Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli

The nucleotide sequence of the promoter region of the melibiose operon of E. coli was determined. Consensus sequences for the -35 region, the Pribnow box and the binding site for cyclic AMP receptor protein were found in this region. The possible secondary structure of this DNA region was very similar to that of the promoter region of the lactose operon. A possible initiation ATG preceded by a Shine-Dalgarno sequence with proper spacing was present just downstream of the promoter region. The possible sequence of 52 amino acid residues in the NH2 terminus of the alpha-galactosidase were determined.     

Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933

The nucleotide sequence of the Shiga-like toxin type II (SLT-II) structural genes cloned from bacteriophage 933W of the enterohemorrhagic Escherichia coli O157:H7 strain 933 was determined. This sequence was compared with the published sequence for the structural genes of the antigenically distinct Shiga-like toxin type I (SLT-I) encoded by bacteriophage 933J. The SLT-I and SLT-II structural genes shared 58% overall nucleotide and 56% amino acid sequence homologies. The A and B subunits of SLT-I and SLT-II were nearly identical in size and had similar secondary structures and hydropathy plots. The regulation proposed for the SLT-II operon is similar to that previously proposed for SLT-I.     

Crystallization of the B chain of Shiga-like toxin I from Escherichia coli

Shiga-like toxin I (SLT-I) is produced by several pathogenic strains of Escherichia coli associated with diarrheal disease. The toxin consists of an A chain, which attacks eukaryotic ribosomes, inhibiting protein synthesis, and multiple copies of a 69 amino acid B chain. The B subunit mediates cell binding and uptake through its interactions with cell surface carbohydrate moieties. Here we report that the B chain has been crystallized in a form suitable for high-resolution X-ray analysis. The space group is P2(1)2(1)2(1), with a = 56.2 A, b = 59.9 A and c = 102.5 A. A rotation function using three-dimensional diffraction data suggests that the asymmetric unit is a tetramer.     

Nucleotide sequence of the mannitol (mtl) operon in Escherichia coli

The nucleotide sequence of the known portions of the mannitol operon in Escherichia coli (mtlOPAD) has been determined. Both the operator-promoter region and the intercistronic region between the mtlA and mtlD genes (encoding the mannitol-specific Enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase, respectively) show parallels with corresponding regions of the glucitol (gut) operon, but neither the mtlA nor the mtlD gene products show obvious homology with the corresponding gene products of the glucitol operon. Five potential cyclic AMP receptor protein binding sites were identified in the mtlOP region, all showing near identity with the consensus sequence. Four regions of dyad symmetry (four to seven bases in length), serving as potential repressor binding sites, overlap with the potential cyclic AMP receptor protein binding sites. Repetitive extragenic palindromic (REP) sequences, forming stem-loop structures in the intercistronic region between mtlA and mtlD and following the mtlD gene were identified. Probable terminator sequences were not found in any of these three regulatory regions. Mannitol-1-phosphate dehydrogenase exhibits two overlapping, potential NAD+ binding sites near the N-terminus of the protein. Computer techniques were used to analyse the mtlD gene and its product.     

Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.

The Shiga-like toxin 1-converting bacteriophage H-19B was recently shown to carry the structural genes for the toxin and was shown to have DNA sequence homology with phage lambda. We present evidence that the linear genome of bacteriophage H-19B has cohesive termini which become covalently associated during prophage integration. Integration occurs through a site on a 4-kilobase-pair EcoRI fragment located near the center of the bacteriophage chromosome. The relationship between bacteriophages H-19B and lambda was examined by Southern hybridization. Homologous regions were mapped on the respective chromosomes which corresponded to the regions of the J gene, the int-xis area, and the O and P genes of phage lambda. The H-19B tox genes were mapped to the right of the O and P gene homology, which was far away from the phage attachment site. We concluded that H-19B is a lambdoid bacteriophage. Unlike other toxin-converting bacteriophages, the toxin genes were not located adjacent to the phage attachment site. It appeared that the Shiga-like toxin 1 genes were not picked up by a simple imprecise prophage excision. H-19B could, however, have acquired chromosomally located toxin genes by a series of events involving deletion and duplication followed by aberrant excision.     

Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli

The nucleotide sequence of a 6.5 kilobasepair chromosomal DNA fragment encoding the anaerobic dimethylsulphoxide (DMSO) reductase operon of Escherichia coli has been determined. The DMSO reductase structural operon was shown to consist of three open reading frames, namely dmsABC, encoding polypeptides with predicted molecular weights of 87,350, 23,070, and 30,789 Daltons, respectively. The DMS A polypeptide displayed a high degree of amino acid sequence homology with the single-subunit enzyme, biotin sulphoxide reductase (bisC) and with formate dehydrogenase (fdhF), suggesting that the active site and molybdopterin cofactor binding site that is common to these enzymes is located in the DMS A subunit. A comparison of the predicted N-terminal amino acids of the dmsA gene product to those of the 82,600 subunit of purified DMSO reductase indicated that post-translational processing of a 16 amino acid peptide at the amino terminus of DMS A had occurred. The DMS B polypeptide contains 16 cysteine residues organized in four clusters, two of which are typical of 4Fe-4S binding domains. The DMS C polypeptide is composed of eight segments of hydrophobic amino acids of appropriate length to cross the cytoplasmic membrane, suggesting that this subunit functions to anchor the enzyme to the membrane.     

Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family

Escherichia coli strain H.I.8 (O128:B12) produces low levels of a Shiga-like toxin (SLT) which we have called SLTIIva because of its close relationship with SLTIIv. The Vero cell cytotoxicity of SLTIIva is neutralized by antisera against SLTII and SLTIIv but not by antisera against SLTI. These data indicate that the SLT of strain H.I.8 is a member of the SLTII family. Since SLTIIva shares with SLTIIv the property of having low cytotoxicity to HeLa cells compared with Vero cells, it is appropriate to consider both toxins as variants of SLTII. SLTIIva differs from SLTIIv in that it is more heat-stable. Further, SLTIIv-producing strains of E. coli have only been isolated from pigs while the SLTIIva-producing E. coli strain examined in this study was isolated from a human infant with diarrhoea. The genes for this SLT were cloned from a cosmid library of total cellular DNA by screening recombinants for Vero cell toxicity and with a DNA probe derived from SLTIIv structural genes. Nucleotide sequence analysis was performed on a 2.0 kb AvaII-HincII fragment which encodes the toxin gene. The nucleotide sequence data confirm the close relationship between SLTIIva and SLTIIv: they have 98% nucleotide sequence homology in the B subunit gene and 70.6% homology in the A subunit gene. Comparison of DNA sequences indicated that SLTIIva was most closely related to SLTIIv, closely related to SLTII and less closely related to SLTI.     

Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon

The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene.