Temporal control of colicin E1 induction.

The expression of the gene encoding colicin E1, cea, was studied in Escherichia coli by using cea-lacZ gene fusions. Expression of the fusions showed the same characteristics as those of the wild-type cea gene: induction by treatments that damage DNA and regulation by the SOS response, sensitivity to catabolite repression, and a low basal level of expression, despite the presence of the fusion in a multicopy plasmid. Induction of expression by DNA-damaging treatments was found to differ from other genes involved in the SOS response (exemplified by recA), in that higher levels of DNA damage were required and expression occurred only after a pronounced delay. The delay in expression following an inducing treatment was more pronounced under conditions of catabolite repression, indicating that the cyclic AMP-cyclic AMP receptor protein complex may play a role in induction. These observations also suggest a biological rationale for the control of cea expression by the SOS response and the cyclic AMP-cyclic AMP receptor protein catabolite repression system.     

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.     

Isolation and characterization of ColE2 plasmid mutants unable to kill colicin-sensitive cells

Summary After transfer from a mutagenized host, twenty one ColE2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene for colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors.Studies with a nonsense mutant producing only a small colicin E2 fragment (ColE2-421) suggest that colicin E2 is not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33°, 37° and 43°. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.     

Isolation of conjugation-constitutive mutants of colicin factor Ib

Summary Colicin factor ColIb-P9 is known to act as a sex factor in E. coli or Salmonella. Although ColIb-P9 confers mating ability on its host bacteria, this ability appears to be repressed since only a small proportion of cells in a culture of a colicinogenic strain are able to pair with, and transmit the factor to recipient bacteria. We have isolated mutants of ColIb-P9 which confer constitutive donor ability on their host. De-repression in these mutants is probably due to failure to produce repressor, rather than to insensitivity to repressor. As the colicin production by the mutants is still repressed, colicin synthesis and conjugation ability are subject to independent systems of regulation.     

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.     

On the interaction of colicin E3 with the ribosome

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone     

Regulation of the SOS response in Bacillus subtilis: evidence for a LexA repressor homolog.

The inducible SOS response for DNA repair and mutagenesis in the bacterium Bacillus subtilis resembles the extensively characterized SOS system of Escherichia coli. In this report, we demonstrate that the cellular repressor of the E. coli SOS system, the LexA protein, is specifically cleaved in B. subtilis following exposure of the cells to DNA-damaging treatments that induce the SOS response. The in vivo cleavage of LexA is dependent upon the functions of the E. coli RecA protein homolog in B. subtilis (B. subtilis RecA) and results in the same two cleavage fragments as produced in E. coli cells following the induction of the SOS response. We also show that a mutant form of the E. coli RecA protein (RecA430) can partially substitute for the nonfunctional cellular RecA protein in the B. subtilis recA4 mutant, in a manner consistent with its known activities and deficiencies in E. coli. RecA430 protein, which has impaired repressor cleaving (LexA, UmuD, and bacteriophage lambda cI) functions in E.coli, partially restores genetic exchange to B. subtilis recA4 strains but, unlike wild-type E. coli RecA protein, is not capable of inducing SOS functions (expression of DNA damage-inducible [din::Tn917-lacZ] operons or RecA synthesis) in B. subtilis in response to DNA-damaging agents or those functions that normally accompany the development of physiological competence. Our results provide support for the existence of a cellular repressor in B. subtilis that is functionally homologous to the E. coli LexA repressor and suggest that the mechanism by which B. subtilis RecA protein (like RecA of E. coli) becomes activated to promote the induction of the SOS response is also conserved.     

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.     

Regulation of expression of the colicin gene of I1 group plasmid TP110.

