the Ends of the DNA - Europe PMC

JOURNAL OF VIROLOGY, Apr. 1989, p. 1587-1594

Vol. 63, No. 4

0022-538X/89/041587-08$02.00/0 Copyright © 1989, American Society for Microbiology

Characterization of the Genome of Pseudomonas aeruginosa Bacteriophage 4PLS27 with Particular Reference to the Ends of the DNA BRENDA J. ALLAN,1 PETER DAVIES,2 ERIC B. CARSTENS,' AND ANDREW M. KROPINSKI1* Departments of Microbiology and Immunology' and Biochemistry,2 Faculty of Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada Received 22 August 1988/Accepted 16 December 1988

The DNA of Pseudomonas aeruginosa rough-specific bacteriophage +PLS27 was studied. The genome size as determined by summing the sizes of restriction fragments was 42.7 kilobase pairs. Of particular interest was the fact that the DNA was insensitive to certain common restriction endonucleases including EcoRI, BamHI, and Hindlll. The ends of the phage DNA were cloned and sequenced, revealing direct repeats of 318 nucleotides. The left end of the genome when cloned into the promoter selection vector pKK232-8 exhibited promoter activity in Escherichia coli. Two promoters bearing >70% sequence homology to the plasmid pNM74 TOL operon and PAK pilin promoters were identified. promoters recognized by the RNA polymerase of E. coli and that of P. aeruginosa. Since many of the characterized Pseudomonas promoter sequences are derived from highly regulated genes, the nucleotide sequences determined may not truly reflect the typical Pseudomonas promoter. In this study we have cloned and sequenced the ends of the (PLS27 genome and characterized two promoterlike sequences.

Phage T7 is the type strain of the genus Podoviridae and is also the best-studied virus of this morphotype. This group shares a common strategy of infection (16). Host RNA polymerase recognizes promoters located near one end of the bacteriophage DNA and transcribes a segment of about 20% of the genome. The early mRNA transcripts are processed and translated into a limited number of polypeptides one of which possesses rifampin-resistant RNA polymerase activity and a molecular weight of approximately 100,000. This phage-encoded RNA polymerase recognizes only specific promoters located on the phage DNA and is associated with late transcription. The mechanism of phage T7 infection has been reviewed in detail by Studier and Dunn (27). Phage 4~PLS27 is a lipopolysaccharide-specific phage that is active on rough mutants of Pseudomonas aeruginosa PAO (13). This phage is very similar to coliphage T7 in a number of properties, including size, buoyant density, mass, and number of structural proteins, but is distinguished from T7 by its host range and the higher mol% G+C content of its DNA. Because of its similarity to T7 we were interested in characterizing the genome of 4XPLS27 to see if it contained elements related to those described for T7. In addition, if XPLS27 shares a similar mechanism of transcription with T7, the left end of the genome should provide a source of promoters recognized by P. aeruginosa RNA polymerase. The structure and function of E. coli promoters have been extensively studied and were recently reviewed by Travers (28). The sequence of more than 200 promoters utilized by RNA polymerase has been determined. On the other hand, only a relatively limited number of Pseudomonas promoters have been characterized (5). Genes originating from Pseudomonas spp. are generally poorly expressed in Escherichia coli (23). However, some exceptions occur. For example, the gene encoding the PAK pilin is well expressed in E. coli (8). Fusion of genes from P. aeruginosa to an E. coli promoter resulted in very high expression of these genes in E. coli (10, 14, 29), suggesting that the expression barrier is essentially at the level of transcription initiation. This suggested that a fundamental difference may exist between the *

