0021-9193/78/0133-0354$02.00/0

Vol. 133, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Jan. 1978, p. 354-363 Copyright © 1978 American Society for Microbiology

Cloning of Beneckea Genes in Escherichia coli HILDEGARD LAMFROM, ANAND SARABHAI, AND JOHN ABELSON* Department of Chemistry, University of California, San Diego, La Jolla, California 92093 Received for publication 31 May 1977

Genes from Beneckea harveyi, a luminescent marine bacterium, were cloned in Escherichia coli. This was done by producing randomly sheared fragments of Beneckea DNA and inserting them into the EcoRI site of plasmid pMB9 by the adenine-thymine joining procedure. The hybrid plasmids were used to transform E. coli C600 SF8. Among the transformants selected for tetracycline resistance, one clone that appeared to complement a leucine B mutation was identified. The transformants were screened for the presence of Beneckea 5S genes. Four of these clones were analyzed in detail by hybridization with 16S, 23S, and 4S Beneckea RNA. The observations suggest that the ribosomal genes in Beneckea are linked, but are present in a different order than those in E. coli. An approach utilizing recombinant DNA techniques is particularly useful in systems in which the techniques for the transfer of genes are limited. We have been interested in the luminous marine bacterium Beneckea harveyi. Although genetic analysis of the luciferase gene is possible in this organism, it has not been possible to transfer the gene. We have undertaken a project to isolate the luciferase gene from Beneckea by cloning it in Escherichia coli. Although we have not as yet been able to do this, we have characterized some of our clones and report here two results from this project. We were able to complement a leucine auxotroph of E. coli with a plasmid-carrying Beneckea DNA, indicating that at least one of the Beneckea genes can be expressed in E. coli. We also isolated several plasmids that carry portions of the ribosomal RNA cluster of Beneckea. Analysis of these plasmids gives us some preliminary clues to the organization of rRNA genes in this species. We observed that in Beneckea the ribosomal genes are linked, but they do not appear to be present in the same order as those in E. coli. MATERIALS AND METHODS Preparation of Beneckea DNA. The luminous marine bacterium B. harveyi 392 was grown and stored at 24°C on nutrient agar plates made with seawater (4). High-molecular-weight Beneckea DNA was isolated from bacteria grown in seawater base medium (4) at 24°C overnight. The cells were suspended in a solution of 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 8.0), 0.1 M NaCl, and 0.01 M ethylenediaminetetraacetate (EDTA), and lysozyme was added to a concentration of 1 mg/ml. After 5 min of incubation on ice, the solution was adjusted to contain 1% sarcosyl and 1 mg of Pronase per ml, and the incubation was continued for 1 h at

37°C. (Pronase had been predigested for 1 h at 37°C.) DNA was separated from proteins by two phenol extractions, concentrated by ethanol precipitation, and banded on a CsCl density gradient. After dialysis against 0.05 M Tris (pH 8.0), 0.1 M NaCl, and 0.01 M EDTA, Beneckea DNA was stored at 4°C. High-molecular-weight Beneckea DNA (8 ,ug of DNA per ml in 1 M NaCl and 0.01 M EDTA) was sheared to fragments of an average size of 5 x 106 daltons in a Virtis homogenizer at 0°C. The sheared DNA was concentrated by ethanol precipitation and suspended in 0.01 M Tris (pH 8.0) and 0.001 M EDTA. The size of the DNA fragments was estimated by centrifugation through a 15 to 30% sucrose gradient as well as by electrophoresis through 0.8% agarose gel, using A DNA restriction fragments as markers. Preparation of 32P-labeled Beneckea RNA: 4S, 5S, 16S, and 23S. B. harveyi 392 was grown for 8 h at 30°C to an optical density at 650 nm of 0.9 in 100 ml of low-phosphate minimal medium (17) containing 1% NaCl and 10 mCi of 32P. The cells were washed with Beneckea minimal medium (11) containing 1% NaCl and suspended in 5 ml of lysis buffer (0.02 M Tris [pH 7.4], 0.2 M NaCl, 0.04 M EDTA, and 1% sodium dodecyl sulfate (SDS). Lysis was achieved by heating for 2 min at 100°C, followed by rapid cooling on ice. After two extractions with phenol, the RNA was precipitated twice with ethanol. RNA was fractionated in one tube in a 15 to 30% sucrose gradient (gradient buffer: 0.01 M Tris [pH 7.4], 0.1 M LiCl, 0.001 M EDTA, and 0.5% SDS) in a Beckman SW27 rotor at 25,000 rpm for 21.5 h at 10°C. By monitoring the radioactivity, it was found that 16S and 23S RNA were well separated, and the 4S and 5S RNA remained at the top of the gradient. The 16S and 23S RNA peak fractions were conservatively pooled, concentrated by ethanol precipitation, dissolved in 0.01 M Tris (pH 7.6), and stored at -20°C. The purity of the 16S and 23S RNA samples was verified by electrophoresis on 1.4% agarose gel and by hybridization to Hae III and Hap II restriction fragments of cloned Beneckea DNA. It was found that some fragments hybrid354

