Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin E1.

The region of the colicin E1 polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin E1 against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical modification showed that the two histidine residues (His 427 and His 440) and the single cysteine residue (Cys 505) present in the COOH-terminal peptide were not necessary for the colicin-immunity protein interaction. The immunity protein was localized in the cytoplasmic membrane fraction, consistent with previous work of others on the colicin Ia immunity protein and the prediction from the immunity protein amino acid sequence that it is a hydrophobic protein. The distribution of hydrophobic residues along the immunity polypeptide was calculated.     

Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate

The complete nucleotide sequence of the structural gene for colicin A has been established. This sequence consists of 1776 base-pairs. According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989. The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured. The rest of the molecule (residues 186 to 592) is very rich in alpha-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A. A similar region has been found in colicin E1, which has the same mode of action as colicin A. Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions. The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.     

Electroinsertion and activation of the C-terminal domain of colicin A, a voltage gated bacterial toxin, into mammalian cell membranes

The C-terminal fragment of colicin, a protein that is highly soluble in aqueous solution, is spontaneously and irreversibly inserted into the membranes of mammalian cells, which are locally permeabilized by a transmembrane voltage increase. Insertion is detected by immunodetection. This is obtained by mixing the protein with electropermeabilized cells. The same result is observed by pulsing the colicin/cell mixture. Electroinsertion is therefore obtained for the first time with a multi-fragment spanning protein. The cell viability is not affected beyond the effect of electropermeabilization. A train of low voltage repetitive transmembrane modulation, which cannot trigger membrane permeabilization, is applied a day after the electroinsertion. This induces no effect on unmodified cells but triggers the lysis of cells in which colicin has been inserted by the first electropulsation. The low-level electrical treatment is high enough to trigger the voltage gated opening of colicin and to induce the associated toxicity. A transmembrane configuration of colicin is therefore obtained by electroinsertion. The toxic effect of their voltage gating is only obtained when a critical number of voltage gated channels are activated.     

Purification of a small receptor-binding peptide from the central region of the colicin E1 molecule

An Mr = 16,000 receptor-binding fragment of colicin E1 has been obtained by cyanogen bromide digestion of colicin E1. The purified 16-kDa fragment shows binding properties similar to those of an Mr = 38,000 colicin E1 receptor-binding fragment generated by thermolysin treatment. Treatment of the 38-kDa fragment with cyanogen bromide also yields the 16-kDa fragment. By comparing the NH2-terminal amino acid sequence of the 16-kDa fragment with the known colicin E1 sequence, the receptor-binding fragment can be shown to occupy the central region of the colicin molecule, extending from residue 231 to 370. It is inferred that the 16-kDa fragment binds efficiently to the colicin receptor because it is able to protect sensitive cells against the lethal effects of colicins E1 and E2 and, when pre-adsorbed to the cell, to physically displace colicin E1. Unlike the 38-kDa receptor-binding fragment, the 16-kDa fragment was found to be devoid of channel-forming ability previously shown to be associated with the COOH-terminal region of the colicin E1 polypeptide.     

A colicin A fragment containing the receptor binding domain can be directed to the periplasmic space in E. coli through gene fusion

The central region of the colicin A polypeptide chain has been fused to the N-terminal part of beta-lactamase through genetic recombination. This region comprising amino acid residues 70-335 confers on the hybrid protein the ability to protect sensitive cells from the lethal action of colicin A. Although colicin A belongs to the cytoplasmic compartment of E. coli, export of the hybrid protein to the periplasmic space was promoted by the signal peptide of beta-lactamase.     

Nucleotide sequences from the colicin E8 operon: homology with plasmid ColE2-P9

The primary structures of the immunity (Imm) and lysis (Lys) proteins, and the C-terminal 205 amino acid residues of colicin E8 were deduced from nucleotide sequencing of the 1,265 bp ClaI-PvuI DNA fragment of plasmid ColE8-J. The gene order is col-imm-lys confirming previous genetic data. A comparison of the colicin E8 peptide sequence with the available colicin E2-P9 sequence shows an identical receptor-binding domain but 20 amino acid replacements and a clustering of synonymous codon usage in the nuclease-active region. Sequence homology of the two colicins indicates that they are descended from a common ancestral gene and that colicin E8, like colicin E2, may also function as a DNA endonuclease. The native ColE8 imm (resident copy) is 258 bp long and is predicted to encode an acidic protein of 9,604 mol. wt. The six amino acid replacements between the resident imm and the previously reported non-resident copy of the ColE8 imm ([E8 imm]) found in the ribonuclease-producing ColE3-CA38 plasmid offer an explanation for the incomplete protection conferred by [E8 Imm] to exogenously added colicin E8. Except for one nucleotide and amino acid change in the putative signal peptide sequence, the ColE8 lys structure is identical to that present in ColE2-P9 and ColE3-CA38.     