MATERIALS AND METHODS Bacteria, bacteriophages, and plasmids. The bacteria, bacteriophages, and plasmids used in these studies are described in Table 1. The bacterial stock cultures were stored at -70°C in 1.5-ml vials containing 1.3 ml of culture and 0.1 ml of dimethyl sulfoxide. The phage lysates were stored at 4°C over chloroform. Plasmids were stored at 4°C in 10 mM Tris hydrochloride (pH 8.0) containing 1 mM EDTA (TE). Purification of bacteriophages and isolation of DNA. XPLS27 was cultivated on P. aeruginosa AK1012 growing in tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) at 37°C. Phages were purified from clarified (10,000 x g, 15 min) lysates by polyethylene glycol precipitation (31), pronase digestion (R. A. J. Warren, personal communication), and equilibrium centrifugation in CsCl (18). Bacteriophage preparations were dialyzed overnight at 4°C against TE buffer to remove the CsCl. EDTA and sodium dodecyl sulfate were added to the final concentrations of 10 mM and 0.2%, respectively, and the phage suspension was heated to 65°C for 10 min or until the solution became viscous, indicating lysis. Proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) was added to a concentration of 100 ,ug/ml, and the mixture was incubated at 37°C for 1 h. The solution was phenol extracted three times by using TEequilibrated phenol, followed by three extractions with chloroform:isoamyl alcohol (24:1 [vol/vol]). The DNA was then dialyzed against TE at 4°C, and its concentration was determined spectrophotometrically at 260 nm (18). Isolation of nucleic acids. Large-scale preparations of plasmid DNA were achieved by using the procedures described by Crosa and Falkow (4), while the procedures of Homes and Quigley (11) were used for minipreparations.

Corresponding author. 1587

1588

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TABLE 1. Bacterial strains, bacteriophages, and plasmids used in this study

Strain, phage, or

Relevant characteristic(s)

plasmid

Strains E. coli HB101 JM83 NM522 P. aeruginosa AK1012

Bacteriophages XPLS27 M13K07 M13mpl8 Plasmids pKK232-8

pUC18 pTZ19R plO1-16-11 plO1-226-1 p83-131-1 p522-138-1

(rB- mB- recA13 ara-14 proA2 lacYl galK2 rpsL20 (Strr) xyl-5 mtl-i supE44; host for pKK232-8 ara A(lac-proAB) rspL lacZAM15 (rK- MK+); host for pUC plasmids hsd-5 A(lac-proAB) supE thi (F' proAB+ lacJPZAMJ5); host for pTZ plasmids Rough mutant of PA01; host for phage XPLS27 F- hsdS20

Rough lipopolysaccharide-specific P. aeruginosa phage Helper phage (Pharmacia)

Source or reference

18 30 21 12

Cloning vector

12 21 32

5.1-kb Apr Cms promoter selection vector 2.6 kb; Apr lacZ' 2.9-kb Apr phagemid (phage fl IGS) Sau3A fragment derived from left end of XPLS27 cloned into pKK232-8; Apr Cmr 4PLS27 HincII M fragment in pKK232-8; Apr Cmr fPLS27 KpnI C fragment in pUC18; Apr JPLS27 KpnI C fragment in pTZ19R; Apr

2 3 21 This study This study This study This study

RNA was extracted by using the procedure developed by Kassavetis and Geiduschek (15) as modified by S. Stoddard (personal communication). The final pellet was dissolved in 100 [lI of 0.1 mM EDTA and stored at -20°C until needed. Restriction digestion of DNA. The high, medium, and low buffer systems described by Maniatis et al. (18) were used for the restriction enzyme digests. The exceptions were Sau3A and PvuI, in which cases the buffers suggested by the manufacturers were substituted. T4 DNA polymerase buffer (Tris-acetate) of Maniatis et al. (18) replaced the usual buffer when the DNA was digested with more than one restriction endonuclease. Complete digestion of phage 4PLS27 DNA usually required a 10-fold excess of restriction endonuclease required to cut lambda DNA. Cloning the ends of the genome of phage 4PLS27. The left end of the genome was cloned into 5.1-kilobase (kb) promoter selection vector pKK232-8. A derivative of pBR322, pKK232-8 contains a promoterless chloramphenicol acetyltransferase (CAT) gene that can be activated by the insertion of a promoter-containing fragment into the multiple cloning site positioned 5' proximally to the CAT gene (2). Partial Sau3A digests of 4PLS27 DNA were cloned into pKK232-8 cut with SmaI and BamHI. The ligated mixture was transformed into competent HB101 cells, and recombinants were selected on TSB agar plates containing ampicillin at 100 Rg/ml and chloramphenicol at 10 pg/ml. For cloning the right end of the phage genome, the KpnI C fragment of XPLS27 was ligated to pUC18 which had been cut with HinclI and KpnI. The ligation mixture was transformed into competent JM83 cells, and the recombinants were selected on TSB agar plates containing ampicillin at 100

jg/ml and 5-bromo-4-chloro-indolyl-3-p-galactoside (X-Gal) at 40 ,ug/ml. Radioactive labeling of DNA. DNA was radiolabeled by nick translation using [ot_32P]ATP, nick translation kit (Bethesda Research Laboratories, Inc., Gaithersburg, Md.), and the instructions provided. Calf intestinal phosphatasetreated DNA was also 5' end labeled by using T4 polynucleotide kinase and the procedures described by Maniatis et al.

