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.     

Mechanism of export of colicin E1 and colicin E3.

The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Colicin E3 and its immunity genes

A DNA segment of plasmid ColE3-CA38 was cloned into pBR328 and its nucleotide sequence was determined. This segment contains the putative promoter-operator region, the structural genes of protein A (gene A) and protein B (gene B) of colicin E3, and a part of gene H. Just behind the promoter region, there is an inverted repeat structure of two 'SOS boxes', the specific binding site of the lexA protein. This suggests that the expression of colicin E3 is regulated directly by the lexA protein. Genes A and B face the same direction, with an intergenic space of nine nucleotides between them. ColE3-CA38 and ColE1-K30 are homologous in their promoter-operator regions, but hardly any homology was found in their structural genes. On the other hand, ColE3-CA38 is fairly homologous to CloDF13 throughout the regions sequenced, with some exceptions including putative receptor-binding regions. By deletion mapping of the immunity gene and recloning of gene B, it was shown genetically that protein B itself is the actual immunity substance of colicin E3. It was also found that the expression of E3 immunity partially depends on the recA function. Thus, we propose two modes of expression of E3 immunity: in the uninduced state, only a slight amount of protein B is produced constitutively to protect the cell from being attacked by the exogenous colicin; and in the SOS-induced state, a large amount of protein B is produced to protect the protein synthesis system of the host cell from ribosome inactivation by endogenously produced colicin E3.     

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3.

Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, ColE8-J was shown to be situated between ColE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids.     

Relationships of the Col plasmids E2, E3, E4, E5, E6, and E7: Restriction mapping and colicin gene fusions

Thirteen ColE plasmids representing the E2-E7 types have been compared by restriction mapping. Over 80% of their restriction sites were found to be similarly positioned, indicating that these plasmids share a common structure. Three variants are ColE2-CA42 and ColE7-K317, both of which contain 1.8-kb DNA segments in place of a 2.5-kb segment common to the other plasmids, and ColE6-CT14, which has an additional 5.0-kb DNA segment compared to the other plasmids. The colicin (col), immunity (imm), and colicin release (hic) genes of these plasmids have been localized to regions corresponding to those known for ColE3-CA38 and ColE2-P9, with the imm and hic genes adjacent to the 3' end of the col gene. Active colicin is produced from hybrid col genes containing 5' and 3' ends from different E-type plasmids. The 3'-termini of the fused col genes specify the colicin type.     

Comparative Study of the Events Associated with Colicin Induction

Colicinogenic factors ColI and ColV, which have been shown to behave as sex factors, could not be induced with mitomycin C. In contrast, the ColE(1), ColE(2), and ColE(3) factors, which do not exhibit any fertility factor characteristics, are inducible by this agent. The induced production of colicins E(1), E(2), and E(3) was accompanied by a loss in viability at a concentration of mitomycin C which was bacteriostatic to noncolicinogenic cells or to cells carrying the ColV or ColI factors. The loss in viability accompanying the mitomycin C induction of the ColE(1), ColE(2), or ColE(3) factors also occurred when colicin synthesis was blocked by chloramphenicol or amino acid starvation. However, chloramphenicol was able to block the loss of viability of a recipient cell after mitomycin C induction of a newly acquired Col factor if the antibiotic was present throughout the mating period. No detectable internal colicin or colicin precursor could be demonstrated during the lag period prior to the appearance of colicin outside the cell 20 to 30 min after the addition of mitomycin C. If chloramphenicol was present during the lag period following the addition of mitomycin C, colicin synthesis began immediately after the removal of these antibiotics. The synthesis of tryptophan synthetase and induced beta-galactosidase proceeded normally throughout the lag period and well into the period of colicin production. Regulation of beta-galactosidase synthesis did not seem to be profoundly affected during the lag period subsequent to mitomycin C addition. Induced colicin synthesis, like bacterial or induced prophage protein synthesis, was subject to inhibition by virulent phage infection.     

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Mutations which affect the structure and activity of colicin E3.

Among 69 ColE3 mutant plasmids selected on the basis of their inability to produce an active colicin, seven (cop-1 to cop-7) were found to bear a mutation affecting the structural gene for colicin. Three of these (cop-1, cop-2 and cop-3) lead to the production of an inactive colicin molecule which has the same molecular weight as wild-type colicin E3 (67,000). These three inactive colicins are still able to interact with the outer membrane receptor. The cop-1 protein retained the ability to inhibit protein synthesis in vitro and therefore seems specifically affected in it ability to penetrate the cell envelope. The cop-2 and cop-3 proteins lost the ability to inhibit protein synthesis in vitro, and activity which is normally associated with the C-terminal part of the colicin molecule. On the basis of this and further evidence, it is suggested that the cop-2 and cop-3 mutations affect the structure of the C-terminal part of the colicin molecule. The other four mutations (cop-4 to cop-7) lead to the production of colicin-related polypeptides of lower molecular weight (29,000 to 45,000) which display a reaction of partial immunological identity with wild-type colicin. These four polypeptides are unable to interact with the cell surface receptor. Three of these mutants are shown to carry a nonsense mutation.     

