Proc. NatL Acad. Sci. USA Vol. 80, pp. 579-583, January 1983 Microbiology

Alternative forms of lethality in mitomycin C-induced bacteria carrying ColE1 (kil /Kil function/endogenous colicin El/cytoplasmic membrane damage) JOAN L. SUIT, M. -L. JUDY FAN, JOSEPH F. SABIK, ROBERT LABARRE, AND S. E. LURIA* Department of Biology, Massachusetts Institute ofTechnology, Cambridge, Massachusetts 02139 Contributed by S. E. Luria, October 28, 1982

ABSTRACT We have studied the physiological effects of mi- MATERIALS AND METHODS tomycin C induction on cells carrying ColEl plasmids with dif- fering configurations of three : the structural gene coding Bacterial Strains and Plasmids. Both bacterial strains used for colicin (cea), a gene responsible for mitomycin C lethality (kil) were derivatives ofEscherichia coli K-12 from the Luria stock that we located as part of an operon with cea, and the immunity collection: A153 is Hfr Hayes thi lac-; A586 is C600 tolC (spe- (imm) gene, which lies near cea but is not in the same operon. kil cifically tolerant to colicin El, tol VIII in ref. 3). ColE1 is close to or overlaps imm. When cea' plasmids are present mi- pDMS630 (4) was obtained from J. Inselburg. It carries a Tn3 tomycin C induction results in 100-fold or greater increases in the transposon that confers resistance to ampicillin. The relevant level of colicin. Within an hour after induction more than 90% of configurations of this plasmid and its derivatives that cells carrying cea' kil' imm' plasmids are killed and macromo- were used in this study are diagrammed in Fig. 1. The isolation lecular synthesis stops, capacity for transport ofproline, thiometh- and mapping of the Tn5 insertion mutants has been described yl -fiD-galactoside, and a-methyl glucoside is lost, and the mem- (2). pRLB6 was constructed by cleaving pDMS630 DNA with brane becomes abnormally permeable as indicated by an increased the restriction endonucleases Sna I and EcoRI (from New En- accessibility of intracellular 13-galactosidase to the substrate o-ni- gland BioLabs), each ofwhich cleaves the plasmid once in the trophenyl .8-D-galactoside. All of these events occur when a cea- cea gene at sites 1,290 base pairs apart (Fig. 1, ref. 5). The two kil+ imm+ plasmid is present and none does when the plasmid is enzymatic digestions were carried out separately, using the con- cea+ kil- imm+, so the damage can be attributed solely to the Kil ditions specified by the supplier. After the EcoRI digestion was function and not to the presence ofcolicin. However, cells carrying a cea+ kil- imm- plasmid are killed upon induction, apparently complete the enzyme was inactivated by heating at 70'C for 5 by action of endogenous colicin on the nonimmune cytoplasmic min and the ends of the DNA fragments were filled in by in- membrane. The pattern of accompanying physiological damage cubation for 1 hr at 370C with the four deoxyribonucleoside tri- is distinguished from the kil+-associated damage by an enhance- phosphates dTTP, dATP, dGTP, and dCTP (each 0.25 mM; ment of a-methyl glucoside uptake and accumulation and efflux from Sigma) and DNA polymerase I (25 units/ml; from New of a-methyl glucoside 6-phosphate and by an absence of the al- England BioLabs). The DNA was then precipitated with ethyl teration in membrane permeability for o-nitrophenyl P-D-galac- alcohol, redissolved in buffer (final concentrations: 0.1 M Tris toside. These features are typical ofcolicin El action on the mem- at pH 7.5, 6 mM MgCl2, 6 mM NaCl, 0.4 mM ATP, 10 mM brane. The induced damage is not prevented by trypsin and occurs dithiothreitol, and bovine serum albumin at 50 Ag/ml), and in cells of a strain specifically tolerant to exogenous colicin E1, ligated by action of phage T4 DNA ligase (400 units/ml; from indicating that the attack is from inside the cell. New England BioLabs) overnight at 40C. The mixture was used to transform recipient cells with selection for ampicillin resis- The ColEl plasmid contains the genes cea and imm, of which tance. Transformants were screened for absence ofcolicin pro- cea codes for colicin E1, a 57,000-dalton protein, and imm codes duction, and one cea plasmid (pRLB6) so identified was found for an immunity protein that specifically protects ColEl-car- to still confer immunity and normal sensitivity to mitomycin C. rying cells from colicin El. The immunity protein has not yet The DNA of this plasmid proved to be 1,200-1,300 base pairs been characterized physically. On the basis of the behavior of shorter than that of pDMS630 when electrophoresed through bacteria carrying ColEl plasmids with deletions or insertions an agarose gel (7). in or near the cea Shafferman et aL the Conditions of Growth and Mitomycin C Induction. Media gene, (1) postulated used were LB broth (8) and Ozeki medium base (3) (OM) sup- existence ofa lethality gene (here called kil) responsible for the plemented with thiamin (1 pkg/ml), Casamino acids (0.2%), and death ofcolicinogenic bacteria when induced with UV light or glucose or lactose (0.4%). When cells contained plasmids re- mitomycin C. We have confirmed this and have presented evi- sistant to kanamycin, ampicillin, or both the medium was sup- dence for the location ofthe kil gene as part ofa cea-kil operon. plemented with kanamycin sulfate (Sigma), sodium ampicillin The genetic experiments are reported in detail elsewhere (2). (Polycillin N, Bristol), or both, each at 20 /ig/ml. When cells In this paper we describe the events after induction of the kil of strain A153 contained pJFS349 the medium was supple- gene that lead to cell death and release of colicin. Our obser- mented with trypsin (Worthington) at 100 ttg/ml. Cultures vations demonstrate that cell death by the product of the kil were grown at 370C. When a cell density of5-7 X 108 cells per gene is biochemically distinct from cell death caused by exog- ml was reached mitomycin C (Sigma) was added to a final con- enous colicin. We also describe conditions in which endogenous centration of 2 pug/ml for strain A153 or 0.3 /ig/ml for strain colicin acts to kill cells just as does exogenous colicin, by action A586, which is more sensitive to the drug. on the nonimmune bacterial envelope. Methods for Physiological Studies. f3-Galactosidase synthe- The publication costs ofthis article were defrayed in part by page charge Abbreviations: aMeGlc, a-methyl glucoside; aMeGlcP, a-methyl glu- payment. This article must therefore be hereby marked "advertise- coside 6-phosphate; NphGal, o-nitrophenyl f-D-galactoside. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 579 Downloaded by guest on September 28, 2021 580 Microbiology: Suit et d Proc. NatL Acad. Sci. USA 80 (1983)

