Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins.

Mannitol-specific enzyme II, or mannitol permease, of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system of Escherichia coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane. We recently reported the importance of a glutamate residue at position 257 in the binding and transport of mannitol by this protein (C. Saraceni-Richards and G. R. Jacobson, J. Bacteriol. 179:1135-1142, 1997). Replacing Glu-257 with alanine (E257A) or glutamine (E257Q) eliminated detectable mannitol binding and transport by the permease. In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to that of the wild-type protein but was severely defective in mannitol uptake. In this study, we have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein. Activities of any active heterodimers resulting from this coexpression were measured. The results show that various inactive mutant permease proteins can complement proteins containing mutations at position 257. In addition, we show that both Glu at position 257 and His at position 195, both of which are in the membrane-bound C domain of the protein, must be on the same subunit of a permease dimer in order for efficient mannitol phosphorylation and uptake to occur. The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphorylated by the subunit containing the E257A mutation (which cannot bind mannitol) and support a model in which there are separate binding sites on each subunit within a permease dimer. Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary for efficient transport by mannitol permease.

A conserved glutamate residue, Glu-257, is important for substrate binding and transport by the Escherichia coli mannitol permease.

The mannitol permease, or D-mannitol-specific enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, both transports and phosphorylates its substrate. Previous analyses of the amino acid sequences of PTS permeases specific for various carbohydrates in different species of bacteria revealed several regions of similarity. The most highly conserved region includes a GIXE motif, in which the glutamate residue is completely conserved among the permeases that contain this motif. The corresponding residue in the E. coli mannitol permease is Glu-257, which is located in a large putative cytoplasmic loop of the transmembrane domain of the protein. We used site-directed mutagenesis to investigate the role of Glu-257. The properties of proteins with mutations at position 257 suggest that a carboxylate side chain at this position is essential for mannitol binding. E257A and E257Q mutant proteins did not bind mannitol detectably, while the E257D mutant could still bind this substrate. Kinetic studies with the E257D mutant protein also showed that a glutamate residue at position 257 of this permease is specifically required for efficient mannitol transport. While the E257D permease phosphorylated mannitol with kinetic parameters similar to those of the wild-type protein, the Vmax for mannitol uptake by this mutant protein is less than 5% that of the wild type. These results suggest that Glu-257 of the mannitol permease and the corresponding glutamate residues of other PTS permeases play important roles both in binding the substrate and in transporting it through the membrane.

Isolation and characterization of a mutation that alters the substrate specificity of the Escherichia coli glucose permease.

We isolated 10 mannitol-positive mutants from a mannitol-negative Escherichia coli strain. These mutations mapped within ptsG, encoding the glucose permease (EIIGlc), and resulted in a G-320-to-V substitution that allows EIIGlc to transport mannitol. Gly-320 lies within a putative transmembrane helix of EIIGlc that may be involved in substrate recognition.

Overexpression, phosphorylation, and growth effects of ORF162, a Klebsiella pneumoniae protein that is encoded by a gene linked to rpoN, the gene encoding sigma 54.

The product of a Klebsiella pneumoniae gene, orf162, may regulate sigma 54-dependent transcription and has sequence similarity to proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). We have overproduced the product of orf162 and demonstrated its PTS-dependent phosphorylation in Escherichia coli extracts. We have also observed moderate growth inhibition of a wild-type, but not a sigma 54-mutant, strain by overexpression of orf162. These results are consistent with the hypothesis that the product of orf162 could be a regulatory link between the PTS and sigma 54 activity in bacteria.

The Escherichia 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 the Escherichia 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.

Role of a conserved histidine residue, His-195, in the activities of the Escherichia coli mannitol permease.

The mannitol permease, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, carries out the transport and phosphorylation of D-mannitol in this organism. Previous studies have shown that His-554 and Cys-384 in the mannitol permease are sequentially phosphorylated in reactions necessary for the transport and phosphorylation of the substrate. These residues are located in a large cytoplasmic domain of the protein. Interaction of the permease with mannitol, and its membrane translocation, however, must involve the N-terminal, transmembrane domain (EIIC domain) of the protein. In this report, we use site-directed mutagenesis and mutant complementation to investigate the role of His-195 in the EIIC domain of the mannitol permease, a residue that is conserved in many PTS permeases. In a previous report [Weng, Q.-P., Elder, J., & Jacobson, G. R. (1992) J. Biol. Chem. 267, 19529-19535], we inferred a role for His-195 that involves its hydrogen-bonding ability. Here we show that His-195 plays a role in high-affinity mannitol binding. Moreover, mutant complementation studies show that a functional His-195 must be on the same subunit as a functional Cys-384 in a permease dimer for phosphotransfer to mannitol to occur. These results and kinetic studies of His-195 mutant proteins imply that His-195 also may play an important role in this phosphotransfer reaction. His-195 is predicted to be in a cytoplasmic "loop" in the EIIC domain of the mannitol permease, in which several other residues have been shown to have roles in mannitol permease activity.