VOL. 133, 1978

CLONING OF BENECKEA GENES IN E. COLI

ized exclusively to 16S 32P-labeled Beneckea RNA and others solely to 23S RNA (data not shown). The mixture of 4S and 5S [32P]RNA was precipitated with ethanol and fractionated by electrophoresis through a 10% acrylamide gel. The 4S and 5S RNA species were located by autoradiography and eluted from excised, crushed gel fragments with 0.3 M NaCl at 37°C. The acrylamide was removed by centrifugation, and the RNA was further clarified through a Swinex filter with glass and membrane filters (Millipore Corp., Bedford, Mass.) back-to-back. Each RNA sample was then concentrated on a diethylaminoethylcellulose 52 column, equilibrated and washed with 0.01 M Tris (pH 7.4) containing 0.3 M NaCl. The samples were eluted with 0.01 M Tris (pH 7.4) containing 1.0 M NaCl. The eluate fractions containing the RNA were located by monitoring the radioactivity. They were pooled, and the RNA was precipitated with ethanol, redissolved in 0.01 M Tris (pH 7.4), and stored at -200C. Preparation of plasmid DNA. The plasmid pMB9 (21) with the selective marker Tcr (tetracycline resistance) (kindly given to us by H. W. Boyer) was grown in E. coli W3110. The DNA from pMB9, as well as from pMB9 carrying Beneckea DNA (hybrid plasmid), was isolated by the method of V. Hershfield based on the procedures described by Clewell (3) and Katz et al. (10). The cells carrying plasmids were grown to late log phase in 1 liter of M-9 minimal medium (14) at 37°C. Chloramphenicol was added to 200 ,ug/ml, and plasmid amplification was allowed to proceed for 12 to 16 h at 37°C. Cells were washed with TES buffer (0.05 M Tris [pH 7.9], 0.05 M NaCl, and 0.05 M EDTA) and suspended in 10 ml of 0.05 M Tris (pH 8.0) containing 25% sucrose, and lysozyme was added to 1 mg/ml. The mixture was incubated for 10 min at 0°C, EDTA was added to 0.06 M, and the incubation was continued for 10 min. After addition of 10 ml of TLM buffer (1% Triton X-100, 0.05 Tris [pH 8.0], and 0.0625 M EDTA), the mixture was incubated for 30 min at 0°C. The chromosomal DNA pellet was removed by centrifugation for 20 min at 48,000 x g. The plasmid DNA was banded twice on a cesium chloride (CsCl)-ethidium bromide (EtBr)-density gradient (19) in TES buffer. EtBr was removed by exhaustive extraction with isopropyl alcohol (saturated with CsCl), and the DNA was subsequently dialyzed against a solution containing 0.01 M Tris (pH 8.0), 0.01 M NaCl, and 0.001 M EDTA. This procedure yielded from 100 to 500 ug of purified plasmid DNA per liter. The plasmid was stored at -20°C. Restriction of DNA by endonucleases. Restriction endonuclease EcoRI (a gift from Donald Helinski and his group) was prepared from E. coli Ryl3 by the procedure of Green et al. (6), as modified by Polisky et al. (18). Endonuclease Hap II was prepared from Haemophilus parainfluenza by the method of Takanami (29) and was donated by Frank Fujimura. The following enzymes were prepared in our laboratory by A. Otsuka, K. Fukada, and B. Druker by published methods: BamHI from Bacillus amyloliquefaciens H. (30); Sma I from Serratia marcescens Sb (Endow and Roberts; Green and Mulder, unpublished observations); Hae III from Haemophilus ae-

