Physiochemical studies on achatininH, a novel sialic acid-binding lectin.

The plasmid pEAP31 contains an alkaliphilic-Bacillus penicillinase gene and a colicin E1 kil gene. Escherichia coli HB101 carrying pEAP31 grown at high temperature released outer-membrane proteins, lipopolysaccharide and phosphatidylethanolamine into the culture medium. Concurrently, penicillinase that had accumulated in the periplasm of the organism was released from the cells. Phospholipase A1-A2 in the outer membrane was not activated in the organism. The results suggest that the release of accumulated periplasmic penicillinase from the producer cells was caused by partial disruption of the outer membrane mediated by the Kil peptide.     

Construction of an excretion vector and extracellular production of human growth hormone from Escherichia coli

A new excretion vector, pEAP8, was constructed to develop an excretion system for Escherichia coli. This plasmid, derived from pEAP37, carried the weakly activated kil gene of plasmid pMB9 [Kobayashi et al., J. Bacteriol. 166 (1986) 728-732] and the penicillinase promoter and signal region of an alkalophilic Bacillus sp. to excrete foreign gene products. A gene for human growth hormone (hGH) was joined to this signal sequence through the HindIII site. The recombinant plasmid p8hGH1 thus constructed, was introduced into E. coli. The hybrid protein which was produced in E. coli carrying p8hGH1 was processed during transport through the inner membrane, with the mature hGH being excreted into the medium through the outer membrane which was made permeable by the action of the kil gene. The N-terminal amino acid sequence and the biological activity of the extracellular hGH were consistent with those of the authentic hGH.     

Nucleotide sequence and gene organization of ColE1 DNA

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).     

Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli

Factors necessary for the export process of colicin E1 across the cytoplasmic membrane of Escherichia coli were investigated. beta-Galactosidase activities from gene fusions between the colicin E1 and lacZ genes were recovered in the inner membrane fraction of E. coli when the region containing the internal signal-like sequence of colicin E1 [M. Yamada et al. (1982) Proc. Natl Acad. Sci. USA 79, 2827-2831] was present, but were found in the soluble fraction when the region was eliminated. The colicin E1 export was reduced upon insertion mutation in a gene that is located downstream from the colicin E1 gene in the same operon and responsible for mitomycin-C-induced killing of the host cell. A frame shift mutation of the colicin E1 plasmid was constructed to direct the protein which had lost the COOH-terminal 13 residues of original colicin E1 and was altered in 6 residues of the new COOH-terminal portion. The aberrant colicin E1 that was inducibly synthesized remained inside the cells. These results indicate that colicin E1 is exported with the aid of a product of the downstream gene and that the COOH-terminal portion is necessary for the export. The binding of colicin E1 to the cytoplasmic membrane through the internal signal-like sequence may be a step in the protein export process.     

Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death

Expression of the kil gene of the ColE1 plasmid in certain classes of Escherichia coli mutants (Kilr) resistant to kil-caused cell death brought about release of periplasmic enzymes and of colicin. Phospholipase A was present but was not activated by kil expression in any of the mutants. This indicates that in these mutants the various effects of kil gene expression have become dissociated.     

Expression of the cloned ColE1 kil gene in normal and Kilr Escherichia coli

The kil gene of the ColE1 plasmid was cloned under control of the lac promoter. Its expression under this promoter gave rise to the same pattern of bacterial cell damage and lethality as that which accompanies induction of the kil gene in the colicin operon by mitomycin C. This confirms that cell damage after induction is solely due to expression of kil and is independent of the cea or imm gene products. Escherichia coli derivatives resistant to the lethal effects of kil gene expression under either the normal or the lac promoter were isolated and found to fall into several classes, some of which were altered in sensitivity to agents that affect the bacterial envelope.     

Detection of large COOH-terminal domains processed from the precursor of Serratia marcescens serine protease in the outer membrane of Escherichia coli.

The Serratia marcescens serine protease gene encoding a 1,045-amino-acid precursor protein of 112 kDa directs excretion of the mature protease of ca. 58 kDa through the outer membrane of Escherichia coli. A typical signal peptide of 27 amino acids and a large COOH-terminal domain of the precursor are both functionally essential for the excretion of the mature protease into the medium. Sequence analysis of the fragment peptides of the mature protease as well as site-directed mutagenesis indicated that the COOH-terminus of the mature enzyme was Asp645. By using the polyclonal antibody against the 112-kDa precursor protein, not only the intact precursor but also two proteins, C-1 (40 kDa) and C-2 (38 kDa), corresponding to the processed COOH-terminal domains were detected in the insoluble fraction of E. coli cells. Further fractionation by sucrose density gradient centrifugation showed that C-1 and C-2 were localized in the outer membrane. The NH2-terminal residues of C-1 and C-2 were determined to be Ala702 and Phe717, respectively. All these data suggest that the precursor is cleaved at three positions, between Asp645-Ser646, Glu701-Ala702, and Gly716-Phe717, probably by the self-processing activity in the normal excretion pathway through the outer membrane.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.     

Plasmid-encoded regulation of colicin E1 gene expression.

A plasmid-encoded factor that regulates the expression of the colicin E1 gene was found in molecular cloning experiments. The 2,294-base-pair AvaII fragment of the colicin E1 plasmid (ColE1) carrying the colicin E1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin E1 synthesis as the original ColE1 plasmid. An operon fusion was constructed between the 204-bp fragment containing the colicin E1 promoter-operator and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida. The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant. These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor. Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent. Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP. The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid. Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function.     