Psychoactive substance use among American anesthesiologists: a 30-year retrospective study.

The purpose of this study was to assess the cumulative incidence of substance use among anesthesiologists during training and practice, the effect of stress on drug use, and deterrent efficacy of institutional prevention programmes. The 260 anesthesiologists who had trained at the Medical College of Wisconsin between 1958-1988 were surveyed by mail regarding psychoactive substance use. Analysis of 183 responses focused on demographic and psychosocial factors. Substances used most frequently included: alcohol (91.6%), marijuana (30.8%) and cocaine (9.4%). Twenty-nine (15.8%) anesthesiologists were identified as being substance-dependent: 19 were alcohol-impaired; six were drug-impaired, and four were dependent on both alcohol and drugs. Impairment was more prevalent in anesthesiologists who had completed their training after 1975. Fifty-eight (32%) anesthesiologists had used illicit drugs to "get high"; 11 acknowledged daily use for two weeks or more, with eight admitting dependency. Substance abuse was more common in parents of impaired anesthesiologists (35.7%) than in unimpaired colleagues (8.1%; P < 0.001). The divorce rate for impaired anesthesiologists (24.1%) was greater than for unimpaired anesthesiologists (5.2%; P < 0.001). Increased stress during training was not reflected by increased substance use. Few recalled any drug counseling whatsoever. Seventy percent assessed hospital drug control policies as fair or poor. Younger respondents (born after 1951) were more critical of drug control programmes than their older cohort. Incidents of substance abuse were reported for both residents and faculty. Psychoactive substance abuse remains a serious problem among anesthesiologists.

Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.

Site-specific mutagenesis of residues in the Escherichia coli mannitol permease that have been suggested to be important for its phosphorylation and chemoreception functions.

The Escherichia coli mannitol permease is an integral membrane protein that catalyzes the concomitant transport and phosphorylation of D-mannitol and also acts as the chemoreceptor for chemotaxis of E. coli to this hexitol. At least 4 aminoacyl residues in this protein have been suggested to be important in these activities: His-195, His-256, Cys-384, and His-554. Previous evidence has implicated His-554 and Cys-384 as residues that are covalently phosphorylated, in sequence, as intermediates in phosphotransfer to mannitol. We have constructed a number of site-specific mutants of the mannitol permease at these positions. The properties of proteins in which His-554 or Cys-384 has been changed are consistent with their essential roles in phosphorylation. We also used these mutants to show that intermolecular phosphotransfer between His-554 and Cys-384 can occur in vivo in membrane-bound heterodimers consisting of different mutant subunits. The properties of proteins with mutations at position 195 suggest an important role for this residue involving hydrogen bonding, while His-256 performs no significant function in the mannitol permease. Finally, the phosphorylation and chemoreception activities for each mutant protein were each roughly in the same proportion to these activities in the wild-type protein, showing that these functions of the mannitol permease are tightly coupled under normal physiological conditions.

A proposed link between nitrogen and carbon metabolism involving protein phosphorylation in bacteria.

We demonstrate that certain phosphoryl transfer proteins of the bacterial phosphotransferase system (PTS), the fructose- and mannitol-specific IIA proteins or domains, are homologous to a class of proteins, one of which is known to affect transcription of some of the nitrogen-regulatory sigma 54-dependent operons in Klebsiella pneumoniae. The phosphorylatable histidyl residue in the homologous PTS proteins and the consensus sequence in the vicinity of the active-site histidine are fully conserved in all members that comprise this family of proteins. A phylogenetic tree of the eight protein members of this family was constructed, and a "signature" sequence that can serve for the identification of new protein members of this family is proposed. These observations suggest that PTS-catalyzed protein phosphorylation may provide a regulatory link between carbon and nitrogen assimilation in bacteria.

Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization.

