Colicin E1 Synthesis Research Articles

Increased production of colicin E1 in stationary phase

The synthesis of colicin E1 is known to be regulated by the SOS response, anaerobiosis, and catabolite repression. The expression of cea-lacZ fusions was also found to be stimulated when cells reached stationary phase. This increase in expression was determined to be due to depletion of nutrients from the medium, since the addition of fresh medium reversed the effect. Expression of the fusion increased when cells were starved in 10 mM MgSO4 and when they were grown in conditioned medium in which cells had been grown previously. The stimulation of expression occurred when the cea-lacZ fusion was present in single-copy as well as in multicopy plasmids. Finally, the data were consistent with this increase being independent of the SOS response, anaerobiosis, catabolite repression, and integration host factor as well as the stationary-phase regulators encoded by rpoS and lrp.

Spontaneous induction of colicin E1 in Escherichia coli strains deficient in both exonucleases I and V.

Colicin E1 synthesis is spontaneously induced in pRSF2124-carrying strains of Escherichia coli deficient in exonucleases I (sbcB) and V (recB recC). In contrast, the specific activity of beta-lactamase, which is also encoded by pRSF2124, is not affected by the absence of these enzymes. These results suggest that colicin E1 induction is specific and does not result either from a significant change in overall plasmid transcription or copy number. Furthermore, the level of spontaneous induction was similar to that obtained with mitomycin C.

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair.

The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated lambda in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5. While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.

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.

Direct participation of lexA protein in repression of colicin E1 synthesis.

Spontaneous colicin E1 production by plasmid RSF2124 in a recA lexA(spr) strain of Escherichia coli was about 10-fold greater than that observed in a wild-type strain. The synthesis was repressed nearly to the level of a recA strain in the presence of the plasmid pMCR551, which carries the lexA gene.

ColE1 vectors for direct selection of cells carrying a hybrid plasmid.

pKY2289, a ColE1::Tn3 derivative, useful for direct selection of cells carrying a hybrid plasmid, was deleted of its mobility functions and parts of the transposon including the left inverted repeat. This deletion mutant, named pKY2700 expresses low levels of colicin E1 synthesis even in recA cells. A nitrosoguanidine mutant of pKY2289 which shows a high level of constitutive colicin E1 synthesis was also deleted of the same sequences as pKY2700. The second plasmid, named pKY2800, has the same molecular weight (3.8 megadaltons) and almost the same structure as pKY2700, but produces colicin E1 at much higher levels and has a copy number 10 times higher. pKY2800 requires no colicin E1 induction for the direct selection of hybrid clones, while pKY2700 requires mitonmycin C at a concentration of 10 ng per ml. These two colE1 derivatives are useful as safe and convenient vectors for cloning DNA fragments at the cleavage sites of EcoRI, XmaI, and SstII of plasmid.

The influence of colicinogenic plasmids ColIb-P9, ColIa-CA53 and ColV-K30 on the repair, mutagenesis and induction of colicin E1 synthesis.

The presence of colicinogenic plasmids ColIb-P9 and ColIa-CA53 in E. coli K-12 cells, wild-type with respect to repair, enhanced the survival of cells after UV irradiation and increased the frequency of UV-induced argE3 and his-4 reversions, while the presence of ColV-K30 negatively affected repair and mutagenesis. The plasmid ColIb-P9 showed a UV-protective effect in E. coli cells carrying mutations in genes uvrA, uvrB, uvrC, polA, recB, recF, though in none of the mutants did cell survival reach the wild-type level. The effect of ColIb-P9 on mutagenesis did not depend on the uvrA or recB genes. The plasmid's protective effect and the enhancement of mutagenesis depended on the recA+ lexA+ genotype. The frequency of 2-aminopurine-induced mutations was not affected by ColIb-P9 or ColV-K30. The presence of ColIb-P9 decreased the ability of ColE1-carrying cells to induce colicin E1 synthesis caused by DNA-damaging agents: UV, MNNG, mitomycin C, whereas ColV-K30 increased the percentage of colicin E1-producing cells. These plasmid effects on the level of induction of colicin E1 synthesis were not observed in the case of induction caused by chloramphenicol which did not depend on the products of recA and lexA genes.

A novel ColEl::Tn3 plasmid vector that allows direct selection of hybrid clones in E. coli

A plasmid, named pKY2289, consists of a whole ColE1 DNA molecule and a complete ampicillin transposon (Tn3). When induced, E. coli K-12 cells which carry pKY2289 promote synthesis of colicin E1, but they are not immune to colicin E1. Inserting a DNA fragment into the Eco RI or the XmaI site of this plasmid abolishes its ability to produce active colicin E1. Thus, cells carrying one of these in vitro recombinant pKY2289 plasmids are able to form normal colonies in the presence of 0.1 μg/ml of mitomycin C and 50 μg/ml of ampicillin, while cells carrying the parental pKY2289 form very tiny colonies under the same conditions. This allows a positive selection for an in vitro recombinant pKY2289 molecule carrying a foreign DNA insertion. The properties of cells carrying the original pKY2289 are described and its potential usefulness as a cloning vehicle is demonstrated by cloning all the EcoRI and XmaI fragments of lambda DNA.

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.

