Analysis of two genes encoding heat-labile toxins and located on a single Ent plasmid from Escherichia coli.

A clinical isolate of Escherichia coli harbored two copies of the heat-labile toxin (LT)-encoding gene (elt) on a 157-kb plasmid. The arrangement of the gene copies is different from the cholera toxin-encoding gene duplication described for some strains of Vibrio cholerae. The nucleotide sequences of the elt alleles are not identical (differing by 2 bp) and the duplicated region flanking the alleles extends 314 bp on one side and 1122 bp on the other side of each copy. Different partial copies of IS600 were identified 280 bp 3' to the stop codon of each allele. Partial and complete copies of other IS were also identified near the elt alleles. The data suggest that the regions surrounding the genes are hot spots for IS which would account for the observed heterogeneity in DNA flanking elt genes.

Functional cloning of the dihydropteroate synthase gene of Staphylococcus haemolyticus.

A 1,7-kilobase fragment of the Staphylococcus haemolyticus chromosome containing the dihydropteroate synthase gene has been cloned by complementation in a temperature-sensitive mutant of Escherichia coli. The gene, designated folP, predicts a gene product of 29613 Da which shares significant amino acid sequence identity with other known bacterial dihydropteroate synthases. Analysis of the DNA sequence upstream and downstream of folP identified two further, incomplete open reading frames, one of which shows predicted amino acid sequence similarity to a second bacterial folic acid synthesis enzyme, dihydroneopterin aldolase.

Monoclonal antibody B9-2 recognizes an 8 amino acid sequence that spans an autocatalytic cleavage site between the two subunits of human procaspase 8.

Caspase 8 is synthesized in a zymogen form and must be proteolytically cleaved to be activated. The catalytically active enzyme is composed of two of the four cleavage products. We have determined that the monoclonal antibody B9-2 recognizes the octomer EMDLSSPQ. This sequence is shared by two of the cleavage products: a decamer linker region released after autocatalysis and the smaller subunit of the active enzyme.

Conformity between heat-labile toxin genes from human and porcine enterotoxigenic Escherichia coli.

The genes encoding the heat-labile toxin of Escherichia coli were isolated by recombinant DNA methods from enterotoxigenic E. coli recovered from a human and a piglet with diarrhea. With restriction endonucleases, a fine-structure map was made for the toxin genes. Both genes were found to be highly homologous within the toxin-coding DNA, but the surrounding DNA sequences were found to be quite divergent. Analysis of in vitro-derived mutants demonstrated that the cistrons for the toxin proteins were located at the same sites on each restriction enzyme map.

The heat-stable toxin I gene from Escherichia coli 18D.

The heat-stable toxin I gene in the human Escherichia coli isolate 18D is the estA1 allele. The gene is not part of a composite transposon, but inspection of the flanking DNA sequences suggests that it was at one time part of a transposon. The hypothetical transposon originated from an event other than the occurrence that formed Tn1681.

Amino acid sequence homology between cholera toxin and Escherichia coli heat-labile toxin.

Cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT) are functionally, structurally and immunologically similar enterotoxins. Both toxins cause the elevation of cyclic AMP levels in gut epithelial cells by catalysing the NAD-dependent ADP ribosylation of membrane proteins. Each toxin is composed of two dissimilar subunits. The A subunit has an enzymatic activity and is the adenylate cyclase-activating component of the enterotoxin. The B subunit recognizes membrane components and binds the holotoxin to the target call juxtaposing the A subunit with its substrates. Binding studies and competition experiments indicate that the membrane receptors for cholera toxin B subunit (CT-B) and LT-B are similar but not identical (these studies were performed before by LT was purified to homogeneity). The monosialosylganglioside GMI has been shown to be the receptor for the cholera toxin, and it probably composes part of the receptor for LT. Gyles and Barnum, first reported that LT and cholera toxin were immunologically related, and it has subsequently been shown that they share common antigenic determinants in both A and B subunits. The primary structure of CT-B has been determined. We report here a comparison between the amino acid sequences of LT-B and CT-B. The nucleotide sequence of the LT-B cistron (eltB) was determined using a recombinant plasmid encoding LT. Translation of this sequence revealed that LT-B and CT-B show significant amino acid sequence homology. In addition, several features of the eltB cistron were revealed by the sequence analysis.