ol E| 1.0 w=El EcoRI SmnI pDMS 630 {i cei..-I + + + /'iA//,

0.10 pJFS 15 -EI=[ ZZZZZ/

pJFS 356-L }- + - + co ZZZZZ3/ cn 0.01

pJFS349 - }- + - - ,7,7ZZ 2

pRLB6 - ED- - + + ,/ZZZ2/ 0.001

200 bp FIG. 1. Schematic representation of the cea-imm regions of ColEl plasmids used in this study. The top diagram represents the parental pDMS630. Indicated on it are the positions of the structural MC genes for colicin El (cea) and immunity (imm); the region in which the kil gene appears to be located; the directions of transcription for cea FIG. 2. Survival after mitomycin C treatment of A153 cells car- and imm (arrows); and the unique recognition sites for the restriction rying pDMS630 or some of its mutants. Cells were grown in LB broth endonucleases EcoRI and Sma I. The size and location of cea indicated at 370C to late logarithmic phase (5 x 10i cells per ml) and mitomycin in the diagram were determined by Yamada et al. (5); those of imm C (MC) at 2 ,ug/ml was added at 0 time. At intervals samples were were determined by Oka et al. (6). The actual terminus of the imm withdrawn, diluted, and plated for viable counts on LB agar contain- gene may in fact be 100 base pairs (bp) or more farther to the left. The ing kanamycin, ampicillin, or both as appropriate. Strain A153 car- other diagrams represent three different insertion mutants and a dele- rying no plasmid was included as control. Survival (S/S.) is expressed tion mutant of pDMS630. 4) andt indicate the points of insertion of as the ratio of viable cells at a given time of treatment to the initial the Tn5 transposon; the closed vs. open circles indicate opposite ori- value. entations of the insertions. ofthe cea gene preserves the lethal response to induction (Fig. sis, constitutive in strain A153, was followed as a measure of 2). Apparently this deletion does not have any polar effect on protein synthesis in these cells. The total ,B-galactosidase level the distally located kil gene. was assayed by measuring the rate of hydrolysis of o-nitrophe- Plasmid pJFS356 affords further evidence for the existence nyl f3D-galactoside (NphGal) in CHCl3/sodium dodecyl sul- of the kil gene. This plasmid has a Tn5 insertion just past the fate-treated cells (9). The rate of NphGal hydrolysis by un- end of the cea gene (Fig. 1). Cells carrying it upon induction treated whole cells was used to measure the accessibility of by mitomycin C synthesize active colicin (Table 1) but are not intracellular enzyme to the substrate. Active transport of pro- killed (Fig. 2). The insertion presumably lies either between line and thiomethyl /3-D-galactoside were measured as de- cea and kil, interrupting the operon, orwithin the kil gene itself. scribed by Plate (10). Uptake of a-methyl glucoside (aMeGlc) The colicin appears to remain intracellular, because most of it and accumulation and efflux ofa-methyl glucoside 6-phosphate can be detected only when the cells are disrupted with chlo- (aMeGlcP) were measured as described by Jetten (11). roform, in contrast with cells containing a cea+ kil+ plasmid (Table 1). This suggests that the Kil function may be involved RESULTS in release of colicin. The insertion in pJFS356 preserves im- We have described (2) the isolation and mapping of a series of munity, suggesting that the imm gene, which is transcribed in Tn5 transposon insertion mutants with alterations in the region the reverse direction from cea (6, 12), ends more than 100 base of the ColEl plasmid that includes the cea (colicin) and imm pairs away from the end of cea. Another possibility is that the (immunity) genes. Three ofthese insertions are depicted in Fig. last part of the imm gene is dispensable (6). 1. Plasmid pJFS15 is representative of a class ofeight different Plasmid pJFS349, with an insertion about 300 base pairs be- mutant plasmids with insertions in the cea gene. All these in- yond cea (Fig. 1), does not confer immunity. Upon induction sertions preserve the immunity property but abolish the pro- by mitomycin C cells carrying this plasmid synthesize active duction of active colicin, as well as the lethality that normally colicin and are killed (Fig. 2). However, the mode of lethality follows induction by treatment with mitomycin C (Fig. 2). We is entirely different from that due to the Kil function: these cells have concluded that this lethality is due not to the colicin itself appear to be killed by the action of endogenous colicin. The but to the product of another gene, kil, located distal to cea in distinguishing patterns ofcell damage are described in the fol- the same operon. Active colicin production is not needed for lowing sections. mitomycin C-induced lethality, as shown, for example, in the Physiological Events Accompanying Mitomycin C Induc- case ofplasmid pRLB6 (Fig. 1), in which a deletion ofabout 80% tion. Several tests were used to characterize the events accom- Downloaded by guest on September 28, 2021 Microbiology: Suit et aL Proc. Natl. Acad. Sci. USA 80 (1983) 581