The control of expression of the colicin Ib gene of the I1 group plasmid TP110 has been investigated. The colicin promoter was fused to the structural gene for beta-galactosidase, using the Mu d(Aprlac) phage, and the plasmid carrying this fusion was introduced into a variety of bacterial strains defective in genes involved in the "SOS" response. Colicin Ib belongs to that group of genes directly controlled by the repressor produced by the lexA gene, and expression was inducible by DNA-damaging agents. Mutations in uvrA, -B, and -C reduced the efficiency of induction by mitomycin C, as did mutations in recB. Mutations in recA and recF effectively prevented induction by mitomycin C, whereas mutations in lexA had contrasting effects, depending upon their effect on the properties of lexA protein. The spr-51 mutation (which inactivates lexA protein) led to constitutive expression, whereas the lexA3 mutation (which makes lexA protein refractory to cleavage by recA protein) completely inhibited inducible expression. In addition to lexA control, a TP110-coded function was identified which appeared able to inhibit colicin expression when the gene responsible was present in high copy number.     

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.     

Two overlapping SOS-boxes in ColE operons are responsible for the viability of cells harboring the Col plasmid

In this study, oligonucleotide-directed site-specific mutagenesis was used to change the consensus sequences of the LexA binding motifs in either one of the two SOS-boxes of the ColE7 operon. The results indicated that both mutants produced larger amounts of colicin than cells harboring the wild-type ColE7 plasmid. This finding would imply that two biologically functional SOS boxes exist in the ColE7 operon. In the non-induced state, no lysis of cells harboring wild-type plasmids occurred at 37°C, whereas, cells harboring recombinant plasmids containing either one of the mutated SOS boxes underwent lysis within 100 min under the same conditions. This result indicated that adaptation of two SOS boxes of the ColE operon would obviously tightly control the expression of ColE operons. In such a way that it may prevent excessive expression of the lysis (cel) gene, thus safeguard the host cells from being lysed in ordinary living conditions.     

Heterogeneity in expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> colicin K activity gene <Emphasis Type="Italic">cka</Emphasis> is controlled by the SOS system and stochastic factors

Phenotypic diversity provides populations of prokaryotic and eukaryotic organisms with the flexibility required to adapt to and/or survive environmental perturbations. Consequently, there is much interest in unraveling the molecular mechanisms of heterogeneity. A classical example of heterogeneity in Escherichia coli is the subset (3%) of the population that expresses the colicin K activity gene (cka) upon nutrient starvation. Here, we report on the mechanism underlying this variable response. As colicin synthesis is regulated by the LexA protein, the central regulator of the SOS response, we focused on the role of LexA and the SOS system in the variable cka expression. Real-time RT-PCR showed that the SOS system, without exogenous DNA damage, induces moderate levels of cka expression. The use of cka-gfp fusions demonstrated that modification of the conserved LexA boxes in the cka promoter region affected LexA binding affinity and the percentage of cka-gfp expressing cells in the population. A lexA-gfp fusion showed that the lexA gene is highly expressed in a subset of bacteria. Furthermore, cka-gfp fusions cloned into higher copy plasmid vectors increased the percentage of cka-gfp positive bacteria. Together, these results indicate that the bistability in cka expression in the bacterial population is determined by (1) basal SOS activity, (2) stochastic factors and possibly (3) the interplay of LexA dimers at cka operator. Other LexA regulated processes could exhibit similar regulation.     

The effect of supercoiling of DNA from colicinogenic plasmids on the expression of col, imm and lys genes

The expression of colicin genes is controlled by the SOS-system (Lex A repressor) and the adenylate-cyclase system (cAMP-CAP complex). The effect of plasmid DNA supercoiling on the expression of the operons of colicins E1, E2, and E3 has been studied by using E. coli minicells. It has been shown for the colicin E1 operon that it is the promoter that is influenced by supercoiling: an increase in negative supercoiling elevates the expression and, vice versa, DNA relaxation reduces the expression. The effect of supercoiling on gene activity of the colicin E1 immunity protein has not been observed, which may be due to the specific orientation of this gene. With the two other colicins supercoiling affects the expression of all genes which constitute the operon. The regulation of the colicin operon expression has been confirmed to occur at three levels: by the LexA protein, by the cAMP-CAP complex, and by the plasmid DNA supercoiling.