Proc. Nail. Acad. Sci. USA Vol. 82, pp. 8389-8393, December 1985 Biochemistry Structural and functional organization of the colicin El operon (gene expression/induction/SOS system/and-sense RNA/molecular cloning) NAHID S. WALEH AND PAUL H. JOHNSON Molecular Biology Program, SRI International, Menlo Park, CA 94025 Communicated by Robert L. Sinsheimer, August 16, 1985 ABSTRACT We analyzed the structural and functional caused by the lys gene product is biochemically distinct from relationships among independently cloned segments of the cell death caused by the action of either exogenous or plasmid ColEl region that regulates and codes for colicin El endogenous colicin on the nonimmune bacterial membrane. (cea), immunity (imm), and the mitomycin C-induced lethality The results presented in this report demonstrate that the function (lys). On the basis of physiological properties, restric- colicin El operon represents a novel gene arrangement in tion endonuclease mapping, and DNA sequence analysis, the Escherichia coli. The discontinuity of the colicin operon and following recombinant plasmids were determined to represent the overlapping nature of individual transcriptional units and three major functional classes: pNP12 (cea+, imm+, lys+), regulatory sequences suggest an unusual economy ofgenetic pNP4 (cea+, imm+, lys-), and pNP6 (cea+, imm-, lys-). Our information that might have several important consequences results have established the order, boundaries, and relative for the control of gene expression in this system. orientation of the three structural genes, the location of the promoter region for imm gene transcription, and the predicted MATERIALS AND METHODS amino acid sequences of the imm and lys gene products. Hydropathicity analysis suggests that both proteins have hy- Strains and Media. A colicin-producing derivative strain of drophobic amino acid segments characteristic of membrane- JC411 (7) was the source of ColEl plasmid. E. coli K-12, associated proteins. A model for the structure and expression strain 294 (endoP hsdR- hsdM' thi-) (16), was the host for of the colicin El operon is proposed in which the cea and lys pBR322 and the recombinant plasmids. Strain CL142 (K-12 genes are expressed from a single inducible promoter that is Row) was used as the indicator strain that was sensitive to all controlled by the lexA repressor in response to the SOS system colicins (17). of Escherichia coli. The imm gene lies between the cea and lys Preparation of Sheared ColEl Fragments. Plasmid DNA genes and is expressed by transcription in the opposite direction was purified as described (18). Approximately 140 ,ug of from a promoter located within the lys gene. This arrangement open-circular ColEl DNA in 0.3 M sodium acetate was of structural genes indicates that the transcriptional units for sheared at 3800 rpm for 20 min at 00C, using the microattach- all three genes overlap. We suggest that the formation of ment of a DuPont Omnimixer. The sheared DNA was anti-sense RNA may be an important element in the coordinate purified by velocity centrifugation using a 5-20% linear regulation of gene expression in this system. sucrose gradient. Colicin Tests. The enhanced production of colicin by Colicin El can be induced by treating cells containing transformants was determined by the procedure ofMales and plasmid ColEl with agents that damage DNA or interfere Stocker (19). Colicin was released from induced cells by with DNA synthesis (1). Previous work has demonstrated using chloroform, according to the procedure of Suit et al. that colicin induction is one of the SOS responses (2-4). The (15). SOS repressor, lexA protein, is also the colicin El gene repressor (5). The colicin El gene (cea) has been sequenced RESULTS (6), and its protein product has been purified (7). Colicin El has been characterized as a protein that forms ion channels Molecular Cloning of the Colicin Gene Region. Sheared in the bacterial membrane, leading to cell death (8, 9). fragments of ColEl DNA were cloned into plasmid pBR322 The size and location of the colicin El immunity gene using the poly(dG-dC) tailing procedure (20). Twenty (6.8%) (imm) have not been well-documented. On the basis of of 368 randomly selected tetracycline-resistant (Tcr) ampicil- transposon mutagenesis, a region of the ColEl plasmid lin-sensitive (Aps) transformant colonies were found to pro- adjacent to the colicin El gene has been proposed for the duce colicin (Col+). Transformant strains were designated immunity function (10-12). Transcriptional studies and DNA NP1-NP20; the corresponding plasmids were designated sequence analysis indicate that an open reading frame exists pNPl-pNP20. in which the transcriptional direction is opposite that of the Physical Mapping. Restriction endonucleases Pst I, EcoRI, colicin El gene (13). The immunity protein has not been and Sma I were used to construct maps of ColEl-derived isolated, however, and there is no direct evidence that the