355

gyptius P. (20); and HindII + III and HindIII from Haemophilus influenza Rd. (23, 24). Digestions to completion were usually carried out at 37°C on 0.5 to 1.0,ug of plasmid DNA in a volume of 0.05 ml for 2 h. The reaction buffer used contained the following components: For EcoRI: 0.1 M Tris (pH 7.4), 0.05 M NaCl, and 0.005 M MgCl2. For all other restriction enzymes Roberts buffer was used, which contained 0.006 M Tris (pH 7.9), 0.006 M MgCl2, and 0.006 M 2-mercaptoethanol. Sequential digestion of DNA by both HindIII and EcoRI was carried out by first treating the DNA with HindIII in the usual buffer and then increasing Tris to 0.01 M and NaCl to 0.05 M for the cleavage by EcoRI. To insure that isolated bacterial DNA was restricted to completion, 10 times the quantity of restriction enzyme was used for 4 times longer than necessary for complete digestion of an equivalent amount of plasmid DNA. Incubation was terminated by heating the reaction mixture for 5 min at 65°C. Restriction was verified by agarose gel electrophoresis, and the size of the DNA fragments estimated relative to A restriction fragments. Gel electrophoresis was carried out on vertical slab gels (27) prepared with 1% agarose (28). Gels (12 by 11 by 0.3 cm) were made and run with the 10-fold diluted Tris-borate buffer described by Peacock and Dingman (16) and subsequently stained with EtBr (0.5,ug/mil in water). Gels were photographed with a long-wave-length transmission source on Polaroid type 52 or 55 film. Tailing of DNA. Extensions of polydeoxythymidylic acid [poly(dT)] were added to the 3' end (EcoRI site) of linear pMB9, and extensions of polydeoxyadenylic acid [poly(dA)] were added to the 3' end of sheared fragments of Beneckea DNA by a procedure based on that of Jackson et al. (8) and Lobban and Kaiser (12) as modified by Louise Clarke (personal communication). To add poly(dT) tails to pMB9, the reaction mixture contained 10 Mg of EcoRI restricted pMB9, 0.2 M K-cacodylate (pH 7.0) (Fisher Scientific Co., Pittsburgh, Pa., freshly made), 0.008 M 2-mercaptoethanol (freshly made), 0.002 M cobalt chloride, 2 x 10-4 M dTTP, 12.5 ,Ci of [3H]dTTP, and 40 U of nucleotidyl transferase (a gift from Winston Salser). The incubation time at 37°C required to add 50 residues of deoxyribosylthymine (dT) per end of DNA was determined in a trial incubation with Yio of the total reaction mixture and appropriately less enzyme. Under these conditions, approximately 100 residues were added per molecule of pMB9 DNA (50 residues per end) in 15 s at 37°C. The reaction was terminated by the addition of EDTA to a concentration of 0.01 M. To add poly(dA) tails to sheared Beneckea DNA fragments, the 50-,lI reaction mixture contained 0.1 M K-cacodylic acid (Fisher Scientific Co., freshly made), 0.008 M MgCl2, 0.002 M 2-mercaptoethanol (freshly made), and 0.0075 M KH2PO4 (adjusted to pH 7.0 with KOH) and 10 Mg of Beneckea DNA (average size, 5 x 106 daltons), 2 x 10-4 M dATP, 12.5 ,Ci of [3H]dATP, and 85 U of nucleotidyl transferase. The

