Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Nucleotide sequence of the staphylococcal enterotoxin C3 gene sequence comparison of all three type C staphylococcal enterotoxins

Summary The structural gene entC3, which encodes staphylococcal enterotoxin C3 was cloned from the genome of Staphylococcus aureus FRI-913 and sequenced. The primary amino acid sequence of the toxin was deduced from the nucleotide sequence data. entC3 contains 801 by and encodes a precursor protein of 266 amino acids. Glutamic acid was found to be the N-terminus of mature enterotoxin C3. Thus, the first 27 residues of the toxin precursor comprise the signal peptide, and the mature toxin contains 239 amino acids with a molecular weight of 27 563 daltons. Enterotoxin C3 differs from enterotoxin C2 by four amino acids and from enterotoxin C1 by nine residues. The 167 C-terminal residues of the three toxins are identical, except for one conservative amino acid substitution in enterotoxin C3. The degree of immunological relatedness among the three Type C enterotoxins is proportional to their molecular relatedness. This study also provides evidence that the N-termini of Type C enterotoxins determine subtype-specific antigenic epitopes, while more conserved C-terminal regions determine biological properties and cross-reactive antigenic epitopes shared with other pyrogenic toxins.     

Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc

The polymerase chain reaction was used to amplify the staphylococcal enterotoxin B and C genes (entB and entC1) and the staphylococcal nuclease gene (nuc). Two sets of primers ("nested primers") were found to be necessary for the detection of low copy numbers of purified DNA in diluent. These allowed detection of ca. 1 fg of purified target DNA, while 100 pg was required before detection of entB, entC1, and nuc with single primer pairs was possible. With nested primers, enterotoxigenic Staphylococcus aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of ca. 10(5) CFU ml-1 within 8 h. No cross-reaction was observed between the highly homologous entB and entC1 genes. The method showed total specificity for entC1 when tested against a wide variety of other bacteria.     

Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc.

The polymerase chain reaction was used to amplify the staphylococcal enterotoxin B and C genes (entB and entC1) and the staphylococcal nuclease gene (nuc). Two sets of primers ("nested primers") were found to be necessary for the detection of low copy numbers of purified DNA in diluent. These allowed detection of ca. 1 fg of purified target DNA, while 100 pg was required before detection of entB, entC1, and nuc with single primer pairs was possible. With nested primers, enterotoxigenic Staphylococcus aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of ca. 10(5) CFU ml-1 within 8 h. No cross-reaction was observed between the highly homologous entB and entC1 genes. The method showed total specificity for entC1 when tested against a wide variety of other bacteria.     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.     

Nucleotide sequence of the type A staphylococcal enterotoxin gene.

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.     

The complete amino acid sequence of staphylococcal enterotoxin C1

This report presents the complete amino acid sequence of staphylococcal enterotoxin C1. It has a total of 239 amino acids and a calculated Mr = 27,500. Reaction of the native toxin with trypsin produced five peptides, designated A through E. Their structures were determined after further fragmentation with cyanogen bromide, trypsin, and chymotrypsin. Overlap peptides were prepared by cleavage at the two half-cystine residues, and by the reaction of enterotoxin C1 with hydroxylamine. The latter procedure was employed when it was found that one of the three asparaginyl-glycine loci in the toxin is refractory to direct sequencing. The sequence is compared to staphylococcal enterotoxin B. Extensive homology is found, particularly in the carboxyl-terminal region. However, the segment spanned by the disulfide bond in enterotoxin C1 is three residues shorter than the corresponding segment in the B variant.     