(18). Gel electrophoresis. DNA restriction fragments were re-

solved by agarose or 5% polyacrylamide (30:0.8, acrylamide: bis) gel electrophoresis in TBE (0.089 M Tris base, 0.089 boric acid, 0.002 M EDTA). RNA molecules were separated by electrophoresis on horizontal agarose gels containing 2.2 M formaldehyde. The running buffer was 0.05 M morpholinepropanesulfonic acid (MOPS), pH 7.0, containing 1 mM EDTA. RNA samples were dried in vacuo and dissolved in 4.5 ,l of 3 x MOPS-EDTA buffer. Formaldehyde and deionized formamide were added to a final concentration of 2.2 M and 50% (vol/vol), respectively. The samples were heated at 70°C for 10 min and quenched on ice. Before the sample was loaded, 3 RI of gel loading buffer (lx MOPS-EDTA buffer containing 40% [wt/vol] sucrose, 50% formamide, 12.4 M formaldehyde, and 0.5% bromphenol blue) was added. RNA size classes were also separated by polyacrylamide gel electrophoresis in the same manner as that used for separation of DNA. RNA was dissolved in gel sample buffer of 10 mM NaH2PO4-Na2HPO4 containing 1.0 M deionized glyoxal, 50% dimethyl sulfoxide, and a small amount of bromphenol blue (20). The samples were denatured at 45°C for 20 min, quenched on ice, and immediately loaded on the gel. Southern and Northern (RNA) blotting. DNA fragments were denatured and blotted on GeneScreen Plus nylon 66 membrane (New England Nuclear Corp., Boston, Mass.). The membranes were prehybridized, hybridized, and washed according to the instructions of the manufacturer. The membranes were exposed to Cronex 4 film (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.) for 1 to 24 h. RNA, separated on formaldehyde gels, was blotted onto GeneScreen Plus according to the instructions of the manufacturer. The membranes were extensively washed: twice in 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at room temperature for 1 min, once in 6x SSC at room temperature for 15 min, once in 2x SSC containing 1% sodium dodecyl sulfate at 47°C for 30 min, once in 0.5 x SSC at room temperature for 15 min, once in 0.1x SSC at 47°C for 30 min, and once in 0.1 x SSC at room temperature for 5 min.

VOL. 63, 1989

PSEUDOMONAS PHAGE 4)PLS27 DNA

TABLE 2. Sizes of fragments generated by digestion of bacteriophage Fragment

A B C D E F G H

4PLS27 DNA with restriction endonucleases

Size (kb)' of digestion product generated with: KpnI PvuI Sall

PstI

SmaI

HpaI

BglII

35.5a 7.15

35.2a 7.48

41.4a 1.33

37.6a 5.10

26.85 10.80 4.52

1589

16.62 14.19 12.05

25.16 11.13 5.89

BstEII

PvuII

HincII

24.88 14.17 3.93

18.25

7.77 6.08 5.15 4.87 3.71

11.98 8.40 4.33

3.27 1.60 1.46 1.14

I J K

1.05 1.05 1.01 0.92 0.74 0.68 0.60

L

M N

0 P

Q

0.36 0.18 0.11

R S a

Size calculated based on a total size of 42.7 kb. The individual sizes are the averages of 5 to 15 independent determinations.