Table 1. Colicin production and release after induction of cells ofactive colicin. The most dramatic changes are the loss in the containing kil+ and kil- plasmids capacity for aMeGlc transport and the change in accessibility Colicin titer by spot test of intracellular /3-galactosidase to NphGal, illustrated in detail in Fig. 3. Cells carrying pJFS15 showed the same pattern of pDMS630 pJFS356 kil- addition of cea' kil+ cea' response as that illustrated for cells with pJFS356 in Fig. 3 Left. mitomycin C, CHCl3- CHCl3- The cea kiNt imm- Plasmid pJFS349. Mitomycin C induc- min Control treated Control treated tion of cells bearing plasmid pJFS349 results in the synthesis 0 10 10 3 3 of active colicin: the titer increases 100- to 500-fold within 45 30 300 1,500 20 300 min after addition of mitomycin C to the cells. Induction leads 60 3,000 3,000 30 1,000 to cell death (Fig. 2) by a mechanism apparently different-from 120 10,000 10,000 300 3,000 the Kil function because the pattern of damage accompanying 210 30,000 10,000 100 3,000 cell death differs markedlyfrom that observed with cells bearing 280 10,000 300 3,000 kil+ plasmids. Induced cells containing pJFS349 exhibit no in- Ten microliters from each of a series of serial dilutions in LB of sam- crease in the accessibility of intracellular 3-galactosidase to ples from induced cultures was spotted onto a lawn of colicin-sensitive NphGal, and the capacity for accumulation of aMeGlc instead indicator cells before and 2 hr after mixing the diluted sample with, ofbeing abolished is increased (Fig. 3 Right). The induced cells chloroform. The chloroform-treated samples were allowed to stand at show an efflux of aMeGlcP whereas cells with a kil+ plasmid room temperature. The indicator cells were tetracycline resistant and do not (Fig. 4). Also, cells containing pJFS349 that were pre- the lawns were poured on LB base agar containing tetracycline at 10 loaded with RbV ions released it earlier after induction than did ,ug/ml to prevent colicin synthesis on the plate in the intact cells being tested. The titer is the reciprocal of the highest dilution.at which kill- kil'-containing cells (data not shown). ing of the indicator cells was detected. Altogether, it appears that induced cells carrying a cea' kil- imm- plasmid are killed by a mechanism similar to the killing ofsensitive cells exposed to exogenous colicin El (see Table 2). panying mitomycin C treatment ofbacteria containing pDMS630 Cell death cannot be ascribed to an exogenous action ofreleased and its derivatives. The results are summarized in Table 2, colicin; the experiments with cells containing pJFS349 were which also lists some of the events that occur when exogenous carried out in the presence of trypsin at 100 pug/ml, which colicin El is added to sensitive cells (11, 13, 14). would rapidly destroy any free colicin. kil+ vs. ki- Plasmids. Induction of the wild-type plasmid Mitomycin C-induced cells of strain C600 toiC carrying leads to cell death the accompanied by cessation ofmacromolecular cea' kil- imm- are synthesis and loss of capacity for active transport, whether plasmid pJFS349, which completely resis- driven by membrane potential, such as proline or thiomethyl tant to exogenous colicin El, are killed in the same way as nor- galactoside accumulation, or by the phosphotransferase system, mal cells carrying this plasmid. Presence of the toiC mutation as for aMeGlk. Also, a change in the cytoplasmic membrane does not alter the physiological response of induced cells to occurs that facilitates access of intracellular 3galactosidase to mitomycin induction. Mitomycin C-induced toiC cells carrying the substrate NphGal. The enzyme is notreleased from the cells the cea' kit imnm- plasmid demonstrated the increased capac- into the medium during this time. Cells carrying the plasmids ity for transport of aMeGlc typical of the response of sensitive with insertions that abolished the mitomycin C-induced le- cells to colicin El. Induced toiC cells containing a cea' kil+ thality-either insertions in the cea gene typified by pJFS15- imm' plasmid, on the other hand, lost the capacity for aMeGlc or beyond cea as in pJFS356, do not exhibit any ofthe alterations transport, a feature that we ascribe to -the Kil function. These just described. Cells carrying pRLB6, with most ofthe cea gene findings indicate that the property of tolerance does not alter deleted but with the killing property preserved, show physio- the response of the cytoplasmic membrane itself to colicin and logical effects ofinduction identical to those carrying pDMS630. lend support to an earlier suggestion (3) that tolerance blocks Thus, the effects observed with the wild-type plasmid can the action of exogenous colicin at some step prior to attack on be attributed solely to the Kil function and not to the presence the cytoplasmic membrane.