带芒草属低分子量谷蛋白基因的克隆及序列分析

在普通小麦中获得了大量的低分子量谷蛋白基因序列, 而在小麦近缘属物种中获得的同源基因则比较少, 导致对麦类低分子量谷蛋白基因家族成员间的关系还不清楚。因此, 进行近缘属物种低分子量谷蛋白基因的研究是非常必要的。此研究通过特殊设计的1对引物, 以小麦近缘属带芒草物种的基因组DNA为模板, 经过PCR和克隆, 从中得到了一条核苷酸序列长度为1 035 bp, 推测的氨基酸序列为343个氨基酸残基的低分子量谷蛋白基因, 该基因序列具有小麦低分子量谷蛋白基因的典型特征, 包括21个氨基酸残基的信号肽、13个氨基酸的N-端和由可重复的短肽单元组成的重复区以及1个C末端。序列比对结果揭示了来自带芒草的低分子量谷蛋白基因与小麦同源基因的差异及相互关系。此研究结果对从带芒草属以及其他小麦近缘属物种中分离未知低分子量谷蛋白基因有参考价值和借鉴意义。     

Amino acid sequence of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis

The complete nucleotide sequence of the gene encoding the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequence are presented. The nucleotide sequence of the gene reveals the existence of a typical bacterial signal peptide of 20 amino acid residues which is not found in the mature cytochrome subunit. The gene encoding the cytochrome subunit is preceded by the gene encoding the M subunit. Both genes overlap by 1 bp. The mature cytochrome subunit consists of 336 amino acid residues; 73% of its amino acid sequence was confirmed by protein sequencing work. The mol. wt of the cytochrome subunit including the covalently bound fatty acids and the bound heme groups is 40 500. The internal sequence homology is low, despite the symmetric structure of the cytochrome subunit previously shown by X-ray crystallographic analysis of the intact photosynthetic reaction centre. Sequence homologies to other cytochromes were not found.     

Complete amino acid sequence of staphylococcal enterotoxin A

The amino acid sequence of staphylococcal enterotoxin A is presented. Staphylococcal enterotoxin A is a single-chain polypeptide which consists of 233 amino acid residues with a molecular weight of 27,078 and has the amino acid composition Cys2, Asp17, Asn19, Thr16, Ser13, Glu15, Gln12, Pro4, Gly15, Ala7, Val13, Met2, Ile10, Leu23, Tyr18, Phe8, His6, Lys24, Arg7, Trp2, with serine as both amino- and carboxyl-terminal amino acids. Automated sequence analysis of intact enterotoxin A, as well as characterization of the peptides obtained from cyanogen bromide treatment and trypsin and chymotrypsin digestion, led to the elucidation of the complete primary structure of this protein. Less structural homology is observed among staphylococcal enterotoxins A, B (Huang, I-Y., and Bergdoll, M. S. (1970) J. Biol. Chem. 245, 3518-3525), and C1 (Schmidt, J. J., and Spero, L. (1983) J. Biol. Chem. 258, 6300-6306) than that seen between enterotoxins B and C1.     

Primary structure of heat-labile enterotoxin produced by Escherichia coli pathogenic for humans

Heat-labile enterotoxin of Escherichia coli pathogenic for humans (LTh) or for piglets (LTp) and Vibrio cholerae enterotoxin (CT) are structurally and functionally similar toxins. We have determined the complete nucleotide sequence of the toxA gene which encodes the subunit A of LTh (LTh A). The deduced amino acid sequence consists of 258 residues including a signal peptide of 18 residues. According to the previously completed LTh B sequence (103 residues), the predicted holotoxin (1A5B) of LTh comprises 755 residues and has Mr = 87,866. With respect to LTh A and LTh B, secondary structures, local hydrophilicity, and sites for antigenic determinants were predicted. Both codon usage and G + C content of the toxA gene and the LTh B gene (toxB) were markedly different from those observed with several E. coli chromosomal genes. Its relatively low G + C content was rather close to that of the V. cholerae chromosome. Although the toxA gene shares a common ancestor with the LTp A gene (eltA), the two genes are apparently distinguishable from each other in their sequences. Like LTh B reported previously, the predicted sequence of the catalytic fragment LTh A1 also showed more homology to that of CT A1 than did that of LTp A1. In contrast, unique sequences were found in LTh A2.     