The Escherichia coli mannitol permease (EIIMtl) of the phosphoenolpyruvate-dependent phosphotransferase system is a 68-kDa membrane protein that carries out the concomitant transport and phosphorylation of D-mannitol. Previous studies indicated that there are ca. 6 membrane-spanning helices within the N-terminal half of the protein, while the hydrophilic C-terminal half was shown to be exposed in the cytoplasm. In the present study, an analysis of C-terminally truncated EIIMtl mutants showed that proteins from which only the cytoplasmic domain has been deleted were present in the membrane at > or = 50% the amount of the intact protein. However, deletion proteins smaller than ca. 34 kDa were present in the membrane at only about 20% the amount of the intact protein. We also constructed a plasmid that encodes the first 43 amino acid residues of ELLMtl fused to residues 378 to 637 (the C-terminal domain). The corresponding protein was associated with the cytoplasmic membrane. These results show that the first 43 amino acid residues of the N terminus are sufficient for membrane localization, although the region comprising the last 2 membrane-spanning helices appears to be important for maximum stability and/or efficient membrane insertion of the complete N-terminal domain. Further studies of these deletion proteins showed that binding of mannitol to the permease occurs even if the entire cytoplasmic domain is absent, but is abolished if the last putative membrane-spanning region is removed. Finally, regions of the protein within the membrane-bound domain were identified that influence the oligomerization state of the protein. These results further define domains of this multifunctional transport protein that are important for membrane insertion, stability, substrate binding and oligomerization.

Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system.

The membrane-bound enzymes II of the bacterial carbohydrate phosphotransferase system (PTS) are multifunctional: they are required for the transport, phosphorylation and chemotactic sensing of their substrates. An oligomer (minimally a dimer) of at least one of these PTS permeases, the Escherichia coli mannitol permease, appears to be necessary for this protein to optimally carry out these functions. Much indirect evidence is consistent with this hypothesis, and recent experiments show that transport and phosphorylation of, and chemotaxis to, mannitol in E. coli involves an intersubunit phosphotransfer reaction, which can only occur in a protein oligomer. Membrane topological studies of the mannitol permease also argue in favour of an oligomeric structure in the membrane which may be necessary to form the hydrophilic channel through which mannitol must traverse the phospholipid bilayer. The possibility that the oligomerization state of the mannitol permease is a target for regulation of its activity in vivo is proposed, but has not yet been explored experimentally.

Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions.

The Escherichia coli mannitol permease catalyzes the concomitant transport and phosphorylation of D-mannitol. This 68-kDa protein consists of a membrane-bound, N-terminal domain involved in mannitol binding and translocation and a C-terminal, cytoplasmic domain responsible for mannitol phosphorylation. Secondary-structure prediction methods suggest that the N-terminal half of the permease spans the membrane approximately seven times in alpha-helical segments, but these data cannot conclusively predict the structure. We have used gene fusions between mtlA (encoding the permease) and 'phoA (encoding alkaline phosphatase lacking its signal sequence) to further investigate the topology of the mannitol permease. Initially, fusions were constructed by using a lambda TnphoA vector and in vitro cloning of 'phoA into naturally occurring restriction sites in mtlA. However, the former method gave severe problems with insertion "hot-spots" in our vector systems, and the latter method was limited by the number of useful restriction sites. Therefore, we developed a nested-deletion method for creating mtlA-phoA fusions. 'phoA was first cloned downstream from the part of mtlA encoding the membrane-bound half of the permease. This construct was then treated with the appropriate restriction enzymes and with exonuclease III to create random fusions. An analysis of greater than 40 different fusion clones constructed by these methods provides strong evidence for six membrane-spanning regions in the mannitol permease with three relatively short periplasmic loops and two large cytoplasmic loops in the membrane-bound half of the protein.

Substance abuse by anesthesiology residents.

A 1989 cross-sectional substance abuse survey of 260 former anesthesiology residents of the Medical College of Wisconsin (MCW) during the previous 30 years yielded 183 responses (70.3%). Over three-fourths (77.2%) of those who responded reported that they had used alcohol when they were residents; 20.0% had used marijuana; and 15.7% had used cocaine. Forty-three of the 178 respondents had used unprescribed psychoactive drugs. Twenty-nine (15.8%) had been self-admitted problematic substance abusers during their residencies: 23, alcohol dependent and six, drug dependent; among the latter were four with a dual (alcohol and drug) dependency. More than 85% considered the drug policy information available during their residencies had been inadequate; institutional drug-control policies were rated "fair-to-poor" by more than 70%. Thirty-five of the residents had observed their teachers using alcohol and/or other drugs to the detriment of their teaching; approximately one-third of these infractions had gone unreported.

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:mannitol phosphotransferase system.

We have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EIIMtl, the phospho group from [(R)-16O,17O,18O]PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a single site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EIIMtl must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EIIMtl intermediates have been demonstrated biochemically, our results imply that EIIMtl is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EIIMtl intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein [Pas, H. H., & Robillard, G. T. (1988) Biochemistry 27, 5835-5839].

Evidence that a low-affinity sucrose phosphotransferase activity in Streptococcus mutans GS-5 is a high-affinity trehalose uptake system.