Table 2. Physiological events accompanying mitomycin C induction of cells carrying ColEl plasmids or accompanying addition of exogenous colicin El to sensitive cells Cellular characteristic Active transport Accessibility of Proline or intracellular Protein thiomethyl Efflux ,&galactosidase Treatment Plasmid synthesis galactoside aMeGlc of aMeGlcP to NphGal Mitomycin C pDMS630 Ceases at Strong reduction Ceases after Not tested Increases at induction cea' kil+ imm' about 60 min after 45-60 min 40-60 min about 45 min pJFS15 Continues Unaffected Unaffected Not tested Unchanged, cea- kil- imm' throughout throughout pJFS356 Continues Unaffected Unaffected None Unchanged cea' kil imm' throughout throughout pRLB6 Ceases at Strong reduction Ceases after None Increases at cea- kil+ imm' 60-75 min after 45-60 min 40-60 min about 45 min pJFS349 Ceases at Ceases after Stimulated Present Unchanged cea' kil imm- about 45 min 30-45 min throughout Exogenous colicin El Ceases Ceases Stimulated Present Unchanged on sensitive cells Downloaded by guest on September 28, 2021 582 Microbiology: Suit et d Proc. Nati Acad. Sci. USA 80 (1983)

No plasmid / UCo pJFS349

pDMS630 pRLB6

pRLB6 pDMS630

0 20

cU / it pJFS349 ~/pJFS356 / / pRLB6 , supernatant 0 60 120 0 60 120 4 Time, min + Time, min MC MC FIG. 3. Effect of mitomycin C induction on the capacity for aMeGlc uptake and on accessibility of intracellular 13-galactosidase to substrate. Cells of A153 carrying the plasmids indicated (genotypes given in Fig. 1) orzcontrol cells with no plasmid were grown in supplementedOzeki medium with lactose as carbon source and treated with mitomycin C (MC). (Upper) At intervals the amount of aMe['4C]Glc (5 jxCi/,umol, 0.125 ,uCi/ml, from New England Nuclear; 1 Ci = 3.7 x 1010 becquerels) taken up in 3 min at 280C by 0.2 .ml of culture was determined. (Lower) Rate of NphGal hydrolysis by whole cells was determined relative to rate of hydrolysis by CHC13/sodium dodecyl sulfate-lysed cells (each in 0.02 ml of culture). In addition, in one experiment (Lower Right) at intervals 1-ml samples were removed from induced A153(pRLB6) cultures and centrifuged, and the rate of NphGal hydrolysis by samples of the supernatant was determined (v). DISCUSSION sumed to be due to a protein, the product ofthe imm gene. The The ColEl plasmid, in addition to the known cea and imm ColEl immunity protein has not been isolated. The immunity genes, appears to contain a kil gene. The cea and kil genes are protein of the ColIa plasmid has been found localized to the presumably part ofa single operon, whose expression is induced cytoplasmic membrane (17). by treatment of plasmid-containing bacterial cells with mito- The present experiments provide information about the in- mycin C. Most interruptions of the cea gene result in loss of terplay among the Kil, Imm, and Cea functions in bacteria coli- function by the kil gene, although some deletions in cea pre- cinogenic for ColEl plasmids. When a ColEl plasmid is cea' serve transcription and expression ofthe kil gene. It should be kit- imm', the bacteria survive mitomycin C induction. C.olicin pointed out that the presence of a ColEl kil gene is postulated is synthesized and accumulates inside the live bacteria, which on the basis of only a small number of genetic observations. It can, however, then proceed to form colonies. How colicin pro- is not excluded that, for example, a protein-protein interaction duction terminates in these induced cells is unknown. between the The Imm function is responsible for protection of induced COOH-terminal regions of the colicin and im- cells from damage by endogenous colicin. If bacteria have a munity substance might simulate the action of a kil gene. Yet plasmid cea' kil- imm-, the cells do die and release colicin. In the kil gene concept is reinforced by analogy with a lethality this case, however, cell death is due not to the Kil function but gene H that has been identified in the genome of plasmid to endogenous colicin itself. The difference between the two CloDF13, in which the H gene, the cloacin gene, and the im- lethal processes-by Kil product vs. endogenous colicin-is munity gene are all members of a single operon that can be in- striking. Killingby endogenous colicin is indistinguishable from duced by mitomycin C treatment (15, 16). killing ofcolicin-sensitive bacteria by exogenous colicin, whose In ColEl the imm gene, responsible for immunity to exog- effects have been explained by formation of transmembrane enous colicin, appears to be in a separate operon transcribed channels and loss ofmembrane potential (18, 19). The similarity in the opposite direction from the cea gene (6, 12); it is not af- of action of exogenous and endogenous colicin confirms that fected by insertions or deletions within the cea gene. It may priorinteraction with outer membrane receptors is not required overlap with the kil gene. The immunity phenotype is pre- for colicin action and reinforces the relevance of experiments Downloaded by guest on September 28, 2021 Microbiology: Suit et aL Proc. Natl Acad. Sci. USA 80 (1983) 583