The structure of a naturally occurring 10K polypeptide derived from the amino terminus of bovine thyroglobulin

A combination of data derived from peptide sequencing and nucleic acid sequencing of cloned cDNA fragments has been used to define the complete amino acid sequence of a 10,000 M.W., thyroxine containing polypeptide derived from bovine thyroglobulin. This fragment, TG-F, which was obtained following reduction and alkylation, has been placed at the amino terminus of the parent protein with hormone located at residue 5 in the primary sequence of the thyroglobulin molecule. The carboxyl terminal sequence of this fragment -Cys-Gln-Leu-Gln is found on the N-terminal side of a lys residue, suggesting that the peptide bond cleavage which occurs to produce this 80 residue fragment from the parent (330K) thyroglobulin chain is a gln-lys. In addition, the amino acid sequence of this 10K fragment contains: No sequence which would be a substrate for glycosylation and no carbohydrate. Several repeated homologous amino acid sequences. A striking number of beta-bends predicted from Chou-Fasman analyses, particularly near its carboxyl terminus.     

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.     

Extracellular release of colicin A is non-specific.

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.     

Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide

A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential.     

Structural and functional properties of colicin B

Colicin B was isolated in pure form from cells of Escherichia coli that contained the colicin activity and immunity genes cloned on a multi-copy plasmid. Active colicin B consisted of a single polypeptide with Mr of about 60,000. The sequence of 44 amino acids from the amino-terminal portion is presented. The isoelectric point of the protein was at 4.5. Colicin B inhibited the membrane potential-dependent transport of proline and enhanced the uptake of alpha-methylglucoside via the phosphoenolpyruvate-dependent phosphotransferase system. Colicin B formed small, ion permeable channels with an average single-channel conductance of 13.7 pS (1 pS = 10(-12) siemens) in 1 M KCl. Channel formation was voltage-dependent in the pH range between 4.5 and 6. At pH 7 the channels were voltage independent. Voltage-dependent channels were only formed when the trans compartment (the protein was added to the cis compartment) was negative by at least 70 mV. Evidence for an asymmetric single channel conductance was obtained. With KCl a hyperbolic conductance-concentration relationship was observed. The conductance for monovalent cations was minimal for Li+ and was maximal for NH+4. The single channel conductance of colicin B was larger than that of colicin A as judged from lipid bilayer experiments under otherwise identical conditions.     

An evolutionary relationship between the ColE5-099 and the ColE9-J plasmids revealed by nucleotide sequencing

The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.     

Thyroglobulin structure-function. The amino acid sequence surrounding thyroxine

A 19-residue, thyroxine (T4)-containing peptide, Tryp-T4, has been isolated from the tryptic digest of a low molecular weight, iodine-enriched fragment derived from 19S bovine thyroglobulin. This tryptic peptide represents the only site of significant iodination in the parent polypeptide fragment. The amino acid sequence of the tryptic peptide has been determined and is NH2-Asn-Ile-Phe-Glu-T4-Gln-Val-Asp-Ala-Gln-Pro-Leu-Arg-Pro-Cys-Glu-Leu-Gln- Arg-COOH. The carboxyl-terminal sequence of this peptide shows a high probability of a beta-turn. These findings establish the involvement of at least a single unique sequence within thyroglobulin in thyroxine biosynthesis and the general nature of a hormonogenic site within this protein. This sequence contains at least 30% of the thyroxine present in 19 S bovine thyroglobulin.     

Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane.

The nucleotide sequence of the yeast chromosomal gene coding for the 70-kd protein of the mitochondrial outer membrane was determined. The deduced amino acid sequence of the protein agrees with the experimentally determined size and amino acid composition of the purified protein and correctly predicts the fragments obtained by cleaving the protein at its single tryptophan residue. The deduced NH2-terminal sequence features an uninterrupted stretch of 28 uncharged amino acids flanked on both sides by basic amino acids. By sequencing a truncated version of the gene it was found that the corresponding polypeptide product lacks the 203 carboxy-terminal amino acids of the authentic 70-kd protein. As shown in the accompanying paper, this protein fragment still becomes attached to the mitochondrial outer membrane in vivo.     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

Isolation and molecular and functional properties of the amino-terminal domain of colicin A

A plasmid was constructed which allowed easy and efficient production and purification of the NH2-terminal domain of colicin A. In only three steps, an homogenous 18-kDa polypeptide was obtained. The NH2- and COOH-terminal sequences of the protein were determined and showed that it corresponded to the NH2-terminal 171 amino acid residues of the 63-kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2-terminal domain (designated AT) has a globular structure. This fragment is not the receptor-binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH-dependent, in contrast to that of colicin A.     

A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.     

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors.

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.