In vitro transcription. Plasmid pKK232-8, plO1-16-11, and plO1-226-1 DNA (1 ,ug; approximately 0.25 pmol) cleaved with HindlIl was preincubated for 5 min at 37°C in a reaction mixture containing 40 mM Tris hydrochloride (pH 8.0), 10 mM MgCl2, 0.4 mM EDTA, 1 U of RNasin ribonuclease inhibitor (Promega Biotec; supplied by BIO/CAN Scientific Inc., Mississauga, Ontario, Canada) per p.l, 10 mM dithiothreitol, and purified RNA polymerase. RNA polymerase from P. aeruginosa was purified as described by Allan and Kropinski (1). The E. coli enzyme was obtained from Pharmacia, Inc., Piscataway, N.J.). Between 25 and 50 ,ug of enzyme was used per assay with an enzyme-to-DNA ratio of 200 to 400:1. RNA synthesis was initiated with unlabeled CTP, GTP, ATP, each to a final concentration of 0.4 mM, cold UTP to a final concentration of 0.02 mM, and 25 ,uCi of [a-32P]UTP (ICN Biomedicals Canada Ltd., Montreal, Quebec, Canada). Heparin (1 ,ug/ml) was added with the nucleotides to prevent reinitiation of RNA synthesis. The mixtures were incubated at 37°C for 25 min. The concentration of UTP was then increased to 4 mM, and the mixture was incubated a further 5 min. RNA synthesis was stopped by the addition of EDTA to a concentration of 25 mM, and the mixture was placed on ice. To recover the RNA, 1 ,ug of carrier RNA was added and the nucleic acid was precipitated with 2 volumes of ethanol. The RNA was stored at -20°C in ethanol until analyzed on polyacrylamide gels. Sequence analysis. (i) Left end. The 1-kb EcoRI-HindIII fragment representing the left end of the 4PLS27 genome from plO1-16-11 (see Fig. 3) was subcloned into M13mpl8 (32) which had been digested with EcoRI and HindIII. For unknown reasons, several attempts to subclone the same fragment into M13mpl9 were unsuccessful, and therefore only one strand was sequenced. Sequencing was performed on purified single-stranded DNA isolated from M13 recombinants by the dideoxynucleotide chain termination method (24). The sequence was completed by using unique synthetic oligonucleotide primers, 15 bases in length, which were purchased from the Department of Biochemistry, Queen's University, Kingston, Ontario, Canada. The complete sequence was compiled with the aid of programs provided by

the National Institutes of Health-sponsored BIONET National Computer Resource for Molecular Biology (17). (ii) Right end. The KpnI C fragment of (PLS27 was electroeluted from a 6% polyacrylamide gel, ligated to the vector pTZ19R (Pharmacia) which had been cut with KpnI and HinclI, and transformed into E. coli NM522. The recombinants were selected on TSB agar containing ampicillin at 150 ,ug/ml and X-Gal at 40 ,ug/ml. The desired clone was grown in 2x YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl per liter) supplemented with 150 ,ug of ampicillin per ml and 0.001% thiamine at 37°C with shaking until an A6. of 0.5 to 0.8 was reached. A sample of 2 ml of this culture was infected with 0.1 ml of phage M13K07 which had a titer of approximately 8 x 1010 PFU/ml. The infected cells were shaken vigorously for 1 h at 37°C in a 50-ml flask. After 1 h, 10 ml of 2x YT broth was inoculated with 0.4 ml of infected cells, kanamycin (Sigma Chemical Co., St. Louis, Mo.) was added to a final concentration of 70 ,ug/ml, and the cells were grown with aeration at 37°C for 8 h. Following centrifugation at 10,000 x g for 15 min, the supernatant fluid was carefully decanted, and the phage was A

PstI BgIl HpoI

A A

B,

SmoI r

A A

Sol I BstE I

C

a A

B

KpnI'

A

B

C C

B

PvuI Pvu Il

A

C

BClI Ili E Kb poirsL %

B c

L

f 5

10 25

I5

D

B C

A 20 50

25

iHiGi

D

30

B 35

40 42.5

75

FIG. 1. Restriction map of bacteriophage (PLS27. The linear genome is oriented so the promoters recognized by the host RNA polymerase are located at the left end. Fragments are indicated by capital letters in decreasing order of size.

Hpi .I Xla I

.

4 db

p

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ALLAN ET AL.

1590

Ps t I 0

a-

a

a

I

Pvu I

-.

BstE 11

Pvu II

-1~~~~~~~f

'fMH_

_ _~~~~~~~~~~~~~~~~~~~7 E

|

dib

..