Effect of guanosine tetraphosphate on in vitro protein synthesis directed by E1 and E3 colicinogenic factors.

The in vitro synthesis of colicin E3 was found to be stimulated by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), while that of several other ColE3 plasmid-specific proteins was reduced in the presence of this nucleotide. The ColE1 plasmid-directed in vitro synthesis of colicin E1 was also found to be stimulated by ppGpp.

Derepression of colicin E1 synthesis in the constitutive tif mutant strain (spr tif sfi) and in a tif sfi mutant strain of Escherichia coli K-12

We show here that expression of the colicin gene of the ColE1 plasmid is greatly derepressed in Escherichia coli K-12 strain DM1187 spr tif sfi, which is a constitutive tif mutant, altered in the lexA gene, and which shows constitutive expression of various pathways of the recA-dependent, lexA-blocked (SOS) repair system. In this strain colicin E1 synthesis is at least 100-fold greater than that observed in uninduced control strains (spr+ tif sfi and spr+ tif+ sfi). This result confirms the regulatory role of the lexA product in colicin E1 synthesis. Colicin yields by the uninduced strain DM1187 are as high as the maximum yields from mitomycin-induced control strains and often are several-fold higher. When the nonconstitutive tif sfi strain GC467 is raised to 43 degrees C to induce the SOS system, a low level of colicin synthesis is observed which is less than one-tenth of the yield obtained by induction with mitomycin C. Addition of adenine at the time of shift-up can increase the colicin yield of tif sfi to about one-third of the yield obtained with mitomycin C. We have also found that colicin overproduction can be detected by altered colony appearance in an overlay assay with colicin-sensitive bacteria. In addition, the lethality of the process of colicin synthesis is observed here without the use of bacteriostatic inducing agents.

Induction of Colicin E1 Synthesis in Recombination-Defective Mutants in Both the RecBC and RecF Pathways

Colicin E1 synthesis was induced in mutants defective in both the RecBC and RecF pathways to the same extent as in Rec(+) strains.

Involvement of NAD in induced synthesis of colicin E1

Escherichia coli K-12 nadC13 (ColE1) was starved for nicotinic acid and cellular NAD levels decreased to less than 10% of the normal. Under these conditions, induction of colicin E1 synthesis decreased to about 1% of the normal value, while 30% of the total protein synthesis remained intact. Addition of nicotinic acid reversed both the ability of the colicin induction and cellular NAD levels. Induced replication of colicin E1 DNA was greatly reduced in the NAD-deprived cells.

Requirements of Glucose and Incubation Under Static Conditions for Optimal Colicin E1 Induction

A rapid rate and large amounts of colicin E1 synthesis were induced by incubating colicinogenic cells without aeration in the presence of glucose. The decrease in ability of colicin induction by aeration was not due to an ample supply of oxygen, but to the simple agitation of the bacterial suspension. Sugars other than glucose that support colicin synthesis were mannose, fructose, glucose-6-phosphate, and mannitol. The effect of sugars in mutants deficient in enzymes of glucose metabolism suggested that some intermediary metabolites between glucose and pyruvate are closely related to the stimulation of colicin synthesis under these conditions. Analyses of deoxyribonucleic acid replication indicated that the induced replication of the colicin E1 plasmid deoxyribonucleic acid occurred even under conditions in which colicin was not actively synthesized.

Synthesis of colicin E1 in a cell-free coupled transcription-translation system

Protein was synthesized in a cell-free coupled transcription-translation (S30) system on plasmid Col E1 and phage T2 and Sd DNA templates. Phage T2 DNA and closed plasmid DNA proved to be the most active templates. The latter controlled the synthesis of eight individual proteinsin vitro, including the biologically active protein antibiotic colicin. The titer of colicin synthesized in this cell-free system reached 1024 units/ml, two orders of magnitude higher than the titer of the antibiotic in populations of colicinogenic bacteria.

Phenotypic difference between uvr A and uvr B mutants of E. coli.

THE synthesis of colicin E1, an antibiotic protein, by cells of Escherichia coli is coded for by a bacterial plasmid, colicinogenic factor E1 (ColE1). The synthesis is induced by agents such as ultraviolet light1 or mitomycin C (mit C)2. Ultraviolet and mit C are known to modify the base portions of DNA to form pyrimidine dimers3 and cross linkages between complementary strands4, respectively. Damage to DNA caused by ultraviolet or mit C is repairable by a similar molecular mechanism5,6, but the relationship between these repair systems and the induction process of ColE1 synthesis has not been established unequivocally. We have analysed the induction of the colicin by ultraviolet and mit C in various ultraviolet-sensitive mutants to which the colicinogenic factor was transferred. Among the mutants tested, the recA mutant did not synthesise the colicin in response to ultraviolet or mit C as reported previously7. In addition, however, we have now found that the induction of the colicin synthesis is greatly delayed in the uvrB mutant, but is normal in the uvrA mutant.

Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids.

An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their beta-lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33 degrees C, but carrier strains grow well at 28 degrees C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.

Synthesis of Colicin E1 in a Cell-Free System

Colicin E1 was synthesized in a cell-free system. The in vitro synthesis was found to be dependent on the Col E1 DNA concentration and was not enhanced by the addition of mitomycin C.