Danièle Cavard

Lysis protein encoded by plasmid ColA-CA31

SummaryA gene, cal, coding for a polypeptide needed for the release of colicin A from Escherichia coli cells has been identified by transposon insertion. The cal gene was located on the ColA plasmid map adjacent to cai, the gene coding for colicin A immunity protein, and therefore 592 bases downstream from caa, the structural gene for colicin A. Transcription of cal is in the same direction as caa, that is in the opposite direction to cai. Its sequence has been determined and the predicted amino acid composition features a basic N-terminal end followed by a serie of hydrophobic residues similar to the signal sequence in precursors of exported proteins. The C-terminal part also contains a core of hydrophobic residues. The overall amino acid sequence of the cal protein is homologous to that of lytic proteins encoded by the related plasmids pColE1, and pCloDF13. The cal protein has been identified on urea-SDS-polyacrylamide gels by selective labelling with various radioactive amino acids and its synthesis is co-induced with that of colicin A. The cal protein undergoes slow processing with loss of the N-terminal “signal” region and the mature form is released into the medium together with colicin A.

High-level expression of the colicin A lysis protein.

SummaryTwo plasmids that overproduce the colicin A lysis protein, Cal, are described. Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed thecal gene near a truncatedcaa gene in such a way that both gene products were synthesized at high levels following induction. Plasmid Ck4 was constructed by insertion of thecal gene downstream from thetac promoter of an expression vector. Overproduction of Cal was obtained after mitomycin C induction of pAT1 cells and after IPTG induction of pCK4 cells. The kinetics of Cal synthesis were examined with [35S] methionine and [2-3H] glycerol inlpp orlpp+ host strains. Each of the steps of the lipid modification and maturation pathway of Cal was demonstrated. The modified precursor form of overproduced Cal was not chased as efficiently as when it is produced in pColA cells. After treatment with globomycin, a significant amount of this modified precursor form accumulated and was degraded with time into smaller acylated proteins, but without release of the signal peptide. Release of cellular proteins and quasi-lysis were observed after about 1 hour of induction for cells containing either plasmid. In addition, in Cal-overproducing cells, the rate of quasi-lysis was increased but not its extent. InpldA cells, quasi-lysis was reduced but not abolished. Lethality of the Cal induction in the overproducing cells was in the same range as that in wild-type cells.

Functioning of the Colicin A Lysis Protein is Affected by Triton X-100, Divalent Cations and EDTA

The colicin A lysis protein, Cal, is synthesized at the same time as colicin A by Escherichia coli harbouring plasmid pColA after induction by mitomycin C. Its function in the induced bacteria involves the release of colicin A, quasi-lysis, the death of the producing cells and the activation of the outer membrane phospholipase A. We have found that these various functions are affected differently by treatment of the induced cells with Triton X-100, divalent cations or EDTA. Triton X-100 and EDTA caused increased quasi-lysis and a higher level of mortality of the producing cells, but while Triton X-100 enhanced the release of colicin A, EDTA reduced it. Divalent cations protected the cells against both killing and quasi-lysis without greatly affecting colicin release. The effects of these agents were similar for both wild-type and phospholipase A mutants and depended only on the presence of a functional cal gene.

Assembly of colicin A in the outer membrane of producing Escherichia coli cells requires both phospholipase A and one porin, but phospholipase A is sufficient for secretion

Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.

Inhibition of colicin synthesis by the antibiotic globomycin

Abstract The antibiotic globomycin, an inhibitor of LspA (the lipoprotein signal peptidase), inhibited synthesis of colicin by Escherichia coli cells grown in rich medium. This inhibition was stronger in cells with mutation(s) within either the colicin operon, which is located on a plasmid, or the host chromosome. This phenotype was called Gbc (globomycin blocks colicin synthesis). The Gbc phenotype was affected by growth conditions since it was partially or totally suppressed in cells subjected to high temperatures, treated with sodium azide, or grown in minimal medium. The Gbc phenotype observed with colicin-A-producing cells was more severe in strains carrying plasmids with a deletion within caa (the first gene of the colicin A operon), which encodes colicin A, than in cells with the wild-type caa gene. The Gbc phenotype was alleviated by a null mutation in the degP gene encoding the DegP/HtrA protease, abolished by a null mutation in the lpp gene encoding the murein-lipoprotein, and enhanced by a mutation in the pldA gene encoding the outer membrane phospholipase A. Transcription of the colicin A operon was blocked in cells exhibiting the Gbc phenotype as evidenced by rifampicin treatment of induced cells. This phenotype suggests that either a lipoprotein or a protein involved in lipoprotein metabolism might be involved in the regulation of the expression of the colicin operons and that the colicin A structural gene might play a role in the regulation of transcription of the colicin A operon.

Role of the colicin A lysis protein in the expression of the colicin A operon

The involvement of the cal gene, which encodes the colicin A lysis protein, in the expression of the colicin A operon is demonstrated. Colicin A synthesis by Escherichia coli was studied at various temperatures in cells containing either the wild-type colicin A operon or the colicin A operon with the cal gene deleted. The amount of colicin A produced was lower in cells containing the colicin A operon devoid of the cal gene than in wild-type cells. In cells treated with the antibiotic globomycin, the synthesis of colicin A was blocked in null cal mutants at all temperatures. It was blocked only at low temperature in cells containing the wild-type colicin A operon, but not in cells subjected to heat shock or azide treatment. The cal gene product may be an activator of colicin A expression and of its own expression. An unidentified product, possibly a heat-shock protein, may also be involved and could complement the cal gene product in some situations.

Mise en évidence par le lauryl-sulfate de quelques modifications précoces de la membrane de E. coli provoquées par les phages T4, les fantômes de phages T4 et la colicine K

Summary The bacterial strain E. Coli BB grows normally in the presence of laurylsulfate (LS) at 0,5 mg per ml. But when colicine K or T4 phage ghosts are fixed to the surfaces of the bacteria, the bacteria, on subsequent exposure to LS, are lysed within 5 min. On the contrary, when T4 phage itself is present alone at the surface of the bacteria, there is no lysis by LS; the proportion of lysed to non lysed cells, in this instance, depends upon the following factors: the medium of the culture, the phase of bacterial growth, the temperature, the presence of metabolic inhibitors and the concentration of LS. Within 2 or 3 minutes, these bacteria acquire a resistance to lysis by phage ghosts in LS and also to lysis by a high multiplicity of phage. The LS by the speed of its action appears to reveal certain modifications of the bacterial membrane. The phospholipids of the bacteria which have fixed to their surfaces phage ghosts or colicine K undergo interconversions: the amount of diphosphatidylglycerol (DPG) increases at the expense of the amount of phosphatidylglycerol (PG), and the amount of lysophosphatidylethanolamine (LPE) increases at the expense of the amount of phosphatidylethanolamine (PE). The phospholipids of the bacteria which have fixed to their surfaces pages T4 do not undergo these interconversions. LS prevents these interconversions, and without notable modification of the composition of the phospholipids of the bacteria, LS provokes the lysis of the bacteria on whose surfaces are fixed either phage ghosts or colicine K. From this work it is possible to suggest hypotheses concerning the modifications of the bacterial membrane induced by phages T4, ghosts of this phage and colicine K, at the onset of their action.

Colicin A multimerizes when unfolded

A purified preparation of colicin A produced by Escherichia coli cells contained various forms of colicin A. Unfolding of the purified colicin A with urea provoked multimerization. Dimers, tetramers and hexamers of colicin A were identified.