I.....: ...... .;....:

A B A B A B A B A B FIG. 2. TR of the genome of bacteriophage 4PLS27. The DNA was digested with various enzymes, run on a 0.8% agarose gel, and stained with ethidium bromide (lanes A). After transfer of the DNA, the membrane was probed with the HpaI B fragment of the genome (lanes B). Symbols: *, fragments from the same end as the HpaI B fragment; *, fragments from the opposite end of the genome; *, internal fragments.

32P]ATP, and used to probe blots of ~PLS27 DNA digested

with a variety of restriction endonucleases (Fig. 2). The autoradiographic results clearly show that two restriction fragments hybridized with the probe and that these correspond to the termini. While the complete restriction map for Hincll is not known, Southern blot analysis revealed that the HincII M fragment and either HincII-C or -D were the terminal fragments. Digestion of the genome of XPLS27, which had previously been 5' end labeled (polynucleotide kinase and [cx-32P]ATP), with HinclI, confirmed that HinclID and HincII-M were the terminal fragments. The 1.3-kb HpaI B fragment which constitutes the left end of the genome was cleaved into 0.78- and 0.52-kb fragments by HinclI, confirming that the HincII M fragment represents the left end of the genome. Cloning the ends of the 4PLS27 genome. (i) Left end. Since phages related to PPLS27 have promoters recognized by the host RNA polymerase clustered near one end of the linear genome, we developed a cloning strategy that would preferentially select for end fragments. Because the genome of the bacteriophage OPLS27 has blunt ends, partial digestion with

precipitated by the addition of 2.5% (vol/vol) polyethylene glycol and 0.3 M NaCl. After 18 h at 4°C the precipitate was collected by centrifugation at 11,000 x g for 30 min, and the phage pellet was dissolved in 0.4 ml of 20 mM Tris hydrochloride (pH 7.5) containing 20 mM NaCl and 1 mM EDTA. The single-stranded DNA was liberated from the phage particles by phenol extraction and was sequenced by the dideoxynucleotide chain termination method of Sanger et al. (24). RESULTS Restriction map of the genome of 4PLS27. The numbers of fragments found upon gel electrophoresis after 4fPLS27 DNA was cut with restriction endonucleases were as follows: PstI, BglII, HpaI, and SmaI, 1; Sall, BstEII, KpnI, and PvuI, 3; PvuII, 4; BclI, 9; and HincIl, 19. The sizes of the fragments calculated from the known Mrs of EcoRI- and Hindlll-generated fragments of lambda DNA and HaeIII digests of XX174 DNA are shown in Table 2. The sum of the sizes of the restriction fragments indicates that the ~PLS27 genome is 42.7 kb. The phage DNA was not cleaved by the following common restriction enzymes: EcoRI, HindlIl, BamHI, XbaI, XhoI, and XorI. In addition, since 4PLS27 DNA was readily cleaved by MboI and Sau3A or HpaII and MspI, the phage DNA did not appear to be modified by dam- or dcm-like methylases. By using single and double digestions, a restriction map for the genomic DNA of ~PLS27 was constructed (Fig. 1). In some cases, restriction fragments were electroeluted from agarose or polyacrylamide gels prior to digestion with a second enzyme. Terminal redundancy. Since the genomes of podoviruses such as coliphages T3 (9) and T7 (6, 27), Salmonella phage SP6 (15), and Klebsiella phage no. 11 (6) are terminally redundant (TR), we have examined 4PLS27 DNA for TR sequences. The HpaI B fragment was electroeluted from gels, 5' end labeled with polynucleotide kinase and [ox-

end

Hinc II

and

Hinc 11 Sau3A PLS27

4PLS27 SmaI

Hinc 11

aamH

FIG. 3. Cloning of the left end of the genome of bacteriophage XPLS27 using the promoter selection vector pKK232-8. T1 and T2 are transcription terminators. Restriction sites in square brackets were lost during construction of the vector. A 0.93-kb Sau3A end fragment with promoterlike activity was cloned into the promoter selection vector pKK232-8, which contained a promoterless chloramphenicol acetyltransferase gene, and recombinants were selected on plates containing chloramphenicol at 5 ,ug/ml (plO1-16-11). Digestion with HinclI confirmed that this was the left end fragment. A HincIl-M subclone of this fragment (plO1-226-1) was produced in a

similar manner.

VOL. 63, 1989

..

*...