Partition of heat-labile-enterotoxin genes between human and animal Escherichia coli isolates.

Heat-labile toxin (LT) genes from human and animal Escherichia coli isolates from a restricted geographical region (Thailand) exhibited a segregated pattern of dissemination that was revealed by a restriction enzyme site polymorphism. The results suggest an exclusion of LT genes from or a low transfer rate for LT genes between E. coli strains infectious for humans and those infectious for animals in natural settings.

Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin.

The nucleotide sequence of the LT-BH cistron (eltBH) from an enterotoxigenic Escherichia coli strain infectious for humans was determined and compared with the LT-B cistron sequence from a porcine E. coli isolate. Both cistrons were shown to comprise 375 nucleotide base pairs, and discrepancies were detected at eight positions. Of the nonhomologous base pairs, six resulted in codon changes that would lead to amino acid variations. The nucleotide sequence distal to both LT-B cistrons was also determined, and only three differences were detected in 197 base pairs. An HhaI site unique to eltBH was shown to be present in all the heat-labile (LT) genes from 31 human isolates surveyed, whereas the restriction enzyme recognition site was absent in the gene from 46 porcine E. coli isolates. The results suggest that two genetically discernable LT groups are identifiable and that the groups are also distinguishable by the isolation source (human or porcine) of the infecting E. coli strains.

Cistrons encoding Escherichia coli heat-labile toxin.

The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter.

Characterization of an Escherichia coli plasmid encoding for synthesis of heat-labile toxin: molecular cloning of the toxin determinant.

P307 is a plasmid isolated from a strain of Escherichia coli that was responsible for an outbreak of diarrheal disease in piglets. This 60 X 10(6)-dalton plasmid was subsequently shown to encode for the synthesis of a heat-labile toxin (LT). Using recombinant DNA technology, we isolated a 7.8 X 10(6)-dalton DNA fragment that contains the LT gene(s). This fragment was generated using an EcoRI partial digestion of P307 DNA, and the fragment was joined to a small multicopy plasmid, RSF2124. E. coli strains harboring the chimeric plasmid produced greater amounts of LT than did the same strains containing P307. The LT genes were also isolated on a 5.8 X 10(6)-dalton DNA fragment made by BamHI digestion, and we identified an EcoRI recognition sequence that is located in a position essential for LT synthesis.

Purification and partial characterization of 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase and 7,8-dihydropteroate synthase from Escherichia coli MC4100.

The enzymes 7,8-dihydroxymethylpterin-pyrophosphokinase (HPPK) and 7,8-dihydropteroate synthase (DHPS), which act sequentially in the folate pathway, were purified to homogeneity from crude extracts of Escherichia coli MC4100. The enzymes represent less than 0.01% of the total soluble protein. HPPK was purified greater than 10,000-fold; the native enzyme appears to be a monomer with a molecular mass of 25 kDa and a pI of 5.2. DHPS was purified greater than 7,000-fold; the native enzyme has an apparent molecular mass of 52 to 54 kDa and is composed of two identical 30-kDa subunits. The amino-terminal sequences for both enzymes have been determined.

Catalytic role of histidine 147 in Escherichia coli thymidylate synthase.