DNA sequencing of the eta gene coding for staphylococcal exfoliative toxin serotype A

We report the nucleotide sequence of a 1.45 kb segment containing the eta gene, coding for staphylococcal exfoliative toxin A (ETA), isolated from the recombinant plasmid pETA-J3. The coding region of 840 bp specified a polypeptide of 280 amino acid residues which included a putative 38 residue signal sequence. The amino acid composition deduced from the structural gene was in agreement with the results of peptide analysis of the ETA molecule reported by others. The sequence of the 35 N-terminal amino acid residues of ETA derived from Staphylococcus aureus strain ZM was also consistent with that deduced from the DNA sequencing.     

Studies on the functional site on staphylococcal enterotoxin A responsible for production of murine gamma interferon

To identify the functional region(s) associated with induction of gamma interferon on the staphylococcal enterotoxin A molecule, native staphylococcal enterotoxin A molecules and 12 various synthetic peptides corresponding to different regions of entire staphylococcal enterotoxin A were compared to induce gamma interferon production in murine spleen cells. The native staphylococcal enterotoxin A molecule induced gamma interferon production, whereas all of the 12 synthetic peptides did not. Pre-treatment of the murine spleen cells with synthetic peptide A-9 (corresponding to amino acid residues 161-180) significantly inhibited the staphylococcal enterotoxin A-induced gamma interferon production, whereas those with other synthetic peptides did not. When native staphylococcal enterotoxin A was pre-treated with either anti-staphylococcal enterotoxin A serum or anti-peptide sera, anti-staphylococcal enterotoxin A serum and antisera to peptides A-1 (1-20), A-7 (121-140), A-8 (141-160), A-9 (161-180) and A-10 (181-200) inhibited the staphylococcal enterotoxin A-induced gamma interferon production. From these findings, the amino acid residues 161-180 on the staphylococcal enterotoxin A molecule may be an essential region for murine gamma interferon production. Furthermore, the neutralizing epitopes may be also located on regions of amino acid residues 1-20, 121-140, 141-160 and 181-200 on the staphylococcal enterotoxin A molecule.     

Biosynthesis of the Iron-Transport Compound Enterochelin: Mutants of Escherichia coli Unable to Synthesize 2,3-Dihydroxybenzoate

Mutants of Escherichia coli K-12 blocked in each of the three enzymatic reactions between chorismate and 2,3-dihydroxybenzoate, in the pathway leading to the iron-sequestering compound enterochelin, have been isolated and biochemically characterized. The three genes concerned (designated entA, entB and entC) have been shown to be clustered on the chromosome between purE and gal and to be located near minute 14 by cotransduction with the purE, lip, and fep genes. entA, entB, and entC were shown to be the structural genes for 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, 2,3-dihydro-2,3-dihydroxybenzoate synthetase, and isochorismate synthetase, respectively.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

The in vitro conversion of chorismate to isochorismate catalyzed by the Escherichia coli entC gene product. Evidence that EntA does not contribute to isochorismate synthase activity

The entC and entA genes, coding for the enzymes isochorismate synthase and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, respectively, were subcloned behind the T7 promoter in the expression plasmid pGEM3Z. Their protein products were overproduced and partially purified for in vitro analysis of the conversion of chorismate to isochorismate. Whereas previous genetic experiments suggested that the EntA enzyme has a role in this conversion, this study clearly indicates that EntC alone catalyzes the reaction. Addition of EntA had no effect on isochorismate synthase activity. As a result, the mutation (previously designated entC401) in strain AN191 was characterized by nucleotide sequence analysis. The lesion is a single base substitution in the entA gene, resulting in a glutamic acid-for-glycine substitution at the penultimate amino acid (residue 247) of the EntA enzyme. The mutant protein was partially purified and shown to be devoid of 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase activity, whereas the entC gene product from strain AN191 exhibited normal isochorismate synthase function. These results conflict with the earlier characterization of the entC401 mutation in a different genetic background. The data presented herein establish that the EntA protein does not contribute to isochorismate synthase activity and that the mutant strain that led to this suggestion harbors a defective allele of entA rather than entC.     

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.