356

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LAMFROM, SARABHAI, AND ABELSON

trial incubation for determining the incubation time was carried out as for dT tailing. Under the conditions described, approximately 100 residues were added per molecule of Beneckea DNA (50 residues per end) in 1.5 min at 37°C. The reaction was terminated by the addition of EDTA (0.01 M). The deoxyribosyladenine (dA)- and dT-tailed DNA samples were purified on a G75 Sephadex column equilibrated with 0.01 M Tris (pH 8.0), 0.1 M NaCl, and 0.001 M EDTA. The eluate fractions were monitored for trichloroacetic acid-precipitable radioactivity, and those containing the tailed DNA were pooled and stored at -20°C. Annealing of tailed DNAs. Equal amounts, by weight, of dT-tailed pMB9 and dA-tailed Beneckea DNA were mixed at 1 to 2 yg/ml in 0.01 M Tris (pH 8.0), 0.1 M NaCl, and 0.001 M EDTA. Annealing was allowed to proceed during three successive incubations: 5 min at 600C, 2 h at 410C, and 3 to 4 h of gradual cooling to room temperature. The annealed DNA was always immediately used for transformation. Transformation of E. coli with annealed hybrid DNA and selection of clones. E. coli C600 SF8 (C600 rk- mk- recBC lop-1l lig+ leuB thr-l thi-6 tonA supE44) (26) was used as the recipient for transformation. Competent cells for transformation were prepared as follows: A 1:20 dilution of an overnight stock solution was inoculated into L broth (14) and grown to 2 x 108 to 5 x 108 cells per ml while shaking for 3 h at 370C. The cells were sedimented by centrifugation and suspended in a ½h volume of cold 0.05 M CaCl2. After 15 min on ice, the cells were resedimented and suspended in l/io of the original volume of cold 0.05 M CaCl2. These competent cells were used immediately. Transformation was carried out in a mixture containing in one tube of annealed DNA (representing 0.01 jig of pMB9 DNA), 0.2 ml of buffer (0.01 M Tris [pH 8.0], 0.01 M CaCl2, and 0.01 M MgCl2), and 0.3 ml of competent cells. Three successive incubations were carried out: 25 min at 00C; 2 min at 39°C; and 10 min at 250C. Then 1 ml of L broth was added, and the reaction was incubated with shaking for 45 min at 37°C. The transformed cells were plated with soft agar containing 10 ,ug of tetracycline per ml on LB plates (14) containing 25 jig of tetracycline per ml (LB tet plates). The plates were incubated at 37°C. Transformants to tetracycline resistance were selected and transferred with toothpicks to LB test plates in a grid array of 50 colonies per plate. These master plates were used for further selection of specific clones. Replica plating of all colonies from a master plate was done in one transfer with a tool (1) whose prongs were mounted on a support plate in the same grid pattern as the clones on the master plate. Between transfers the tool was sterilized in an alcohol flame and cooled in sterile, distilled water. Screening of clones by in situ hybridization. Clones containing Beneckea DNA coding for 5S ribosomal RNA were selected from the collection of transformed E. coli colonies by in situ hybridization with 32P-labeled Beneckea 5S RNA. The method was a modified procedure described by Grunstein and Hogness (7). The colonies were transferred from the master plates to nitrocellulose (NC) filters (Schleicher