PSEUDOMONAS PHAGE XPLS27 DNA

1591

T

.........

.. ...... .

.... .oo*o....

...

...oo

VC..X.....

60

CCCACTACCG TGAAGGACCG CGCGCCCCCA TGCCCCTACG TGCGCGATTT CATTCGCATG .X . ....... X X.......... .......... ..... ......... ... .X... CGCGGGGTAT TGCTCGCGTG CGCTACGTGC GCGTACTAGC CTCGCGGGTG TGCACCTGCG 120 ..

....... .0.. ......... ......... .. CCGAGAGGAT TGCACGCACG GCGCGTGGAG AGGTTCTAGC GCGGTTCAAT CTCCCGTTGC 180 ...

...

.. .oG...... ...... .......... oosoo..... .......... CTAGTACACC GGAACGGGTG GCCCTGGCTC TCCCTGGTGG CGGCTCGGTT CCCTGGTGCG 240 ooew.....

.. ......................... .......... ............. ....... ....... ... ....... CCTGGTGCGT TCCCTCCTCC CTTCCCTGGT ATGTTCTCTC CCTGGTGGGT TCGGCTCTCG 300 .

.

TCGTCCTGGT GTAGGTGGGG CGCTGCCTTA CCTCTTTTGC CGCCCTACCT CTTTTGCGGG 360 GAGGGTACGC GGGGCCGCCT TGCCTCTTTT GCTGGCTTGC CTCTTTCGCT CCCGGTATCC 420

CTAGTCACTA CCTGGGCAGC ATCCTCTCCT ACCTGGGTAC TACCTTGCTG GGTTTCGTCT 480 TCGACGGGTG GGGCCCCCGT TCCCCACGCC TTCGAGCGCT CAGTCTGGCC CTGCCGCCTT 540

CGCTTCTCTC ATGCTAGTGC GACAATTCTT TTAGGCATAA GCAGGTACGG TTGATATAAC 600 PROMOTER P1

GAATTGGTAT TGACGGAGCT TCGGTAGGTC TGTAGAGTTC GCCACATGGT TAGGCCATAA 660 GCAACACGGA CTAGCCAACG CAAGGCATGG TGACGACTCT AGGCTAGGGG CCTAGGTCAG 720

ACGGACAACG GTACACGTCA GTCGTCACCA GTGAACCAAG GCATGGCGAA GGCTAGCTGA 780 PROMOTER P2 GGTTGACAAG CCAAGCGGAT GCTGTAGAGT GCGACACCAG CAACACGTTT CACCGCTCTT 840 S/D BOX Q S K A M TAACAAGTCG AGTCTGCTGT AGTGTATCCA CATAGGAGGC ACTACTATGC AAAGCAAAGC 900 V I A Y R D Q A H ACAAGCGCAC GTTATCGCCT ATCGTGATC 929 FIG. 4. DNA sequence of the left end of (PLS27 DNA, showing the positions of the two putative promoters (P1 and P2), the Shine-Dalgarno (S/D) box, and the possible amino acid sequence downstream from the Shine-Dalgarno box. The dotted line corresponds to the sequence of the right terminally repetitious region. Differences in the sequences are indicated. V, Insertion; X, deletion.

Sau3A yielded end fragments with one blunt end and one Sau3A end. The promoter selection vector pKK232-8, containing the promoterless CAT gene, was cut with BamHI and SmaI to yield compatible ends. The internal fragments containing two Sau3A ends would not be cloned. Selection on chloramphenicol yielded clones with inserts ranging from approximately 1 to 4 kb (data not shown). Since the Sau3A restriction map for XPLS27 is not known, it was not possible to determine if the clones containing internal Sau3A fragments had been rearranged during cloning. To overcome this problem, the smallest clone, plO1-16-11, which has a 0.93-kb insert and contains no internal Sau3A sites, was used in our studies. Digestion with HincII cut the insert into two fragments of 0.78 and 0.15 kb and confirmed the fact that this Sau3A fragment arose from the left end of the phage