fragment inserts carried by Col' recombinant plasmids pNP1- presumed polypeptide product of this DNA sequence is pNP20. Fig. 1 presents a summary of these results in which responsible for immunity to colicin El. the individual physical maps are oriented to the known Upon induction, death of the bacteriocinogenic cells oc- organization and direction oftranscription ofthe colicin gene, curs concomitantly with the overproduction of colicin. Cell the proposed immunity region, and the origin of ColEl death occurs in the absence of active colicin molecules (14). replication (11). Recombinant plasmids are ordered by in- A gene (here designated lys) that is close to or overlaps the creasing length of the Pst I fragment that contains the immunity region ofColEl plasmid has been postulated for the COOH-terminal coding region ofcolicin and extends through mitomycin C-induced lethality function (15). Cell death the proposed immunity region toward the ColEl replication origin. Arrows indicate the orientation of the inserts to the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: cea, colicin gene; imm, immunity gene; lys, lysis in accordance with 18 U.S.C. §1734 solely to indicate this fact. gene. 8389 Downloaded by guest on September 25, 2021 8390 Biochemistry: Waleh and Johnson Proc. Natl. Acad. Sci. USA 82 (1985) Plasmid Physical Map NP12, NP6, and NP4 are presented in Fig. 2 and Table 1. Plasmids pNP12 and pNP6 contain fragments of ColEl DNA Cea 1mm that extend, respectively, 580 and 50 base pairs beyond the 600bp ' COOH-terminal end of the colicin gene. Strains carrying Col El these two plasmids, however, exhibit similar sensitivity ' PstI Pst I patterns to mitomycin C. A decrease by a factor of 10 in the pNP6 ~~~~~~~~~~Ori pNP77 viability of both cultures is observed within 2 hr after the pNP1 _----- addition of the inducing agent (Fig. 2). pNP13 --_ __ _i_(_ ) C induction, cells carrying plasmids with pNP 8 s-___ Upon mitomycin pNP 4 a functional lys gene are killed and colicin is released into the pNP14 H medium (15). Moreover, plasmid-carrying cells lacking a pNP12 (.) functional lys gene but protected from the action of endog- pNP16 ' .--- enous are not pNP17 (4) colicin by the immunity function killed, and the pNP99() colicin is not released into the medium. The results presented pNP 5 (+) in Table 1 indicate that most of the colicin synthesized by pNPl1 ( pNP155 , -------- - NP12 cells is released, whereas colicin made by NP6 cells is pNP10 >___ + released only when the cells are disrupted with chloroform. pNP 2 - --- These results, together with the observation that NP12 cells pNP19 (__- pNP 3 are immune to colicin El and yet are killed upon induction pNP20 with mitomycin C, indicate that pNP12 carries a functional lys gene. On the basis of a similar argument, we conclude that pNP6 lacks a functional lys gene and that the observed FIG. 1. Physical maps ofColEl-derived fragments. Wavy arrows represent the direction of transcription of the colicin and immunity lethality is due to the intracellular toxicity of colicin in the genes. Arrowheads indicate the orientation ofthe inserts with respect absence of a functional immunity gene. Plasmids pNP1, to the direction oftranscription from the A-lactamase promoter ofthe pNP7, and pNP8, which also lack the immunity function, cloning vector pBR322. Ori represents the replication origin of confer upon their host the same properties with respect to ColEL. Heavy lines indicate the Pst I fragments containing the mitomycin C-induced lethality and colicin release as does carboxyl-terminal coding region of the colicin gene. The presence pNP6 (data not shown). and absence of immunity function is indicated by (+) and (-), respectively. bp, Base pairs. Plasmid pNP4 contains a region that extends 400 base pairs beyond the COOH-terminal end of the colicin gene. Strain NP4, in contrast to NP6 and NP12, shows only a partial loss direction of transcription from the p-lactamase promoter of ofcell viability upon mitomycin C induction. Phenotypically, the pBR322 cloning vector. The Col+ fragments were insert- ed in both orientations with approximately equal frequency. NP4 is imm'. However, when the 600-base-pair Pst I Plasmid pNP18 was not mapped because it produced ambig- fragment of pNP4 (Fig. 1) is isolated and recloned into uous results upon double digestion with endonucleases Pst I and EcoR. 100 Functional Characterization of Transformants. To distin- guish between transformant strains that were immune to 50 colicin El and those that had lost their outer membrane btuB protein and had therefore become resistant to all E colicins, we tested strains NPl-NP20 for immunity to colicins El-E7. The btuB protein is essential as a receptor-binding site for E colicins, vitamin B-12, and phage BF23 (21-23). The immu- 10 nity properties of the transformed strains are represented in Fig. 1 by a (+) for recombinant plasmids that were immune 0.50 to colicin El and a (-) for those that were not.