Alternative forms of lethality in mitomycin C-induced bacteria carrying ColE1 plasmids

Abstract

We have studied the physiological effects of mitomycin C induction on cells carrying ColEl plasmids with differing configurations of three genes: the structural gene coding for colicin (cea), a gene responsible for mitomycin C lethality (kil) that we located as part of an operon with cea, and the immunity (imm) gene, which lies near cea but is not in the same operon. kil is close to or overlaps imm. When cea' plasmids are present mitomycin C induction results in 100-fold or greater increases in the level of colicin. Within an hour after induction more than 90% of cells carrying cea' kil' imm' plasmids are killed and macromolecular synthesis stops, capacity for transport ofproline, thiomethyl -fiD-galactoside, and a-methyl glucoside is lost, and the membrane becomes abnormally permeable as indicated by an increased accessibility of intracellular 13-galactosidase to the substrate o-nitrophenyl .8-D-galactoside. All of these events occur when a ceakil+ imm+ plasmid is present and none does when the plasmid is cea+ kil-imm+, so the damage can be attributed solely to the Kil function and not to the presence ofcolicin. However, cells carrying a cea+ kil-imm-plasmid are killed upon induction, apparently by action of endogenous colicin on the nonimmune cytoplasmic membrane. The pattern of accompanying physiological damage is distinguished from the kil+-associated damage by an enhancement of a-methyl glucoside uptake and accumulation and efflux of a-methyl glucoside 6-phosphate and by an absence of the alteration in membrane permeability for o-nitrophenyl P-D-galactoside. These features are typical of colicin El action on the membrane. The induced damage is not prevented by trypsin and occurs in cells of a strain specifically tolerant to exogenous colicin E1, indicating that the attack is from inside the cell. transposon that confers resistance to ampicillin. The relevant genome configurations of this plasmid and its derivatives that were used in this study are diagrammed in Fig. 1. The isolation and mapping of the Tn5 insertion mutants has been described (2). pRLB6 was constructed by cleaving pDMS630 DNA with the restriction endonucleases Sna I and EcoRI (from New England BioLabs), each of which cleaves the plasmid once in the cea gene at sites 1,290 base pairs apart (Fig. 1, ref. 5). The two enzymatic digestions were carried out separately, using the conditions specified by the supplier. After the EcoRI digestion was complete the enzyme was inactivated by heating at 70'C for 5 min and the ends of the DNA fragments were filled in by incubation for 1 hr at 370C with the four deoxyribonucleoside triphosphates dTTP, dATP, dGTP, and dCTP (each 0.25 mM; from Sigma) and DNA polymerase I (25 units/ml; from New England BioLabs). The DNA was then precipitated with ethyl alcohol, redissolved in buffer (final concentrations: 0.1 M Tris at pH 7.5, 6 mM MgCl2, 6 mM NaCl, 0.4 mM ATP, 10 mM dithiothreitol, and bovine serum albumin at 50 Ag/ml), and ligated by action of phage T4 DNA ligase (400 units/ml; from New England BioLa...

Characterization of the ColE9-J Plasmid and Analysis of its Genetic Organization

“…1). Transposon TnlOOO inserts which inactivated colicin E9 production also abolished MC sensitivity (data not shown), a result which has previously been found with ColEl (Suit et al, 1983), ColE2-P9 (Pugsley & Schwartz, 1983), ColE3-CA38 (Chak & James, 1984;Jakes & Zinder, 1984) and ColE8-J (Lawrence & James, 1984) plasmids.…”

“…The genetic organization of the two immunity genes of the ColE9-J plasmid is however very different to that found with ColE3-CA38. The only other E colicin plasmid in which the immunity gene is transcribed in the opposite orientation to the respective colicin structural gene is ColE 1 (Suit et al, 1983). Colicin E9 can be distinguished from colicin El by the insensitivity of tolA and tolD mutants to the former but not to the latter.…”

“…The specificity of the interaction between immunity proteins and their homologous colicins make this a valuable system for studying protein-protein interaction. E. coli K12 strains carrying an E colicin plasmid are sensitive to mitomycin C (MC) compared to plasmid-free isogenic strains, due to the induction of a plasmid-coded lysis gene (lys) which may be involved in colicin release from the producing cell (Shafferman et al, 1979;Pugsley & Schwartz, 1983;Suit et al, 1983;Chak & James, 1984;Jakes & Zinder, 1984;Lawrence & James, 1984). Since immunity is constitutive, whilst colicin production and MC sensitivity are inducible by DNA damage (Herschman & Helinski, 1967), the regulatory control of these three genes is of considerable interest.…”

Competitive interactions in Escherichia coli populations: the role of bacteriocins

“…The cultures were treated with crude lysates of (i) the ancestral control strain (for PE2lum and PE7lum); (ii) ColE7 (for PE2lum); and (iii) ColE2 (for PE7lum)-producing strains. All lysates were prepared according to the procedure described in Suit et al (1983) and stored at À80 1C until use. A twofold dilution series of the appropriate lysates was added to 96-well microtiter plates (Grainer, Frickenhausen, Germany) and equal volumes of appropriate cells were added.…”

The nucleotide sequence of cea* and the region of origin of plasmid pKY-1.

“…2,390) which has almost the same sequence as the kil of Co1E1. kil is supposed to be important for the release of colicin E 1 and to be responsible for the killing of the host cell (13). This frame is indicated as kil * in Fig.…”