and Schuell Co., Keene, N.H.; BA 85, 0.45 ,m, diameter 90 mm, boiled three times in water and autoclaved for 10 min. Whatman no. 540 paper may be used instead of NC). The dry NC filter was carefully placed on the master plate and then immediately peeled off. The filters with the adhering colonies were air-dried, and the colonies were fixed on the filters by successively exposing them to the following solutions: 0.5 M NaOH for 10 min; 1.0 M Tris (pH 7.4) two times each for 2 min; 0.5 M Tris (pH 7.4) containing 1.5 M NaCl for 5 min; and 95% ethanol. The filters were then dipped into 0.3 M NaCl for 15 min, air-dried, and baked in a vacuum oven for 2 h at 800C. Hybridization was carried out individually for each filter, which was wetted with 1.0 ml of buffer (50% deionized reagent grade formamide, 4 x SSC [SSC is 0.15 M NaCl-0.015 M sodium citrate] and 0.4% SDS) containing 2 x 105 cpm of 32P-labeled Beneckea 5S RNA. Filters were incubated overnight at 37°C and then washed at room temperature for 20 min. Washing was performed twice in hybridization buffer made with technical grade formamide while agitating gently for 20 min and rinsed twice with 2 x SSC. The filters were then incubated in RNAse A solution (20 ,tg/ml in 2 x SSC) for 1 h at room temperature, followed by onewa8 g in 2 X SSC containing 0.5% SDS. The air-iiedb<ers were autoradiographed. Those colonies which appeared to have hybridized to the 32P-labeled Beneckea 5S RNA probe were purified, and the plasmid DNA was isolated as described above. Hybridization of DNA restriction fragments with RNA. The DNA restriction fragments were transferred from the agarose gel to an NC sheet (Schleicher and Schuell, B6), following the method described by Southern (25). They were then hybridized to the appropriate 32P-labeled Beneckea RNA species as follows. The NC sheets were wetted with a minimal volume of a solution containing 50%o deionized formamide, 4 x SSC, 0.5% SDS, and 4 x 105 cpm/ml of the particular 32P-labeled Beneckea RNA species, sealed into a plastic bag (Sears boilable cooking pouch), and incubated for 18 h at 370C. While remaining in the same bag, the filters were then treated with the following solutions at room temperature: twice with hybridization buffer (but containing technical formamide) for 2 h with gentle agitation; twice with 2 x SSC; once with RNAse (20 ,g/ml in 2 x SSC) for 1 h; and twice with 2 x SSC containing 0.5% SDS. The filters were then air-dried and analyzed by autoradiog-

raphy.

Electron microscopy. Double-stranded plasmid DNA was spread by the formamide or aqueous technique of Davis et al. (5). Contour lengths were measured relative to reference ColEl relaxed circles. Single-stranded "snap back" DNA was examined by the following procedure. The hybrid plasmid was treated with Bam, which cuts pMB9 once, but does not cleave the inserted Beneckea DNA sequence. The strands of the linear hybrid DNA were separated by alkali denaturation. Subsequent neutralization allowed pairing of inverted repeated sequences in the singlestranded DNA. The DNA was spread by the formamide method (5) and measured relative to singlestranded OX 174 DNA on the same grid. Luciferase assay. Colonies were transferred from

VOL. 133, 1978

CLONING OF BENECKEA GENES IN E. COLI

the master plates onto LB tet plates, as described above, and grown overnight at 30°C. Lysates were prepared by pooling the 50 colonies from one plate as follows. The colonies were washed off the plate with 2.0 ml of buffer (0.01 M Tris [pH 8.0], 0.05 M NaCl, 0.01 M EDTA, and 0.001 M 2-mercaptoethanol). The solution was brought to 0.023 mg of phenyl methane sulfonyl fluoride (2) and 0.75 mg of lysozyme per ml, incubated for 5 min at 0°C, and frozen and thawed twice. DNAse was added to 0.025 ,g/ml, and MgCl2 was added to 0.005 ,g/ml. After 15 min at room temperature, the supernatant was collected by centrifugation and stored at -20°C. The assay for luciferase was carried out by the procedure of Cline and Hastings (4). It involved the rapid addition of reduced flavin mononucleotide to a solution containing 0.5 ml of lysate (representing approximately 1.2 x 108 cells), decanal (a ClO aldehyde), and oxygen. The luciferase activity was measured as the initial maximum light intensity, using a photomultiplier photometer (SAI ATP photometer, model 2000). Using this instrument, it is possible to measure light emission from luciferase extracted from as few as 10 fully induced Beneckea cells (10' copies of luciferase). If 1 clone among 50 contained luciferase and each cell of that clone (107 cells) contained one enzyme copy, we could expect to have 6 x 106 copies of enzyme per assay. It was shown that E. coli extracts do not inhibit luciferase when lysates were prepared and assayed from pooled colonies of B. harveyi and E. coli C600 SF8.