High-affinity sucrose uptake in the oral pathogen Streptococcus mutans is mediated by the phosphoenolpyruvate-dependent phosphotransferase system. In this report, we provide evidence that a lower-affinity sucrose phosphotransferase system in S. mutans GS-5, previously described by others, is in fact a high-affinity trehalose uptake system that also recognizes sucrose as a substrate.

Molecular cloning of the C-terminal domain of Escherichia coli D-mannitol permease: expression, phosphorylation, and complementation with C-terminal permease deletion proteins.

We have subcloned a portion of the Escherichia coli mtlA gene encoding the hydrophilic, C-terminal domain of the mannitol-specific enzyme II (mannitol permease; molecular mass, 68 kilodaltons [kDa]) of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system. This mtlA fragment, encoding residues 379 to 637 (residue 637 = C terminus), was cloned in frame into the expression vector pCQV2 immediately downstream from the lambda pr promoter of the vector, which also encodes a temperature-sensitive lambda repressor. E. coli cells carrying a chromosomal deletion in mtlA (strain LGS322) and harboring this recombinant plasmid, pDW1, expressed a 28-kDa protein cross-reacting with antipermease antibody when grown at 42 degrees C but not when grown at 32 degrees C. This protein was relatively stable and could be phosphorylated in vitro by the general phospho-carrier protein of the phosphotransferase system, phospho-HPr. Thus, this fragment of the permease, when expressed in the absence of the hydrophobic, membrane-bound N-terminal domain, can apparently fold into a conformation resembling that of the C-terminal domain of the intact permease. When transformed into LGS322 cells harboring plasmid pGJ9-delta 137, which encodes a C-terminally truncated and inactive permease (residues 1 to ca. 480; molecular mass, 51 kDa), pDW1 conferred a mannitol-positive phenotype to this strain when grown at 42 degrees C but not when grown at 32 degrees C. This strain also exhibited phosphoenolpyruvate-dependent mannitol phosphorylation activity only when grown at the higher temperature. In contrast, pDW1 could not complement a plasmid encoding the complementary N-terminal part of the permease (residues 1 to 377). The pathway of phosphorylation of mannitol by the combined protein products of pGJ9-delta 137 and pDPW1 was also investigated by using N-ethylmaleimide to inactivate the second phosphorylation sites of these permease fragments (proposed to be Cys-384). These results are discussed with respect to the domain structure of the permease and its mechanism of transport and phosphorylation.

Inhibition of Streptococcus mutans by the antibiotic streptozotocin: mechanisms of uptake and the selection of carbohydrate-negative mutants.

The antibiotic streptozotocin [2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranoside], an analog of N-acetylglucosamine (NAG), has been shown to be useful for the selection of carbohydrate-negative and auxotrophic bacterial mutants. We have adapted this method for use with the oral pathogen Streptococcus mutans, a gram-positive, aerotolerant anaerobe that uses predominantly carbohydrates as carbon sources for growth. Streptozotocin selectively kills growing cells of S. mutans GS-5, and under appropriate conditions it can reduce the number of viable cells in actively growing cultures by a factor of 10(3) to 10(4). However, unlike in enteric bacteria, which take up this antibiotic by a single NAG-specific transport system, streptozotocin appears to be taken up in S. mutans by both a NAG-specific system and a relatively nonspecific system that is also involved in glucose, fructose, and mannose uptake. Combining streptozotocin selection and a screening procedure involving indicator plates containing triphenyl-tetrazolium chloride, we developed a general method for the isolation of carbohydrate-negative and auxotrophic mutants of S. mutans. A preliminary characterization of both pleiotropic and specific carbohydrate-negative mutants isolated by using this procedure is presented.

Carbohydrate uptake in the oral pathogen Streptococcus mutans: mechanisms and regulation by protein phosphorylation.

Streptococcus mutans is the primary etiological agent of dental caries in man and other animals. This organism and other related oral streptococci use carbohydrates almost exclusively as carbon and energy sources, fermenting them primarily to lactic acid which initiates erosion of tooth surfaces. Investigations over the past decade have shown that the major uptake mechanism for most carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), although non-PTS systems have also been identified for glucose and sucrose. Regulation of sugar uptake occurs by induction/repression and inducer exclusion mechanisms in S. mutans, but apparently not by inducer expulsion as is found in some other streptococci. In addition, ATP-dependent protein kinases have also been identified in S. mutans and other oral streptococci, and a regulatory function for at least one of these has been postulated. Among a number of proteins that are phosphorylated by these enzymes, the predominant soluble protein substrate is the general phospho-carrier protein of the PTS, HPr, as had previously been observed in a variety of Gram-positive bacteria. Recent results have provided evidence for a role for ATP-dependent phosphorylation of HPr in the coordination of sugar uptake and its catabolism in S. mutans. In this review, these results are summarized, and directions for future research in this area are discussed.