genome. In contrast to E. coli HB101 which is sensitive to chloramphenicol (c5 ,ug/ml), the MIC for clone plO1-16-11 was 320 ,ug/ml as determined by the tube dilution method. We have arbitrarily oriented the genome of (PLS27 so that, like the genome of T7, the promoters recognized by the host RNA polymerase are located at the left end. The 0.78-kb Hinc M fragment also displayed promoterlike activity when cloned into pKK232-8 cut with SmaI, yielding a chloramphenicol-resistant clone, plO1-226-1 (Fig. 3). (ii) Cloning the right end of the genome of bacteriophage 4PLS27. The KpnI C fragment which represents the right end of the genome of 4PLS27 was ligated to pUC18 cleaved with HinclI and KpnI to yield compatible ends. When this construct (p83-131-1) was transformed into E. coli JM83 and plated on agar containing ampicillin and X-Gal, the colonies

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ALLAN ET AL.

B C

A

D

*

kb -7.76 -608 -'-5.15 -4.87

-3.71 -3.27

_ 1.60 1.46

K-

-1.14 1.05 1.00 -0.92 _.;

lUl -0.79

cloned DNA from 4)PLS27 was transcribed in E. coli, total cellular RNA was isolated from clones plO1-16-11 and plOl226-1 and analyzed by Northern blotting using the 32p_ labeled XPLS27 HpaI B fragment as a probe. Two transcripts of 1,200 and 1,050 bases were observed in clone plO1-16-11, while the transcripts from clone plOl-226-1 were approximately 150 bases shorter (Fig. 5). This difference is similar to the difference in the sizes of cloned DNA. It is not possible to determine if the two transcripts arose from independent promoters or if the smaller transcript was produced by processing the larger transcript. (ii) In vitro runoff transcripts. The recombinant plasmids plO1-16-11 and plO1-226-1, as well as the vector pKK232-8, were linearized with HindIII, which cuts the DNA 20 base pairs (bp) downstream of the DNA insert. Runoff transcription using either purified RNA polymerase from E. coli or P. aeruginosa produced a similar pattern. Two new transcripts of 330 and 125 bases were observed from plO1-16-11, while a single new transcript of 165 bases was observed when plO1-226-1 was transcribed (Fig. 6).

-0.68

-0.60 -0.36

FIG. 5. Northern blot analysis of clones containing 4)PLS27 DNA. RNAs isolated from E. coli HB101 containing plO1-16-11 (A), pKK232-8 (B), and plO1-226-1 (C) and XPLS27 DNA cut with HinclI (D) were run on a 1.0% agarose gel containing formaldehyde. After transfer, the membrane was probed with the HpaI B fragment of XPLS27. The molecular masses indicated were obtained by probing 4PLS27 DNA digested with HinclI with total fPLS27 genomic DNA.

containing the recombinant plasmid displayed variable colony morphologies. White or cream-colored colonies were observed. However, both types of colonies appeared to contain the recombinant plasmid as detected by minipreparation analysis. The host JM83, containing pUC18, did not display this colony variation. When the KpnI C fragment was ligated to the vector pTZ19R and transformed into E. coli NM522, a similar variation in colony morphology was observed. The reason for this observation is not known. Sequence analysis of the ends of the genome. The first 929 nucleotides of the left end of the genome were sequenced by the dideoxynucleotide chain termination method (Fig. 4). Visual inspection as well as computer analysis of this sequence indicates that it contains many repetitive sequences (A. M. Kropinski and B. J. Allan, manuscript in preparation). Southern blot analysis under stringent conditions revealed that the ends of the genome of (PLS27 share homology (Fig. 1). The extent and nature of this homology were determined by sequencing the right end of the genome and comparing it with the left end. The phage ends contained a direct repeat of 318 nucleotides. The TR was highly conserved; the right end differs from the left in only seven places. It would appear that insertions or deletions occurred at or adjacent to nucleotides 51, 55, 75, and 80. An A->T transition occurred at nucleotide 47, and an A-*G transversion occurred at nucleotide 194. The direct repeats also differ in the terminal nucleotide, with the left end terminating in a C while the right end terminates in a T. Transcription analysis. (i) In vivo. To demonstrate that the