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.     

Cloning and sequencing of the metallothioprotein beta-lactamase II gene of Bacillus cereus 569/H in Escherichia coli

The structural gene for beta-lactamase II (EC 3.5.2.6), a metallothioenzyme, from Bacillus cereus 569/H (constitutive for high production of the enzyme) was cloned in Escherichia coli, and the nucleotide sequence was determined. This is the first class B beta-lactamase whose primary structure has been reported. The amino acid sequence of the exoenzyme form, deduced from the DNA, indicates that beta-lactamase II, like other secreted proteins, is synthesized as a precursor with a 30-amino acid N-terminal signal peptide. The pre-beta-lactamase II (Mr, 28,060) is processed in E. coli and in B. cereus to a single mature protein (Mr, 24,932) which is totally secreted by B. cereus but in E. coli remains intracellular, probably in the periplasm. The expression of the gene in E. coli RR1 on the multicopy plasmid pRWHO12 was comparable to that in B. cereus, where it is presumably present as a single copy. The three histidine residues that are involved (along with the sole cysteine of the mature protein) in Zn(II) binding and hence in enzymatic activity against beta-lactams were identified. These findings will help to define the secondary structure, mechanism of action, and evolutionary lineage of B. cereus beta-lactamase II and other class B beta-lactamases.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterisation of the colicin E2 operon and identification of its products

Summary The DNA sequence of the entire colicin E2 operon was determined. The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells. A potential LexA binding site is located immediately upstream from ceaB, and a rho-independent terminator structure is located immediately downstream from celB. A comparison of the predicted amino acid sequences of colicin E2 and cloacin DF13 revealed extensive stretches of homology. These colicins have different modes of action and recognise different cell surface receptors; the two major regions of heterology at the carboxy terminus, and in the carboxy-terminal end of the central region probably correspond to the catalytic and receptor-recognition domains, respectively. Sequence homologies between colicins E2, A and E1 were less striking, and the colicin E2 immunity protein was not found to share extensive homology with the colicin E3 or cloacin DF13 immunity proteins. The lysis proteins of the ColE2, ColE1 and CloDF13 plasmids are almost identical except in the aminoterminal regions, which themselves have overall similarity with lipoprotein signal peptides. Processing of the ColE2 prolysis protein to the mature form was prevented by globomycin, a specific inhibitor of the lipoprotein signal peptidase. The mature ColE2 lysis protein was located in the cell envelope. The results are discussed in terms of the functional organisation of the colicin operons and the colicin proteins, and the way in which colicins are released from producing cells.     

Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9.

Most of the cloned penicillinase from alkalophilic Bacillus sp. strain 170 and alkaline phosphatase were released into the culture medium by Escherichia coli strains bearing plasmid pEAP1 or pEAP2 (T. Kudo, C. Kato, and K. Horikoshi, J. Bacteriol. 156:949-951, 1983). We analyzed the basis for excretion of periplasmic enzymes in the cells bearing these plasmids. Several experiments such as subcloning, insertion of a chloramphenicol acetyltransferase cartridge, and DNA sequencing were done. A dormant kil gene in plasmid pMB9 was expressed by a promoter of the inserted DNA fragment of alkalophilic Bacillus sp. strain 170, and as a result, the outer membrane of E. coli became permeable, allowing the proteins to be excreted without cell lysis.     

Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein.

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [35S]cysteine or [3H]lysine. This 3-kDa protein was acylated, as shown by [2-3H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants.     

Secondary structure of the pore-forming colicin A and its C-terminal fragment. Experimental fact and structure prediction

Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A (Mr 60 000), and a C-terminal bromelain fragment (Mr 20 000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly alpha-helical with an enrichment of the alpha-helical content in the C-terminal domain carrying the ionophoric activity. The non-negligible beta-sheet structure in the C-terminal domain is unstable and is easily transformed into alpha-helix upon decreasing the polarity of the solvent. No evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane alpha-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic movement calculations. Similar calculations on the C-terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.     

Plasmid ColE3 specifies a lysis protein.

Tn5 insertion mutations in plasmid ColE3 were isolated and characterized. Several of the mutants synthesized normal amounts of active colicin E3 but, unlike wild-type colicinogenic cells, did not release measurable amounts of colicin into the culture medium. Cells bearing the mutant plasmids were immune to exogenous colicin E3 at about the same level as wild-type colicinogenic cells. All of these lysis mutants mapped near, but outside of, the structural genes for colicin E3 and immunity protein. Cells carrying the insertion mutations which did not release colicin E3 into the medium were not killed by UV exposure at levels that killed cells bearing wild-type plasmids. The protein specified by the lysis gene was identified in minicells and in mitomycin C-induced cells. A small protein, with a molecular weight between 6,000 and 7,000, was found in cells which released colicin into the medium, but not in mutant cells that did not release colicin. Two mutants with insertions within the structural gene for colicin E3 were also characterized. They produced no colicin activity, but both synthesized a peptide consistent with their map position near the middle of the colicin gene. These two insertion mutants were also phenotypically lysis mutants--they were not killed by UV doses lethal to wild-type colicinogenic cells and they did not synthesize the small putative lysis protein. Therefore, the lysis gene is probably in the same operon as the structural gene for colicin E3.