Discadenine Distribution in Cellular Slime Molds and Its Inhibitory Activity on Spore Germination

We constructed a hybrid plasmid to allow controlled expression of a gene and the subsequent secretion into the culture medium of the gene product in Escherichia coli. This was achieved by the use of five trp promoter-operator regions in tandem followed by the DNA fragment coding for the signal peptide and the N-terminus of the OmpF protein, and the trpR gene coding for the Trp repressor. Multiplication of the trp promoter-operator appreciably enhanced expression of the gene that followed. A single copy of the trpR gene on the chromosome was insufficient for controlling the enhanced expression. The expression was, however, completely controlled when the trpR gene was cloned onto the same plasmid. When the multiple trp promoter-operator was followed by the DNA fragment coding for the signal peptide and the N-terminus of the OmpF protein that was further followed by the gene for human β-endorphin, a β-endorphin-containing polypeptide was synthesized under the complete control of the trp promoter-operator, and secreted to the culture medium across both the cytoplasmic membrane and the outer membrane. Controlled expression of a foreign gene and subsequent secretion into the medium of the product were thus achieved.     

Excretion of human beta-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli

Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage lambda to construct E. coli MBEL-BL101. For the excretion of human beta-endorphin as a model protein, the beta-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-beta-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-beta-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield beta-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of beta-endorphin was obtained.     

Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin.

Recently, we have identified a novel topogenic sequence at the C terminus of Escherichia coli haemolysin (HlyA) which is essential for its efficient secretion into the medium. This discovery has introduced the possibility of using this secretion system for the release of chimeric proteins from E. coli directly into the medium. We have now successfully fused this C-terminal signal to a hybrid protein containing a few residues of beta-galactosidase and the majority of the E. coli outer membrane porin OmpF lacking its own N-terminal signal sequence. We find that this chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium. In addition, we have further localized the HlyA secretion signal to the final 113 amino acids of the C terminus. In fact, a specific secretion signal appears to reside at least in part within the last 27 amino acids of HlyA.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

Specialized transducing bacteriophage lambda carrying the structural gene for a major outer membrane matrix protein of Escherichia coli K-12.

A specialized transducing phage lambda carrying the structural gene for the OmpF protein, an outer membrane matrix protein, was isolated. The phage carries the 20.5--21-min region of the Escherichia coli K-12 chromosome and carries asnS, ompF, and aspC genes.     

Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1 beta

Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment.     

Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

The secretion of the Klebsiella oxytoca cell surface lipoprotein pullulanase involves translocation across the cytoplasmic and outer membranes of the Gram-negative bacterial cell envelope. A variant of pullulanase was created by fusing the signal peptide-encoding 5' region of the Escherichia coli gene for periplasmic MalE protein to the 3' end of the pulA gene encoding almost the entire mature part of pullulanase. When produced in E. coli carrying the malE-pulA gene fusion on a high copy number plasmid and the complete set of genes specifically required for pullulanase secretion on a second plasmid, the hybrid protein differed from wild-type pullulanase as follows: (i) it was not fatty-acylated; (ii) it was apparently processed by LepB signal peptidase rather than by LspA lipoprotein signal peptidase; (iii) it was released into the periplasm and was only slowly transported across the outer membrane, and (iv) it was released directly into the medium rather than via the usual surface-anchored intermediate. The hybrid protein was secreted more rapidly when malE-pulA was expressed from a low copy number plasmid. The two steps in the secretion pathway could be totally uncoupled by expressing first the malE-pulA gene fusion and then the cognate secretion genes. These results show that fatty-acylation of wild-type PulA is not essential for secretion but may improve its efficiency when large amounts of the protein are produced, that the two steps in secretion can occur quite independently and that the periplasmic intermediate can persist for long periods under certain circumstances.     

Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli

The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.     

Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium.

The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.     

Protein secretion controlled by a synthetic gene in Escherichia coli

The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain.     

Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: Fusions with secretable peptides

The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.     

Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein)

A new secretion vector, pEAP84 which contained a unique restriction site (BglII) at the 3' end of the penicillinase gene to produce a fused protein, and the Ex-kil region to make the outer membrane permeable, was constructed from pEAP82. A recombinant plasmid p84h06, which contained a synthetic gene for human calcitonin with a cyanogen bromide cleavage site at the junction site of the fused protein, was constructed and introduced into Escherichia coli. The hybrid protein produced in E. coli carrying p84h06 was secreted into the culture medium. The amino acid composition of this product was consistent with that deduced from the DNA sequence. Mature calcitonin was obtained following cyanogen bromide cleavage of the fused protein.     

A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene

The DNA sequence of the ompC gene which encodes one of the outer membrane porins has been determined. The gene appears to encode a secretory precursor of OmpC protein consisting of a total of 367 amino acid residues with a signal peptide of 21 amino acid residues at its NH2-terminal end. The 5' end noncoding region including the promoter of the ompC gene is extremely [A-T]-rich, and the codon usage in the ompC gene is unusual as are those in genes for other abundant outer membrane proteins. The promoter sequence of the ompC gene was compared with that of the ompF gene, both of which are controlled by the osmoregulatory operon, ompB. The deduced amino acid sequence of the OmpC protein showed extensive homology with that of the other porins (OmpF and PhoE proteins). The homology in the primary amino acid sequences, as well as the coding DNA sequences among the porins, indicates that the structural genes for the three porins evolved from a common ancestral gene. Comparison of the amino acid sequences among the OmpC, OmpF, and PhoE porins will be discussed with regard to structure and function.     

Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli

The OmpR protein is a positive regulator involved in osmoregulatory expression of the ompF and ompC genes, which respectively code for major outer membrane proteins OmpF and OmpC of Escherichia coli. The OmpR protein has been purified to homogeneity from an overproducing strain harboring an ompR gene-carrying plasmid. Throughout the purification the OmpR protein behaved as a single entity. The molecular weight determined on sodium dodecyl sulfate-polyacrylamide gel, the total amino acid composition, and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the ompR gene. Molecular weight determination and cross-linking study on the native protein revealed that the purified protein exists as a monomer. The purified OmpR protein was specifically bound to the promoter regions of the ompC and ompF genes. Experiments with a series of upstream deletions of the ompC and ompF promoters revealed that the region upstream from the -35 region was indispensable for OmpR binding to both the ompC and the ompF promoters. Although it has been proposed that depending on the medium osmolarity the OmpR protein may exist in two alternative structures, which respectively regulate functioning of the ompC and the ompF promoters, the purified OmpR protein appeared to be homogeneous and interacted with both promoters to the same extent.     

Trimeric structure and localization of the major lipoprotein in the cell surface of Escherichia coli

A hybrid gene consisting of the ompF promoter, the coding regions for the signal peptide, and the Ala-Glu residue of the OmpF NH2 terminus and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal cysteine residue was constructed. Escherichia coli carrying the cloned gene produced the predicted hybrid protein that is the same as the major lipoprotein except that the diacyl glycerylcysteine residue at the NH2 terminus is replaced by the Ala-Glu residue. The hybrid protein was localized in the periplasmic space as a trimer with a noncovalent interaction in addition to the previously known covalent interaction with the peptidoglycan. These results strongly indicate that the major lipoprotein exists as a trimer in the periplasmic space with covalent and noncovalent interactions with the peptidoglycan layer through the protein domain on one side and with the hydrophobic interaction with the outer membrane through the lipid domain on the other side. The trimeric structure of the lipoprotein was directly demonstrated by the chemical cross-linking of the native lipoprotein with both cleavable and uncleavable reagents. The cross-linking study also revealed interaction between the lipoprotein and the OmpA protein, a major outer membrane protein.