Nine mutant thymidylate synthases were isolated that only differed in sequence at position 147. The wild-type enzyme (which had a histidine residue at 147) and mutant enzymes were purified to near homogeneity and their kinetic properties were compared. Although the kcat values for the mutant enzymes were 10-10,000-fold lower than for the wild-type enzyme, the Km values for both 2'-deoxyuridylate and 5,10-methylenetetrahydrofolate were nearly identical for all the enzymes indicating that His-147 is not significantly involved in initial substrate binding. By comparing the wild-type (His-147) to the glycine (Gly-147) enzyme, the side chain of His-147 was estimated to lower the activation energy of the catalytic step by 1.6-2.9 kcal mol-1. In contrast to the wild-type enzyme, the activity of the Gly-147 enzyme decreased when the pH was raised above 7.5. The activity loss coincided with the deprotonation of a residue that had a pKa of 9.46 +/- 0.2 and an enthalpy of ionization (delta Hion) of 12.1 +/- 0.9. These values are consistent with the involvement of a lysine or an arginine residue in the catalytic process. An inspection of the rates of ternary complex formation among enzyme, 5-fluoro-2'-deoxyuridylate, and 5,10-methylenetetrahydrofolate for the mutant enzymes indicated that His-147 is not needed for the proton removal from C-5 of 2'-deoxyuridylate but rather participates in an initial catalytic step and alters the pKa value of a catalytically important lysine or arginine residue.

Identification, cloning, and purification of protein antigens of Treponema pallidum.

Difficulties in culturing the bacterium Treponema pallidum have greatly hindered syphilis research. In recent years, several laboratories have begun applying recombinant DNA technology to the study of this organism. Recent work is summarized concerning the expression of T. pallidum DNA in Escherichia coli. A number of E. coli clones expressing treponemal protein antigens have been identified. In one instance, a recombinant protein was purified to homogeneity and shown to be identical to a highly immunogenic, native T. pallidum membrane protein of molecular weight 39,000, which was designated the basic membrane protein (BMP) of this organism. In addition, recent experiments are described that were designed to identify cell-surface proteins that would serve as the primary focus of our cloning efforts. Results obtained with use of several different approaches strongly suggest that the outer membrane of T. pallidum is an antigenically inert structure largely devoid of protein. However, a class of low-molecular-weight protein antigens have been identified that are actively secreted into the extracellular medium. Attempts currently are being made to clone these secreted proteins and investigate their roles in the pathogenesis and immunobiology of syphilis.

Functional role of cysteine-146 in Escherichia coli thymidylate synthase.

Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.

Epitopes of the cholera family of enterotoxins.

Hybridoma-derived monoclonal antibodies were raised to enterotoxins of the cholera family and to chimeric B-subunit proteins in which individual amino acid residues of a heat-labile, cholera-related enterotoxin from an Escherichia coli strain of porcine origin (P-LT) were substituted with corresponding residues from such an enterotoxin from an E. coli strain of human origin (H-LT). Single amino acid substitutions were found to have profound effects on the physicochemical behavior of the proteins and on their immunologic reactivity. With the use of enzyme-linked immunosorption assays (ELISAs) with and without the GM1 ganglioside receptor for these toxins, several distinct epitopes in GM1-binding domains were identified by different monoclonal antibodies. Polyclonal rabbit antisera to synthetic peptides of the cholera enterotoxin B subunit were cross-reactive to various degrees with the proteins in our library, which include two different cholera enterotoxins, two H-LTs, P-LT, and four chimeric proteins. Some of these reactions were blocked by GM1 ganglioside but not by the oligosaccharide of GM1, a finding suggesting that the peptides generated antibodies to epitopes near, but not in, a GM1-binding domain. A hypothetical evolutionary tree based on the reported amino acid sequences of the various enterotoxins is constructed. As additional enterotoxins are described, it will be interesting to determine if and where they fit in this scheme.

Antigenic determinants of the cholera/coli family of enterotoxins.

Hybridoma-derived monoclonal antibodies were raised to enterotoxins of the cholera family and to chimeric B-subunit proteins in which individual amino acid residues of a heat-labile, cholera-related enterotoxin from an Escherichia coli strain of porcine origin (P-LT) were substituted with corresponding residues from such an enterotoxin from an E. coli strain of human origin (H-LT). Single amino acid substitutions were found to have profound effects on the physicochemical behavior of the proteins and on their immunologic reactivity. With the use of enzyme-linked immunosorption assays (ELISAs) with and without the GM1 ganglioside receptor for these toxins, several distinct epitopes in GM1-binding domains were identified by different monoclonal antibodies. Polyclonal rabbit antisera to synthetic peptides of the cholera enterotoxin B subunit were cross-reactive to various degrees with the proteins in our library, which include two different cholera enterotoxins, two H-LTs, P-LT, and four chimeric proteins. Some of these reactions were blocked by GM1 ganglioside but not the oligosaccharide of GM1, a finding suggesting that the peptides generated antibodies to epitopes near, but not in, a GM1-binding domain. A hypothetical evolutionary tree based on the reported amino acid sequences of the various enterotoxins is constructed. As additional enterotoxins are described, it will be interesting to determine if and where they fit in this scheme.

Mode of binding of folate analogs to thymidylate synthase. Evidence for two asymmetric but interactive substrate binding sites.

Human thymidylate synthase is a polymeric protein composed of two subunits with identical primary structures. In this study we determined the binding affinities of 5,10-methylene tetrahydropteroyltetraglutamate (folate substrate) and a group of close structural folate analog inhibitors. Thymidylate synthase bound both mono and polyglutamylated folate substrates and analogs more tightly in the presence of deoxyuridylate. These results and product inhibition studies confirmed that the orders of substrate addition and product release from thymidylate synthase were similar for mono and polyglutamylated substrates. Equilibrium dialysis studies showed that the folate substrate in a ternary complex with deoxyuridylate bound to one of the subunits (site A) with a Kd of 720 nM. The binding of the substrate to the second subunit (site B) was much weaker, and the Kd could not be determined by this method. However, dissociation constants for each subunit could be measured for the folate analog inhibitors, and, depending on the inhibitor, the relative Kd value for each subunit varied substantially. For example, formyl-5,8-dideazafolate and tetraglutamylated 10-propargyl-5,8-dideazafolate bound to both sites with similar Kd values, whereas D1694Glu4 bound to subunit A with a higher affinity (Kd = 1.0 nM) than to subunit B (Kd = 30 nM). In contrast, 1843U89 (mono or diglutamylated form) had a much higher affinity for subunit B (Kd approximately 0.1 nM) compared with subunit A (Kd approximately 400 nM). Enzyme inhibition kinetic analyses showed that the Ki values of 1843U89 were quite low (0.1 nM) and that the inhibition was noncompetitive. In contrast, the other folate analogs inhibited the enzyme via mixed inhibition (i.e. both the Km for the folate substrate and the Vmax were altered). We conclude that the two subunits of thymidylate synthase bind folate substrates and analogs differently and that the asymmetric binding of the ligands is the major factor that determines the inhibition kinetics of each folate analog inhibitor.

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.

Solution studies of recombinant human stromal-cell-derived factor-1.

Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence. The protein was denatured, refolded, and further purified by reversed-phase HPLC. SDF-1alpha was shown to be >95% pure as judged by SDS-PAGE. The final yield of purified and refolded SDF-1alpha was 1-2 mg per gram of wet cell paste. The refolded protein is a ligand for the CXCR4 receptor and has been used to block HIV-mediated cell fusion and downmodulates the CXCR4 receptor. Our ability to purify hundreds of milligrams of refolded protein allowed us to conduct detailed studies of the biophysical properties of the protein. We have used a combination of biophysical techniques to study the solution properties of SDF-1alpha. The average mass of SDF-1alpha, as determined by static light scattering, gave us the first indications that the chemokine may self-associate. Further investigation with sedimentation velocity ultracentrifugation confirmed the existence of two species. The measured s(20, W) values defined two masses corresponding to monomer and dimer. Finally, sedimentation equilibrium ultracentrifugation and dynamic light scattering yielded a composite value of 150 +/- 30 microM for the dimerization constant. We conclude that SDF-1alpha exists in a monomer-dimer equilibrium.

Application of monoclonal antibodies and genetically-engineered hybrid B-subunit proteins to the analysis of the cholera/coli enterotoxin family.

Single amino acid substitutions, introduced by genetic engineering, significantly modify the behavior of the B-subunits of the cholera/coli enterotoxin family in SDS-PAGE and also markedly affect the reactivity of the proteins with mouse hybridoma-derived monoclonal antibodies raised against H-LT. The results indicate that single amino acids play an important role in defining epitopes in these proteins.