It seems possible that the lethality genes in plasmids con- 24 cea. imm- ferring bacteriocinogenicity are homologous as well as func- 18 jA pJFS349 tionally analogous to each other. A further suggestive analogy 15 is to certain lysis genes of bacteriophages (gene s of A; gene t ofT4) whose products are needed to allow endogenous phage Total External lysozyme to reach the peptidoglycan layer and induce lysis (22, aMeGlcP a]MeGlcP 23). Experiments to be reported elsewhere have shown that the 6 kil gene product of ColE1 and the s gene product of phage A complement each other efficiently. The action of the A s gene as recently reported (24) is very similar to that of the kil gene 3 on induced colicinogenic cells. Whether this is a significant or accidental similarity shall await sequence analysis of the cor- Internal responding genes and purification ofthe respective gene prod- x 0 ° /aMeGlc + aMeGlcP ucts. 0A AI - We thank Robert T. Sauerforamino acid sequence analysis ofcolicin kil+ pRLB6 a El and Leonard Guarente for helpful discussion of the manuscript. Researchreported in this paper was supported by grants to S.E.L. from the National Science Foundation (PCM-81-08866) and the National In- stitute ofAllergy and Infectious Diseases (5-R01-A103038) and by fund- ing awarded to. J.F.S. and R.L. by the Undergraduate Research Op- 0- portunities Program at Massachusetts Institute ofTechnology. No plasmd a~~~- 1. Shafferman, A., Flashner, Y. & Cohen, S. (1979) Mol. Gen. Ge- net. 176, 139-146. 3 2. Sabik, J. F., Suit, J. L. & Luria, S. E. (1983) J. BacterioL, in press. 3. Nagel de Zwaig, R. & Luria, S. E. (1967)J. BacterioL 94, 1112- 1123. 01Lo. -I 1I 4. Hashimoto-Gotoh, T. & Inselburg, J. (1979) J. Bacteriol 139, -0 30 60 90 597-60. Time, min 5. Yamada, M., Ebina, Y., Miyata, T., Nakazawa, T. & Nakazawa, aM~e[14C]GIC MC A. (1982) Proc. NatL Acad. Sci. USA 79, 2827-2831. 6. Oka, A., Nomura, N., Morita, M., Sugisaki, H., Sugimoto, K. FIG. 4. Effect of mitomycin C induction on aMeGlc retention. & Takanami, M. (1979) Mol Gen. Genet. 172, 151-159. Cells of A153 carryingthe plasmids indicated in the figure or control 7. Sharp, P. A., Sugden, B. & Sambrook, J. (1973) Biochemistry 12, cells with no plasmid were grown in supplemented Ozeki medium with 3055-3063. lactose as carbon source. aMe[(4C]Glc (1 juCi/pumol, 0.1 juCi/ml) was 8. Luria, S. E., Adams, M. J. & Ting, R. C. (1960) Virology 12, addedto the culture 10 min prior to mitomycin C. At intervals samples 348-390, were removed and assayed for the uptake of aMeGIc and accumula- 9. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold tion and efflux of aMeGlcP (11). Total (A), internal (o), and external Spring Harbor Laboratory, Cold Spring Harbor, NY). (.) values refer to amounts of radioactivity associated. with the total 10. Plate, C. A. (1976)J. Bacteriol 125, 467-474. cell suspension, the filtered cells, and the filtrate, respectively. 11. Jetten, A. M. (1976) Biochim. Bophys. Acta 440, 403-411. 12. Patient, R. K. (1979) Nucleic Acids Res. 6, 2647-2665. with colicin on artificial membranes (14, 18). 13. Fields, K. L. & Luria, S. E. (1969)J. Bacteriol 97, 57-63. 14. Death by Kil remains a Kayalar, C. & Luria, S. E. (1979) in Membrane Bioenergetics, function biochemical mystery. Loss eds. Lee, C. P., Schatz, G. & Ernster, L. (Addison-Wesley, ofaccumulation and phosphorylation ofaMeGlc and increased Reading, MA), pp. 297-306. accessibility of bacterial /-galactosidase to its exogenous sub- 15. Hakkart, M. J. J., Veltkamp, E. & Nijkamp, H. J. J. (1981) Mol strates. suggest a nonspecific membrane damage, which is in- Gen. Genet. 183, 318-325. dependent of colicin action because it occurs with cea- plas- 16. Hakkart, M. J. J., Veltkamp, E. & Nijkamp, H. J. J. (1981) Mol mids. Delayed leakiness ofcellular enzymes (20) and some late Gen. Genet. 183, 326-332. 17. Weaver, C. A., Redborg, A. H. & Konisky, J. (1981)J. Bacteriol phospholipid hydrolysis (unpublished observations) are pre- 148, 817-828. sumably secondary effects of the Kil gene function. 18. Luria, S. E. & Suit, J. L. (1982) in Membranes and Transport, The Kil function appears to determine release ofcolicin from ed. Martonosi, A. (Plenum, New York), Vol. 2, pp. 279-283. induced cells. In the CloDF13 plasmid the lethality gene H is 19. Cramer, W. A., Dankert, J. R. & Uratani, Y. (1983) Biochim. reported to code for a small protein predominantly located in Biophys. Acta, in press. the cellular envelope (15) and required for export ofcloacin (21). 20. Jakes, K. S. & Model, P. (1979)J. Bacteriol 138, 770-778. 21. Oudega, B., Steghuis, F., van liel-Menkveld, G. J. & de Graaf, Processing of the ColEl cea product does not appear to be in- F. K. (1982)J. Bacteriol 150, 1115-1121. volved in the export ofcolicin because colicins El synthesized 22. Reader, R. W. & Siminovitch, L. (1971) Virology 43, 623-637. in vivo or in vitro are chemically identical (20). Intracellular and 23. Josslin, R. (1971) Virology 44, 101-107. released colicin have the same NH2-terminal sequence (18). 24. Wilson, D. B. (1982)J. Bacteriol 151, 1403-1410. Downloaded by guest on September 28, 2021