Isolation and characterization of a mutant ColE1 plasmid that allows constitutive colicin E1 synthesis.

It has been possible to isolate a ColE1 mutant which synthesizes colicin E1 constitutively. This result shows that there must be a gene(s) responsible for the regulation of colicin E1 synthesis.     

Incompatibility between E colicin plasmids

We have tested the ability of pairs of colicin E plasmids to replicate stably in the same cell line. Although many of the pairs of E colicin plasmids were compatible, plasmids ColE3-CA38, ColE7-K317 and ColE8-J were mutually incompatible, as were ColE5-099, ColE6-CT14 and ColE9-J. Incompatibility between ColE6-CT14 and ColE5-099 or ColE9-J was asymmetrical, whereas incompatibility between the other plasmid pairs was symmetrical.     

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.     

Characterisation of the ColE8 plasmid, a new member of the group E colicin plasmids

We have determined the restriction map of the ColE8-J plasmid after cloning it into the pBR322 vector. By subcloning and transposon mutagenesis we have localized the colicin immunity gene, the colicin structural gene, and lys, the region that determines MC sensitivity. In contrast to the ColE3-CA38 plasmid, the genes coding for colicin E8 production and immunity cannot be cloned on a single EcoRI fragment. Insertion of Tn5 transposons into the colicin structural gene region of the recombinant plasmid inactivated colicin production and MC sensitivity. Insertion of transposons into the lys region reduced colicin E8 production and MC induced lysis, the extent of which was dependent upon the precise site of insertion. We propose that the colicin E8 structural gene and lys must be transcribed from a common promoter situated proximal to the structural gene, whilst the colicin E8 immunity gene is transcribed from a second promoter. The lys region is responsible both for cell lysis after MC induction and positive regulation of colicin E8 synthesis.     

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.     

Mutants of ColE1 plasmid with altered expression of the colicin E1 immunity gene

Mutagenesis with Tn1 transposon was used to isolate mutants of ColE1 plasmid with inactivated gene responsible for immunity to colicin E1. Cells containing such mutants synthesized active colicin E1 and were sensitive to its action. Spontaneous and UV-induced colicin synthesis was strongly changed in the mutants, as compared to the control. Mutations occurring outside the immunity gene, including those within the structural gene for colicin E1, could also affect the immunity gene expression.     

Molecular structure and immunity specificity of colicin E6, an evolutionary intermediate between E-group colicins and cloacin DF13.

The primary structure of a 3.1-kilobase E6 or E3 segment carrying colicin and related genes was determined. Plasmid ColE6-CT14 showed striking homology to ColE3-CA38 throughout this segment, including homology to the secondary immunity gene, immE8, downstream of the E6 or E3 immunity gene. The ColE3-CA38 and ColE6-CT14 sequences, however, contained an exceptional hot spot region encoding both the colicin-active domain (RNase region) and the immunity protein, reflecting their different immunity specificities. On the other hand, some chimeric plasmids were constructed through homologous recombination between colicin E3 and cloacin DF13 operons. The resulting plasmids were deduced to produce chimeric colicins with a colicin E3-type N-terminal part, a cloacin DF13-type C-terminal-active domain, and the DF13 immunity protein. The killing spectra of the chimeric colicins and the immunities of the plasmids were identical to those of colicin E6 and ColE6-CT14, respectively, showing that the colicin E6 immunity specificity is completely equivalent to that of cloacin DF13. Nevertheless, colicin E6 has been found to show a sequence diversity from cloacin DF13 almost to the same extent as that from colicin E3 in their RNase and immunity regions, indicating that only a small number of amino acids defines the immunity specificity for discrimination between colicins E3 and E6 (or cloacin DF13).     

Escherichia coli mutant containing a large deletion from relA to argA.

A mutant of Escherichia coli has been isolated that contains a large deletion (about 3 X 10(7) daltons of deoxyribonucleic acid) encompassing argA, fuc, and relA. This mutant strain (AA-787) is also cold sensitive for growth at 18 degrees C. Strain AA-787 was obtained fortuitously as a cold-sensitive pseudorevertant of a strain having a heat-sensitive peptidyl-transfer ribonucleic acid hydrolase. Genetic analysis, using transduction and interrupted mating, showed the cold sensitivity mutation to be located adjacent to relA. Further analysis demonstrated loss of relA, fuc, and argA gene functions but retention of eno and recB, closely linked genes adjacent to relA and argA, respectively. Unusually high cotransduction of flanking markers (cysC and thyA) indicated loss of approximately 1 min of the E. coli genetic map in strain AA-787. Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) was synthetized in mutant strain AA-787 at basal levels, and ppGpp synthesis was stimulated by carbon-source downshift. No ppGpp synthesis could be obtained using ribosomes isolated from strain AA-787. These findings, taken together, show that deletion of relA in E. coli does not completely abolish ppGpp synthesis and suggests that another enzyme system must also be responsible for ppGpp synthesis.     

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1.

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.