binding protein

Proc. NatL Acad. Sci. USA Vol. 80, pp. 4422-4426, July 1983

Genetics

F sex factor of Escherichia coli K-12 codes for a single-stranded DNA binding protein (replication/plasmid/ssb gene/asf gene)

ALEX L. KOLODKIN*, MIKE A. CAPAGEt, EFIM I. GOLUB, AND K. BROOKS Low Radiobiology Laboratories, Yale University School of Medicine, New Haven, Connecticut 06510

Communicated by Edward A. Adelberg, March 28, 1983

ABSTRACT In Escherichia coli K-12 strains that carry the mutation ub-1 in the gene for single-stranded DNA binding protein, the presence of the F sex factor partially reverses the temperature-sensitive growth phenotype caused by the mutation. The region of F (EcoRI fragment 3) responsible for this compensation has been identified and subcloned onto pBR322. A BamHI cleavage site has been found to intersect the essential coding region for this F function. By using this site, mutational blocks in the function have been constructed and used to identify a protein product (Mr approximately 22,000, slightly larger than the E. coli K-12 singlestranded DNA binding protein) which is correlated with the ssb1-complementing activity. Labeled extracts from maxicells were used to show that this protein binds tightly to single-stranded DNA. The gene on F that codes for this protein is denoted ssf and is located at approximately 55.2 kilobases on the standard map of F, in the region transferred very early during bacterial conjugation.

Escherichia coli K-12 produces a number of DNA binding proteins, including the single-stranded DNA binding protein (SSB) coded for by the ssb gene (for a review, see ref. 1). Analysis of conditional lethal mutants of ssb, such as ssb-1 and ssb-113, has indicated that SSB (the ssb product) is essential for replication of the chromosome and certain small bacteriophages (2), for the SOS response to DNA damage (stimulation of DNA repair and prophage induction) (3), and for genetic recombination in certain types of crosses (unpublished data). In addition, a number of in vitro systems for reactions related to replication, recombination, and SOS induction have been shown to depend on SSB (for a review, see ref. 4). We report here that the F sex factor of E. coli codes for its own single-stranded DNA binding protein, and that this protein can partially complement the ssb-1 defect in the chromosomal SSB. This represents a case in which a given DNA polymerase (from E. coli) can interact with more than one single-stranded DNA binding protein. As such, it contrasts with the previously apparent unique correlation of DNA polymerases with single-stranded DNA binding proteins (for a review, see ref. 1).

gifts of A. Sancar. Other protein molecular weight standards (transferrin, 81,000; bovine serum albumin, 66,000; ovalbumin, 43,000; DNase I, 31,000; myoglobin, 17,600; lysozyme, 14,400; and bacteriophage fd gene 5 protein, 9,070) were the gift of K. R. Williams. Genetic Procedures. Conjugation, transduction, and transformation procedures were as described (10-12). Because of residual growth of ssb-J cells upon shift to high temperature, routine tests of this Ssb- phenotype were carried by a modified ("double velvet") replica plating technique as follows. A master grid of candidate recombinant patches was grown and replica plated (10) onto a plain LB or minimal medium plate. This was then used, without further incubation, as a master plate to replicate onto two other minimal medium plates, containing 0.4% Casamino acids (Difco) plus 0.3% glucose as well as any other required growth factors. These second (much diluted) replicas of the original master grid were then incubated overnight, one at 350C and one at 420C. Growth at 420C was clearly absent for ssb-1 strains and strong for ssb-1/F+ or ssb+ strains, whereas all these strains grew well at 350C. Plasmid Analysis. Routine size analysis of plasmid DNA was carried out by using the procedure of Birnboim and Doly (13). Larger-scale isolation of plasmid DNA, routine digestions with restriction enzymes, and gap-filling (Klenow enzyme) and ligation reactions all were done as described (12, 14). Plasmid Protein Identification. The maxicell procedure of Sancar et al. (6) was used, with slight modifications, to identify protein products from the plasmids. Protein standards were run on the NaDodSO4/10% polyacrylamide gels so that the Mr of the SSB could be determined. In order to check for binding to single-stranded DNA, the final maxicell extract was applied to a 1 x 13 cm denaturated DNA-cellulose column and eluted as described in the text and figure legends. RESULTS Partial Suppression of ssb-1 by F and by Certain Cloned F Fragments. In the course of constructing various E. coli K-12 strains carrying the ssb-1 mutation, it was found that the introduction of an F factor caused a partial reversal of the usual temperature-sensitive growth phenotype characteristic of ssb1. Whereas F- ssb-1 strains failed to grow at 420C on Casamino acid-containing minimal medium, F+ ssb-1 cells grew almost normally. A more quantitative measure of this effect was obtained by assaying the survival of both types of strains as a function of the incubation temperature of plates onto which ex-