RESULTS A bank of E. coli clones containing Beneckea DNA was formed by the adenine-thymine (AT) joining procedure. Random sheared fragments of Beneckea DNA (5 x 106 daltons) were tailed with dA residues, using the enzyme nucleotidyl transferase, and EcoRI-cleaved plasmid (pMB9) DNA was tailed with dT residues (about 50 residues were added to each end). These DNAs were then annealed to form the recombinant plasmid. Transformation of E. coli C600 SF8 with hybrid DNA, equivalent to 0.9 jig of pMB9, produced 8,930 transformants selected for tetracycline resistance. We assume that most of these contain Beneckea DNA because the dT-tailed plasmids alone did not have the capacity to transform E. coli to tetracycline resistance. Search for a luciferase gene. The 8,930 clones were pooled in groups of 50 clones (around 180 groups), and luciferase assays were performed on cell extracts from each of the pooled cultures. The assays were performed in collaboration with Kenneth Nealson of the Scripps Institution of Oceanography. The sensitivity of the method would have allowed us to detect one molecule of luciferase per cell had it been present in any one of our clones. We failed

357

to detect any activity. Expression of a Beneckea gene in E. coli. It is possible that our failure to detect the luciferase gene in our collection was because the gene was not expressed in E. coli. For this reason we decided to find out whether another Beneckea gene could be expressed in E. coli. A convenient gene for this purpose was the leuB gene in E. coli, because the recipient strain C600 SF8 of our collection of transformants is a leucine auxotroph. (leuB is the structural gene for the enzyme 3-isopropyl malate dehydrogenase.) In our bank one clone (37) had lost its requirement for leucine. That the ability to complement the leuB gene is carried by the hybrid plasmid was confirmed as follows: plasmid DNA was purified from clone 37, and strain C600 SF8 was again transformed with this DNA. From the tetracycline-resistant clones, 100 random colonies were tested and all of these had lost the requirement for leucine. That the DNA carried by the hybrid plasmid in clone 37 is from Beneckea was substantiated by these observations. The plasmid DNA from clone 37 could hybridize to Beneckea DNA, but it did not hybridize to E. coli DNA; pMB9 DNA hybridized to neither Beneckea DNA nor E. coli DNA (data not shown). Analysis of clone 37 by restriction mapping and electron microscopy showed the Beneckea DNA insert to be 2.5 x 106 daltons. Thus it appears that at least one Beneckea gene can be expressed in E. coli although there remains the possibility that the Beneckea DNA could be coding for a missense suppressor or a polypeptide that complements the leuB mutation. Identification of a ribosomal transcription unit in Beneckea. The recombinant DNA technology can be useful in the study of comparative gene organization. We have been interested in RNA synthesis and processing and, therefore, decided to study the Beneckea ribosomal RNA genes. Using a modification of the Grunstein and Hogness method, 5,520 clones were screened for the presence of DNA sequences complementary to Beneckea 5S RNA. Nineteen clones showed definite hybridization, and four of these (25, 29, 106, and 114) were chosen for further study. Hybrid plasmid DNA was isolated from each clone, and the DNA was characterized by restriction enzyme analysis and electron microscopy. Table 1 gives the size of the insert in each of the plasmids as determined by the two techniques. Surprisingly, the size of the insert is small in all of the plasmids. The average size of the sheared Beneckea DNA used to construct the bank was 5 x 106 daltons, but the largest of these inserts was 0.85 x 10' daltons,

LAMFROM, SARABHAI, AND ABELSON 358 or approximately 1,275 base pairs. In E. coli the 16S and 23S rRNA genes are linked to the 5S gene. (For reviews see Pace [15] and Jaskunas et al. [9]). We performed

J. BACTERIOL.

hybridization experiments to test whether this principle of gene organization is also found in Beneckea. Plasmid DNA was cleaved with either Hindll + III or BamHI + Sma I restriction endonucleases. The DNA fragments were separated by gel electrophoresis and transferred to NC paper by the method of Southern (25). Three such filters were prepared for each set of restriction enzymes to measure hybridization of each of the fragments to 32P-labeled Beneckea 5S, 16S, or 23S RNA. The results of this experiment are shown in Fig. 1 and 2 and Table 1. We derive several conclusions from them. (i) In general, in Beneckea the 5S, 16S, and 23S RNA genes are linked, as they are in E. coli. (ii) In clones 25, 106, and 114, the 5S gene is linked to the 23S and 16S RNA genes. (iii) In clone 29, there are two BamHI-Sma I fragments that hybridize to 5S RNA, indicating that there may

TABLE 1. Properties of Beneckea DNA inserts in pMB9

Hybridization to Beneckea

Size of insert

RNA

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0.85 0.43 0.54 0.57

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be two 5S genes in this clone. 16S RNA also hybridizes to both the DNA fragments, but there is very little, if any, 23S RNA hybridizing to the clone 29 DNA. Let us assume that the ribosomal RNA genes are repeated in Beneckea, as they are in E. coli, and that, as in E. coli, there is a fixed order of transcription in each of the clusters. There are three possible orders of the three genes (excluding permutations): 5 (a) 23 16 5 (b) 16 23 5 23 (c) 16 The order in E. coli is (b), but it does not appear that this is so in Beneckea. As previously mentioned, the size of the Beneckea inserts in these plasmids is small, and none of them could contain a complete 16S or 23S RNA gene. Our hybridization experiments indicate that the 5S rRNA gene is linked to both the 16S and 23S rRNA genes in clones 25, 106, and 114. In clone 29, we have evidence that there are two 5S rRNA genes, each linked to a 16S RNA gene. These results could be explained if there were both tail-to-tail and head-to-tail arrangements of ribosomal RNA genes in the order given in (a) and illustrated in Fig. 3. The tail-to-tail arrangement ... 16 ... 5 ... 5 ... 16 ... is required to explain the results for clone 29. We propose that there may be an inverted repeated sequence in the clone 29 DNA, and we have provided

some supportive evidence for this in the electron micrographs shown in Fig. 4. In clone 29 a larger fraction of the inserted DNA seems to be in double-stranded form than in clones 25, 106, and 114. This may represent Beneckea self-complementing sequences in addition to paired A-T tails. If this is the case, the loop would be in the spacer region between them. Because the segments of DNA are small, we are nearing the limit of resolution of this technique. ARBITRARY ORDER OF TRANSCRIPTION

_

BENECKEA 16 s5 RBSM GEE I23 RIBOSOMAL GENES I23 I11

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359

CLONING OF BENECKEA GENES IN E. COLI

VOL. 133, 1978

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360

LAMFROM, SARABHAI, AND ABELSON

J. BACTERIOL.

FIG. 4. Electron micrographs of single-stranded plasmid DNA showing self-complementing structures. (25, 106, and 114) DNA from clones 25, 106, 114 with double-stranded stem from A-T tailed regions and bubble from Beneckea DNA insert (arrow). (29) Structures seen in DNA from clone 29. A larger fraction of the inserted DNA seems to be in double-stranded forrn (arrow). It is possible that the inserted Beneckea DNA contains self-complementary sequences in addition to the A-T tails.

In E. coli there is a tRNA gene in the spacer region between the 16S and 23S rRNA genes (13) so that the order of genes is 16S ... tRNA ... 23S ... 5S. We, therefore, were interested to determine whether any of our clones contained tRNA genes. Beneckea 32P-labeled 4S RNA, purified by acrylamide gel electrophoresis, was hybridized to four NC filters containing

20 ,ug each of the plasmid DNAs. The hybridized RNA was eluted from the filters and analyzed by acrylamide gel electrophoresis. Only one of

the plasmids (114) showed significant hybridization (Fig. 5). The 4S RNA that had hybridized to plasmid 114 DNA was characterized by Ti RNase oligonucleotide mapping and was shown to be a unique species of complexity of approxi-

CLONING OF BENECKEA GENES IN E. COLI

VOL. 133, 1978 -z

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BPB FIG. 5. Polyacrylamide gel electrophoresis of 32Plabeled Beneckea 4S RNA purified by hybridization to cloned Beneckea DNA. 20 pg each of clone DNA 25, 29, 106, and 114 and a control experiment with no added DNA were immobilized on NC filters and hybridized with 4.35 x 10i cpm of 3P-labeled Beneckea 4S RNA. The RNA was eluted with formamide and fractionated by electrophoresis through 10%o acrylamide. The autoradiogram shows hybridized RNA and RNA markers (5.8S and 5S RNA from yeast; serine tRNA from E. coli).

