Vibrio cholerae enterotoxin genes: nucleotide sequence analysis of DNA encoding ADP-ribosyltransferase.

The nucleotide sequence of the DNA encoding the ADP-ribosyltransferase (A1) fragment of cholera enterotoxin was determined. A putative precursor of the A1 peptide contains an 18-amino acid leader peptide, and the mature A1 peptide contains 194 amino acids. The primary structure of the A1 fragment from cholera enterotoxin is more related to that from a human enterotoxigenic Escherichia coli than to that from a porcine enterotoxigenic E. coli.     

The nucleotide sequence of the promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of Escherichia coli

The promoter and the amino-terminal region of phoA, the structural gene for alkaline phosphatase of Escherichia coli K12, was cloned by using a promoter cloning vector pMC1403. The nucleotide sequence of the cloned fragment has been determined. A sequence encoding the amino-terminal portion of mature alkaline phosphatase is found and it is preceded by a sequence encoding the signal peptide. The signal peptide consists of 21 amino acids; Met-Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala. The translation initiation codon is GUG, which is preceded by the Shine-Dalgarno sequence GGAG. Upstream to these sequences, there is a typical procaryotic promoter. TATAGTC for the Pribnow box. Around the Pribnow box, there are several dyad symmetrical sequences, which may probably be concerned with the regulation of this gene.     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

人产肠毒素大肠杆菌ST、LT—B肠毒素基因融合的研究

将人产肠毒素大肠杆菌(ETEC),编码耐热肠毒素(ST)的基因片段与编码不耐热肠毒素B亚基(LT-B)的基因进行融合,并在此基础上进行不同数目ST基因的串联,ELISA检测融合基因表达蛋白产物观察到ST与LT-B之间存在着相互影响。ST的检测滴度随基因串联个数增加而逐渐升高,而LT的ELISA滴度则减弱。说明了ST可以通过基因串联提高表达产物抗原活性,这为产肠毒素大肠杆菌多价疫苗的研制提供了重要的研究基础。     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

人毒素原性大肠杆菌肠毒素ST、LT-B基因的融合

将毒素原性大肠杆菌(ETEC)编码耐热肠毒素(ST)的基因片段与编码热敏肠毒素B亚基(LT—B)的基因进行融合,并在此基础上进行不同数目ST基因的串联。ELISA检测融合基因表达蛋白产物,观察到ST与LT-B之间存在着相互影响。ST的检测滴度随基因串联个数增加而逐渐升高,而LT的ELISA滴度则减弱。本研究说明ST可以通过基因串联提高表达产物抗原活性。这为毒素原性大肠杆菌多价疫苗的研制提供了重要的研究基础。     

Molecular cloning and sequencing of a pectate lyase gene from Yersinia pseudotuberculosis.

A pectate lyase gene (pelY) from Yersinia pseudotuberculosis was cloned in Escherichia coli DH-5 alpha. The gene was expressed in either orientation in pUC plasmids, indicating that the insert DNA carried a Y. pseudotuberculosis promoter which functioned in E. coli. However, when cloned in the orientation which placed the coding region downstream of the vector lac promoter, expression of pelY was nine times higher than it was in the opposite orientation and the growth of E. coli cells was inhibited. Nucleotide sequence analysis of the pelY gene disclosed an open reading frame of 1,623 base pairs (PLY). The peptide sequence at the amino-terminal end of the protein contains a typical signal peptide sequence, consistent with the observation that the mature PLY protein accumulated largely in the periplasmic space of E. coli. The pI of PLY produced in E. coli cells was 4.5, and its activity was inhibited 90% or more by EDTA. The enzyme macerated cucumber tissue about 1,000 times less efficiently than did PLe from Erwinia chrysanthemi EC16. The pelY gene has no sequence similarity to the pel genes thus far sequenced from Erwinia spp.     

Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi

Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low-molecular-weight, heat-stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellular protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escherichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 condons. The N-terminal amino acid sequence of the purified inhibitor was also determined. It indicated the existence of an amino-terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli.     

Signal peptide determinants of SecA binding and stimulation of ATPase activity

A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.     