MATERIALS AND METHODS Plasmids. The basic strains and plasmids used and Strains are listed in Table 1. Media and Reagents. Luria broth without glucose and modified minimal medium 56 were used as described (10). BamHI, T4 DNA ligase, and DNA polymerase large fragment (Klenow fragment) were from New England BioLabs. EcoRI and A DNA were from Bethesda Research Laboratories. Denatured DNAcellulose was from P-L Biochemicals. [3S]Methionine was from New England Nuclear. Purified SSB and recA protein were

Abbreviations: SSB, single-stranded DNA binding protein; SSF, singlestranded DNA binding protein produced by ssf (a gene on the F factor of E. coli K-12). * Present address: Institute of Molecular Biology, Univ. of Oregon, Eugene, OR 97403. t Present address: Synergen, Boulder, CO 80301.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4422

Genetics: Kolodkin et al.

Proc. Natl. Acad. Sci. USA 80 (1983)

Table 1. Bacterial strains and plasmids Strain or plasmid Genotype KL450 KL451 KL452

CSR603

pBR322 pRS5 pRS15 pRS21 pRS26 pRS27

pRS53 pKL1 pKL2 pKL3

pKL4

E. coli K-12 strain ssb-1 (ref. 2) derivative of W1485F4 (ref. 5) cured of F ssb-1 (ref. 2) derivative of W1485F+ (ref. 5) F+ ssb-1 KL450 transformed with ligation mixture pKLl/ssb-1 (F-) from pBR322 + pRS53 (each cut with EcoRI) -* TetR at-420C A. Sancar; (ref. 6) F- recAl uvrA6phr-1 thi-1 thr-1 leu-6proA2 argE3 ara-14 lacYl galK2 xyl-5 mtl-i rpsL31 tsx-33

Plasmid (tet+ bla+) As plasmid pSC101 + EcoRI F fragments 3, 5, 6, & 7 As plasmid pSC101 + EcoRI F fragments 1, 2, 4, 9, 10, 12, 13, 14, 16, 17, & 19 As plasmid pSC101 + EcoRI F fragment 5 As plasmid pSC101 + EcoRI F fragments 1, 2, 12,15, 17, & 19 As plasmid pSC101 + EcoRI F fragment 6 As plasmid pSF2124 + EcoRI F fragment 3 As pBR322 + EcoRI F fragment 3 tet-; has smaller EcoRI-BamHI fragment from pKL1 tet-; has larger EcoRI-BamHI fragment from pKL1 Similar to pKL1 As pBR322 but BamHIAs pKL1 but BamHI- on portion from pBR322

pEG303

As pEG302 but BamHI- on F fragment As pKAC50 but BamHI- on F fragment See Fig. 1 See Fig. 1; vector portion of pKAC50

pKAC50 pKAC51

Source (ref.)

F- ssb-1

pEG301 pEG302

pEG310

4423

A. Sancar; (ref. 7) A. J. Clark; (ref. 8) A. J. Clark; (ref. 8) A. J. Clark; (ref. 8) A. J. Clark; (ref. 8) A. J. Clark; (ref. 8)

A. J. Clark; (ref. 9) See KL452 EcoRI/BamHI digest of pKL1: ligation and transformation of KL450 -- AmpR, 350C EcoRI/BamHI digest of pKL1; ligation and transformation of KL450 -* AmpR, 350C EcoRI/BamHI digest of pKL2 + pKL3; EcoRI digest of pBR322; ligation and transformation of KL450 + TetR, 420C See Materials and Methods EcoRI digest of pEG301 + pKL1; ligation and transformation of KL450 -* AmpR, 420C See Materials and Methods

See Materials and Methods J. W. Chase J. W. Chase

TetR, tetracycline resistant; AmpR, ampicillin resistant.

ponentially growing cells had been spread. The resulting survival (Fig. 1) confirmed the initial finding and showed that the presence of an F factor increases the highest survival temperature of an ssb-1 strain by about 2°C. However, even the F+ ssb-1 strain had a low survivability at 440C. [Ssb+ cells plated with approximately 100% efficiency at 440C (data not shown).] When the region of F that caused this phenotype was cloned onto a high-copy-number vector (see below), the resulting plasmid (pKL1) was found to reverse the Ssb- temperature-sensitive growth phenotype even at 440C (Fig. 1). In order to find the F factor gene(s) responsible for ssb-1 suppression (or complementation), use was made first of the series of clones of EcoRI restriction fragments of F DNA isolated and analyzed by R. A. Skurray, A. J. Clark, and colleagues (8, 9, 15). Each of six plasmids with cloned EcoRI fragments from F (see pRS plasmids, Table 1) was transformed into strain curves

KL450 (ssb-1) and the resulting strains were tested for temperature resistance. Two of the plasmids tested, pRS5 and pRS53, caused suppression of the Ssb- phenotype in a manner similar to the F factor, whereas the other four plasmids (pRS15, pRS21, pRS26, and pRS27) did not (data not shown). This suggested that the ssb-l-suppressing function(s) is coded for by EcoRI fragment 3 which is the only EcoRI fragment carried by pRS53 (8, 9) and is also carried by pRS5 but not any of the other pRS

plasmids tested. EcoRI fragment 3 was subeloned from pRS53 into the EcoRI site of plasmid pBR322 (Table 1; Fig. 2) to obtain plasmid pKLl, which suppresses the ssb-1 temperaturesensitivity even better than the F factor. Genetic Analysis of the Subcloned Fragment That Suppresses ssb-1. In order to localize further the ssb-1 -suppressing gene(s) on pKL1, use was made of a BamHI restriction site known to lie near the middle of EcoRI fragment 3 (16). Because pBR322

Genetics: Kolodkin et al.

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Proc. Natl. Acad. Sci. USA 80 (1983)

lies in the gene(s) responsible for ssb-l-suppressing (or complementing) activity. To ensure that plasmids pKL2 and pKL3 were not inactive (for ssb-1 suppression) because of undetected deletions formed during their construction, they were used to reconstruct a plasmid similar to pKL1 such that the original sequence in the middle of EcoRI fragment 3 of F was restored. The resulting plasmid, pKL4, was found to suppress the Ssb- temperature sensitivity as well as pKLL did (data not shown), thus supporting the indication that the BamHI site in pKLI lies within a coding region that is essential for the ssb-1 suppression. (Alternatively, there could be two or more ssb-1-suppressing genes, one on the portion cloned on pKL2 and another on pKL3, so that neither plasmid alone would cause suppression of ssb-1. However, see evidence to the contrary given below.) The importance of the BamHI site per se in KLF1 was further established by mutating this site specifically by filling in the single-stranded ends of the cleaved site and religating it (Table 1; Fig. 2). The resulting plasmid, pEG303, was found to have lost ssb-1-suppressing activity (data not shown). This indicates that the BamHI site is indeed within a critical coding region for a function that is involved in reversal of the temperature-sensitive growth phenotype of the ssb-1 defect. This procedure of mutation at the BamHI site was repeated with a smaller subclone of pKL1, on plasmid pKAC50 to produce plasmid pEG310. As in the former case, mutation of the BamHI site led to a loss of ssb-1-suppressing activity (data not shown). Correlation of a Protein Product with the ssb-1-Suppressing Activity. In order to search for a product of the pKL1 plasmid that is responsible for suppression of ssb-1, the maxicell method (6) was used to label radioactively the protein products of pKL1 and its derivatives. At least three protein products were produced from pKL1 which were not produced from the vector pBR322-namely, the bands labeled A, B, and SSF in Fig. 3. Bands A and B were produced by plasmid pKL3 which did not suppress the Ssb- phenotype. However, band SSF was missing from pKL3 (and also from pKL2) and thus is correlated with ssb-1 suppression. This same correlation holds for plasmids pKAC50 and pEG310 (Fig. 4). The Mr of SSF estimated from

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also contains one BamHI site located within the tetracyclineresistance determinant (7), it was possible to subclone each half of the EcoRI fragment from pKL1 by using pKL1 DNA that had been cleaved with BamHI and EcoRI, as indicated in Fig. 2 and Table 1. The two resulting plasmids, pKL2 and pKL3, were tested for ssb-l -suppressing activity; neither plasmid provided suppression of the temperature-sensitive growth defect. This suggested that the BamHI site (on the portion of F on pKL1)

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Proc. Natl. Acad. Sci. USA 80 (1983) DNTA cellulose

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