Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli

A DNA fragment containing genes for both large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-beta-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures.     

Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2).

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.     

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.     

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins.

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.     

Immunochemical characteristics of the subunits of cholera enterotoxin and of thermolabile Escherichia coli enterotoxins of various origins

Antigenic determinants of subunits A and B of cholera enterotoxin (CT), heat-labile enterotoxin from the human E. coli strain (hLT) and heat-labile enterotoxin from the porcine E. coli strain (pLT) were analysed by Ouchterlony double gel-diffusion test against antisera to B subunits of three toxins and antisera to three holotoxins. The results have shown the existence of the following antigenic determinants: in subunits B-1. antigenic determinants, common for B subunits of all three enterotoxins-B(chp); 2. group antigenic determinants, common for B subunits of two toxins in the pair-B(ch), B(hp); 3. antigenic determinants, unique for B subunits of each CT, hLT, pLT-(B(c), B(h), B(p); in subunits A.-1. antigenic determinants, common for A subunits of all three enterotoxins-A.(chp); 2. group antigenic determinants, common for A subunits of two enterotoxins (hLT and pLT/-A(hp); 3. antigenic determinants, unique for A subunit of CT-A(c). On the basis of these results antigenic formulas for subunits of CT, hLT, pLT were proposed.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.     

Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Methylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli. A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7-5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, E. coli expressed protein B which was fully functional after purification. The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7-7 (a plasmid similar to pT7-5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in E. coli was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.     

Preprotein translocation by a hybrid translocase composed of Escherichia coli and Bacillus subtilis subunits

Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B. subtilis SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E. coli and B. subtilis secY, secE, and secG genes were expressed in E. coli. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits. E. coli SecA, but not B. subtilis SecA, supported efficient ATP-dependent translocation of the E. coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E. coli SecY and either E. coli SecE or E. coli SecG were present. Translocation of B. subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to E. coli SecA, B. subtilis SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.     

Primary structures of and genes for new ribosomal proteins A and B in Escherichia coli

We determined the partial primary structures of and identified the genes for new basic proteins A and B in Escherichia coli ribosomal 50S subunits, found by means of an improved two-dimensional gel electrophoresis method. The sequence up to the 17th amino acid of protein B was in agreement with that of the X gene in the spc operon. The gene for protein A was searched for in the GenBank data base using the sequence up to the 35th amino acid, and was found at a locus between infC and rplT. The base sequence indicated that protein A contained 64 amino acids and had a molecular weight of 6,984. We conclude that proteins A and B are intrinsic ribosomal proteins, and propose calling their genes, rpmI and rpmJ, respectively.     

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli.

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Purification and Partial Characterization of the B Subunit of Serratia marcescens Tryptophan Synthetase

A trpE mutant of Serratia marcescens (E-7) was isolated, and the multimeric enzyme tryptophan synthetase (EC 4.2.1.20) was purified to homogeneity from derepressed cells. The A and B subunits were resolved, and the B subunit was partially characterized and compared with the Escherichia coli B subunit as part of a comparative evolution study of the trpB cistron of the trp operon in the Enterobacteriaceae. The S. marcescens B subunit is a dimer (beta(2)), and its molecular weight was estimated to be 89,000. The separate subunits (beta monomers) had molecular weights of approximately 43,000. The B subunit required pyridoxal phosphate for catalytic activity and had an apparent K(m) of 9 x 10(-6) M. The N terminus of the B subunit was unavailable for reaction with terminal amine reagents (blocked), whereas carboxypeptidase digestion released a C-terminal isoleucine. Using S. marcescens B antiserum in agar immunodiffusion gave an almost complete reaction of identity between the B subunits of S. marcescens and E. coli. The antiserum was used in microcomplement fixation, allowing for a comparison of the overall antigenic surface structure of the two B subunits. The index of dissimilarity for the heterologous E. coli enzyme compared with the homologous S. marcescens enzyme was 2.4, indicating extensive similarity of the two proteins at their surfaces. Comparative antiserum neutralization of B-subunit enzyme activity showed the E. coli enzyme to cross-react 85% as well as the S. marcescens enzyme. With regard to the biochemical and immunochemical parameters used in this study, the S. marcescens and E. coli B subunits were either identical or very similar. These findings support the idea that the trpB cistron of the trp operon is a relatively conserved gene in the Enterobacteriaceae.     

Comparison of the tryptophan synthetase alpha-subunits of several species of Enterobacteriaceae

Creighton, T. E. (Stanford University, Stanford), D. R. Helinski, R. L. Somerville, and C. Yanofsky. Comparison of the tryptophan synthetase alpha subunits of several species of Enterobacteriaceae. J. Bacteriol. 91:1819-1826. 1966.-The tryptophan synthetase alpha subunits of Escherichia coli K-12, E. coli B, Shigella dysenteriae, Salmonella typhimurium, and Aerobacter aerogenes have been purified and their structures compared. Each of these alpha subunits exhibits a sedimentation coefficient of about 2.7S. Peptide patterns of trypsin plus chymotrypsin digests of the alpha subunits have indicated that all of the alpha subunits have peptide regions in common. The patterns of E. coli K-12, E. coli B, and S. dysenteriae alpha subunits appear to be nearly identical, whereas the alpha subunits from S. typhimurium and A. aerogenes differ from those of E. coli and from each other. It has also been shown that the E. coli structural gene for the alpha subunit is translated identically in E. coli and S. typhimurium.     

Cloning and expression of the gene for diphtheria toxin and its subunits in Escherichia coli

The results of cloning Corynebacterium diphtheriae phi 984 tox gene and its A and B subunits in Escherichia coli are presented. Regulatory sequences of tox gene are capable to promote effective expression in E. coli cells. A set of recombinant plasmids has been obtained which can determine the synthesis of A and B individual subunits and are suitable for constructing immunotoxins by gene engineering. The diphtheria toxin of 62 kDa synthesized in E. coli has enzymatic activity and reacts with antitoxin sera. Some sites for E. coli proteases are present in tox-specific polypeptides.     

Nature of the free radical in ribonucleotide reductase from Escherichia coli.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The active site of the enzyme is made up from both subunits. Protein B2 contributes inter alia an organic free radical which gives a characteristic EPR signal. This radical was now located by isotope substitution experiments to the beta position of a tyrosine residue. The EPR spectrum of protein B2 from bacteria grown in a completely deuterated medium was drastically changed. The change was reversed by the addition of other protonated amino acids. The involvement in radical formation of the beta position of tyrosine was demonstrated from EPR spectra of protein B2 from bacteria grown in the presence of specifically deuterated tyrosine.     

Identification of Escherichia coli proteins cross-reacting with antibodies against region 2.2 peptide of RNA polymerase sigma subunit.

Antisera against a synthetic tetradecameric peptide with the sequence DLIQEGNIGLMKAV, which is present in region 2.2 of both sigma 70 and sigma 32 subunits of Escherichia coli RNA polymerase, cross-reacted with more than 10 E. coli proteins including these two sigma subunits. Four major species of these cross-reacting proteins (SCRPs) were purified. N-Terminal amino acid sequence analysis revealed that one of them (SCRP-27A) was an as yet unidentified protein while the other three (SCRP-34, SCRP-27B and SCRP-23) were thioredoxin reductase, ribosomal protein S2, and alkyl hydroperoxide reductase, respectively. Immunological competition experiments with various fragments of this sigma region 2.2 peptide indicated that the anti-sigma peptide serum contained at least three different species of antibodies. All the four SCRPs analyzed here reacted with an antibody against a C-terminus-proximal epitope.     

Synthesis of nitrate reductase components in chlorate-resistant mutants of Escherichia coli.

Specific antibody to purified nitrate reductase from Escherichia coli was used to identify enzyme components present in mutants which lack functional nitrate reductase. chlA and B mutants contained all three subunits present in the wild-type enzyme. Different peptides with a broad range of molecular weights could be precipitated from chlCmutants, and chlE mutants contained either slightly degraded enzyme subunits or no precipitable protein. No mutants produced significant amounts of cytoplasmic enzyme. The chlA and B loci are suggested to function in the synthesis and attachment of a molybdenum-containing factor. The chlC locus is suggested to be the structural gene for nitrate reductase subunit A and chlE is suggested to be involved in the synthesis of the cytochrome b1 apoprotein.