mately 75 nucleotides. Insufficient material was available for sequence analysis, so we cannot yet conclude that this RNA is in fact a tRNA. DISCUSSION We were able to clone Beneckea DNA in E. coli. Among the 8,930 clones that were screened, we identified one that can complement the leuB function of an E. coli auxotroph. We have not yet characterized the functional DNA, which could be the structural gene or code for a missense suppressor or for a complementing peptide. It is interesting that a Beneckea DNA

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product, originating from an extracellular environment of 3% NaCl and with a temperature optimum of 24°C, can be expressed in E. coli with a milieu of 0.9% NaCl and 370C optimal temperature. We also analyzed four clones containing three ribosomal genes and demonstrated that the three ribosomal genes are linked, but appear to be arranged in a different order than in E. coli. Our results suggest that the ribosomal transcription unit 23 ... 16 ... 5 is joined to an inverted repeated sequence. This conclusion rests heavily on the proposed arrangement of DNA sequences for clone 29 DNA. There are undoubtedly two BamHI-Sma I fragments in this plasmid that hybridize to 5S RNA. It is unlikely that these result from an Sma I cleavage in the 5S gene, since such a site does not exist in the 5S genes of the other three ribosomal clones (although heterogeneity cannot be ruled out) and both fragments hybridize to 16S RNA as well as to the 5S RNA. It is more likely that the Sma I site is in the spacer region between the two rRNA clusters. The cloned segment in plasmid 29 is quite small, and, although the structures seen in Fig. 4 suggest a self-complementary sequence in the insert, they do not prove it. One of the clones (114) contains a sequence complementary to a unique species of 4S RNA (Fig. 5). This gene is closely linked to 5S RNA and may be in a space between rRNA clusters (Fig. 3) rather than be included in the ribosomal transcription unit. We have some evidence that certain common similar DNA sequences occur in E. coli and Beneckea. In situ hybridization experiments with Beneckea 4S RNA indicated cross-hybridization to E. coli DNA. Some hybridization was also observed between Beneckea rRNA probes and E. coli DNA restriction fragments. We would still like to understand why we failed to clone the luciferase gene; among possible explanations are: (i) The gene is not expressed in E. coli. We have suggestive evidence that a Beneckea gene can be expressed in E. coli, since the leuB gene of E. coli can be complemented by the product of a Beneckea DNA fragment. We also showed that luciferase activity is not inhibited by E. coli lysate. We did not include the Beneckea luciferase inducer in the E. coli growth media, but the assays were sensitive enough to detect the Beneckea basal level of luciferase. (ii) The two subunits of the luciferase are not linked. Were this the case, it would probably be impossible to transfer both genes to a single plasmid, and one would have to devise a screening method for detecting each of the subunits. (iii) The presence of the enzyme is

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lethal in E. coli. Beneckea luciferase may represent a substantial drain on cellular reducing power (FMNH2), and perhaps its presence would be deleterious. (iv) We did not look at enough clones. The number of clones to be examined to achieve a high probability of success is dependent on the size of the inserted fragment. To accommodate an 80,000-molecular-weight enzyme would require a DNA sequence of only 1.6 x 106. Since the average size of our sheared Beneckea fragments was 5 x 106 daltons, we would require a collection of 2,770 clones to achieve a 99% probability of finding a particular gene. We actually examined 8,900 clones. However, the two kinds of Beneckea DNA inserts (0.45 x 10' to 0.85 x 10' and 2.5 x 106 daltons) we have identified and examined involved shorter sequences of DNA than the average size of the sheared Beneckea DNA. We cannot explain this result. Since we were able to clone and identify Beneckea DNA in E. coli and have found suggestive evidence that it can be expressed, it appears that cloning will be a promising technique to study gene organization of Beneckea arid possibly other marine bacteria. ACKNOWLEDGMENTS We acknowledge the interest and participation of Ken Nealson in some of these experiments. We are very grateful to Marcia Hilmen and Lisa Goossens for the electron microscopy. We thank Tony Otsuka for his many helpful discussions and suggestions. This work was supported by research grant PCM 74-21089 from the National Science Foundation Public Health Service grant CA 10984-09 from the National Cancer Institute.

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