The nucleotide sequence of the gene encoding the K99 subunit of enterotoxigenic Escherichia coli

Abstract The nucleotide sequence of the gene encoding the K99 fimbrial subunit of enterotoxigenic Escherichia coli was determined. It appeared that the subunit is composed of 159 amino acid residues preceded by a N-terminal signal sequence of 22 amino acid residues. The secondary structure of the mature K99 polypeptide and the location of potential antigenic determinants were predicted. A comparison was made between the amino acid sequence of the K99 subunit and the subunits of other fimbrial adhesins.     

Molecular cloning and sequence analysis of human placental alkaline phosphatase

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.     

Cloning and nucleotide sequence of a gene for Actinomyces naeslundii WVU45 type 2 fimbriae.

A genomic library of Actinomyces naeslundii WVU45 DNA in Escherichia coli was screened for antigen expression with rabbit antibody against A. naeslundii fimbriae. Western blotting (immunoblotting) of one recombinant clone carrying a 13.8-kilobase-pair insert revealed a 59-kilodalton (kDa) immunoreactive protein. A protein of similar electrophoretic mobility was detected from the isolated fimbrial antigen. Expression of the 59-kDa cloned protein in E. coli was directed by a promoter from the insert. The DNA sequence of the subunit gene was determined, and an open reading frame of 1,605 nucleotides was identified which was preceded by a putative ribosome-binding site and followed by two inverted repeats of 14 and 17 nucleotides, respectively. The reading frame encoded a protein of 534 amino acids (calculated molecular weight, 57,074), and the N-terminal sequence resembled that of a signal peptide. The presence of a 32-amino-acid signal peptide was indicated by amino-terminal sequencing of the fimbriae from A. naeslundii. The sequence, as determined by Edman degradation, was identical to that deduced from the DNA sequence beginning at predicted residue 33 of the latter sequence. Moreover, the amino acid composition of the predicted mature protein was similar to that of the isolated fimbriae from A. naeslundii. Thus, the cloned gene encodes a subunit of A. naeslundii fimbriae.     

5′端非翻译区对LT-B基因表达的影响

mRNA5＇端非翻译区的不同结构可影响基因表达,为了改善编码人毒素源性大肠杆菌热敏感肠毒素B亚单位的LT-B基因的表达水平,我们把该基因置于pBV220载体的P_RP_L串联启动子下游,构建了带有不同核苷酸组成的5＇端非翻译区的重组体。这些重组体分别在大肠杆菌HB101和DH5α中表达。结果表明,起始密码前有两个连续串联SD序列的LT-B基因的表达水平低于只有单个SD序列下的表达水平,而翻译偶联可使表达改善;用不同的SD序列LT-B基因的表达水平也有所不同,用基因本身SD序列可能要比用pBV220P_L启动子下游的SD序列好;在只含单个LT-B基因SD序列的重组体中,5＇端非翻译区序列的长短对LT-B基因表达没有什么影响;重组体在HB101中的表达水平高于在DH5α中的表达水平。     

Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli

Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli. The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain. We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins. We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein. Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized. This signal peptide was previously shown to translocate lipoprotein efficiently. Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase. Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase. These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein. On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed.     

Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB

We have determined the nucleotide sequence of the Escherichia coli fepA gene, which codes for the outer membrane receptor for ferrienterochelin and colicins B and D. The predicted FepA polypeptide has a molecular weight of 79,908 and consists of 723 amino acids. A 22-amino acid leader or signal peptide preceded the mature protein. With respect to overall composition, hydropathy, net charge and distribution of nonpolar segments, the FepA polypeptide was typical of other E. coli outer membrane proteins, except that FepA contained 2 cysteine residues. Comparison of the deduced amino acid sequence of FepA with that of three other TonB-dependent receptors (BtuB, FhuA, and IutA) revealed only a few regions of sequence homology; one of these included the amino-termini. An amino acid substitution within the conserved amino-terminal region of BtuB resulted in production of a receptor that had normal binding functions but was incapable of energy-dependent transport of vitamin B12. This result suggests that the amino-terminal end of these four polypeptides is involved in interaction with the TonB protein or another step of energy transduction. Three other regions of homology were shared among the four proteins: one near residues 50 to 70, another at about residue 100 to 140, and the last between 20 and 40 amino acid residues from the carboxyl terminus. The function of these three regions remains speculative.     

Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R₁ kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R₁ kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R₃ seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R₃ seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens.