairs which encompasses the operator-promoter of the pts operon, the entirety of the ptsH gene, encoding HPr of the phosphotransferase system (PTS), the first 29 nucleotides of the ptsI gene, encoding Enzyme I of the PTS, and the intercistronic region between the ptsH and ptsI genes was determined and compared with the corresponding sequence from Escherichia coli (De Reuse et al., 1985). The two sequences showed 91% overall identity, with some regions showing sequence conservation and others exhibiting relative divergence. Two open reading frames were identified in both species: one encoded HPr on the 'sense' strand (255 nucleotides; 12 nucleotide differences, no amino acid differences); the other, on the anti-sense strand, consisted of 291 nucleotides (13 nucleotide differences, 13 amino acid differences). While HPr bears a net negative charge, the putative protein encoded by the open reading frame on the anti-sense strand is strongly basic. Computer analyses of HPr proteins from five different bacterial genera revealed four regions which show strong sequence identity and therefore are presumed to be critical for maintenance of biological activity. Two of these regions were specific to Gram-positive bacteria. Proposed functions for each of these regions are discussed. Relative evolutionary distances between the HPr proteins were also computed.     

The phosphoenolpyruvate:glucose phosphotransferase system of Salmonella typhimurium. The phosphorylated form of IIIGlc

Enzyme IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Salmonella typhimurium can occur in two forms: phosphorylated and nonphosphorylated. Phosphorylated IIIGlc (P-IIIGlc) has a slightly lower mobility during sodium dodecyl sulphate/polyacrylamide gel electrophoresis than IIIGlc. In bacterial extracts both phosphoenolpyruvate (the physiological phosphoryl donor of the PTS) as well as ATP can phosphorylate IIIGlc. The ATP-catalyzed reaction is dependent on phosphoenolpyruvate synthase, however, and is due to prior conversion of ATP to phosphoenolpyruvate. The phosphoryl group of phosphorylated IIIGlc is hydrolysed after boiling in sodium dodecyl sulfate but phosphorylated IIIGlc can be discriminated from IIIGlc if treated with this detergent at room temperature. We have used the different mobilities of IIIGlc and P-IIIGlc to estimate the proportion of these two forms in intact cells. Wild-type cells contain predominantly P-IIIGlc in the absence of PTS sugars. In an S. typhimurium mutant containing a leaky ptsI17 mutation (0.1% enzyme I activity remaining) both forms of IIIGlc occur in approximately equal amounts. Addition of PTS sugars such as glucose results, both in wild-type and mutant, in a dephosphorylation of P-IIIGlc. This correlates well with the observed inhibition of non-PTS uptake systems by PTS sugars via nonphosphorylated IIIGlc.     

Phosphocarrier proteins in an intracellular symbiotic bacterium of aphids.

A GroEL homolog produced by Buchnera, an intracellular symbiotic bacterium of aphids, is not only a molecular chaperone but also a novel phosphocarrier protein, suggesting that this protein plays a role in a signal transducing system specific to bacteria living in an intracellular environment. This prompted us to look into phosphocarrier proteins of Buchnera that may be shared in common with other bacteria. As a result, no evidence was obtained for the presence of sensor kinases of the two-component system in Buchnera, which are found in many bacteria. It is possible that the lack of sensor kinases is compensated for by the mulitifunctional GroEL homolog in this symbiotic bacteria. In contrast, we successfully identified three phosphotransferase system genes, ptsH, ptsI, and crr in Buchnera, and provide evidence for their active expression. While the deduced amino acid sequences of these gene products, histidine-containing phosphocarrier protein, Enzyme I, and Enzyme III were similar to their counterparts in Escherichia coli, the predicted isoelectric points of the Buchnera proteins were strikingly higher. It was also suggested that Buchnera Enzyme I, when produced in E. coli, is able to accept the phosphoryl group from phosphoenolpyruvate, but not from ATP.     

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and protein sequencing of the Staphylococcus carnosus histidine-containing protein, and cloning and DNA sequencing of the ptsH gene

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr-expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy-chain-termination technique. Upstream, the 264-bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.     

Diversity of Streptococcus salivarius ptsH mutants that can be isolated in the presence of 2-deoxyglucose and galactose and characterization of two mutants synthesizing reduced levels of HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase system

In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His approximately P), HPr(Ser-P), and HPr(Ser-P)(His approximately P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for alpha-galactosidase, beta-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of alpha-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and the cellular levels of HPr(Ser-P).     

Sugar transport by the bacterial phosphotransferase system. Reconstitution of inducer exclusion in Salmonella typhimurium membrane vesicles

The accompanying articles (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253; Mitchell, W.J., Saffen, D. W., and Roseman, S. (1987) J. Biol. Chem. 262, 16254-16260) show that "inducer exclusion" in intact cells of Escherichia coli is regulated by IIIGlc, a protein encoded by the crr gene of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The present studies attempt to show a direct effect of IIIGlc on non-PTS transport systems. Inner membrane vesicles prepared from a wild type strain of Salmonella typhimurium (pts+), carrying the E. coli lactose operon on an episome, showed respiration-dependent accumulation of methyl-beta-D-thiogalactopyranoside (TMG) via the lactose permease. In the presence of methyl-alpha-D-glucopyranoside or other PTS sugars, TMG uptake was reduced by an amount which was dependent on the relative concentrations of IIIGlc and lactose permease in the vesicles. The endogenous IIIGlc concentration in these vesicles was in the range 5-10 microM, similar to that found in whole cells. Methyl-alpha-glucoside had no effect on lactose permease activity in vesicles prepared from a deletion mutant strain lacking the soluble PTS proteins Enzyme I, HPr, and IIIGlc. One or more of the pure proteins could be inserted into the mutant vesicles; when one of the two electrophoretically distinguishable forms of the phosphocarrier protein, IIIGlc Slow, was inserted, both the initial rate and steady state level of TMG accumulation were reduced by up to 40%. The second electrophoretic form, IIIGlc Fast, had much less effect. A direct relationship was observed between the intravesicular concentration of IIIGlc Slow and the extent of inhibition of the lactose permease. No inhibition was observed when IIIGlc Slow was added to the outside of the vesicles, indicating that the site of interaction with the lactose permease is accessible only from the inner face of the membrane. In addition to the lactose permease, IIIGlc Slow was found to inhibit both the galactose and the melibiose permeases. Uptake of proline, on the other hand, was unaffected. The results are therefore consistent with an hypothesis that dephosphorylated IIIGlc Slow is an inhibitor of certain non-PTS permeases.