DISCUSSION Phage 4~PLS27 is a member of the Podoviridae, a family of phage with isometric heads and short tails (19). Its genome shares several characteristics with the type Podoviridae, coliphage T7, including TR, blunt ends, and clustering of promoters recognized by the host RNA polymerase at one end of the genome. Indirect evidence suggesting that the linear genome of 4PLS27, like that of T7, is blunt ended, includes the fact that the ends of the 4PLS27 DNA can only be labeled with polynucleotide kinase by using the protocol for blunt ends and that the ends can be ligated to other blunt-ended fragments of DNA (unpublished data). Southern blot analysis revealed that the ends of 4PPLS27 are highly homologous (Fig. 2). Sequence analysis of both 1

1419

2

3

1

2 3

1

2 3

b

51 7* 396

214 S

*

75 65 M

A

B

C

FIG. 6. Analysis of clones plO1-16-11 and plO1-226-1 by in vitro runoff transcription. pKK232-8 (1), plO1-16-11 (2), and plO1-226-1 (3) were transcribed in vitro with purified RNA polymerase from E. coli (A) and P. aeruginosa (B and C), and the transcripts were denatured in glyoxal and run on an 8% polyacrylamide gel. Molecular mass markers (lane M; in base pairs) were generated by digesting pUC18 DNA with Hinfl and end labeling the fragments.

PSEUDOMONAS PHAGE ~PLS27 DNA

VOL. 63, 1989

PUTATIVE 0PLS27 P1 PROMOTER T AACG

T

C

AATT

GG

TATTGACGGA

GC

! I I I I 11 11 11 I I It AACG ATTT GG CATGGTAAGT GC G GIr PAK PILIN PROMOTER PUTATIVE

OPLS27 P2

C

TT GGTAGG

111111

II

TT GGTAGG

G

PROMOTER

TA

AAGC

1111 AAGC G

GG II

GG

ATGCTG II

I

ATACAG

GAGT GC 1111 II GAGT GC

C

GA I AA

A pNM74 PROMOTER FIG. 7. The nucleotide sequence of putative phage promoters compared with promoters for the PAK pilin gene (8) and the TOL operon on plasmid pNM74 (22).

ends confirmed the presence of a highly conserved 318-bp direct repeat. The terminal repeats in coliphage T7 and T3 DNA are 160 and 230 bp (7, 9, 27), respectively. Many other phages with linear double-stranded genomes have terminal repeats of a wide variety of lengths. For example Klebsiella phage 11 and Bacillus subtilis phage 2C have terminal repeats of 181 bp and greater than 1,000 bp, respectively (3, 6). The left termini of the genomes of bacteriophage qPLS27 and T7 contain many direct repeats both outside and within the direct terminal repeats (Fig. 4). The sequence between the left end of the T7 DNA and the first early promoter does not appear to specify any protein. It has been suggested that the terminal repeat of T7 in conjunction with its repetitive sequences may be involved in concatemer formation, production of mature ends, packaging of DNA into phage particles, or injection of DNA into the cell (7). Since the end structure of the genome of (PLS27 is similar to that of T7, it may serve a related purpose. The left end of the genome of 4PLS27, like that of T7, contains promoters recognized by the bacterial RNA polymerase. Two fragments from (PLS27 (929 and 774 bp) with promoter activity were cloned into the promoter selection vector pKK232-8 (Fig. 3). This was a somewhat surprising result, since genes from P. aeruginosa are often poorly expressed when cloned in E. coli and it seemed unlikely that promoter selection vectors developed for E. coli could function effectively with promoters recognized by the RNA polymerase from P. aeruginosa. However Northern blot analysis confirmed that the DNA from XPLS27 was transcribed in E. coli. The resulting transcripts were large enough to encode the complete chloramphenicol acetyltransferase of 219 amino acids (25). In vitro, the larger clone (plO1-16-11) contained two promoters that gave rise to runoff transcripts of 340 and 145 bases when the recombinant was cut with HindIII. When treated in a similar fashion, the smaller clone (plO1-226-1) gave rise to a transcript of 185 bases. Because the insert DNAs in the two clones differ in length by 145 bp, it is probable that the transcripts of 185 and 340 bases have a common initiation site. When the recombinant was cut with Hindlll, a 20-bp section of the multiple cloning site was attached to the end of the XPLS27-inserted DNA. By subtraction of this value from the length of the

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transcripts, it is possible to localize the area of the initiation of transcription to approximately nucleotides 811 and 615. The amount of RNA polymerase used for the in vitro transcription assay was standardized to incorporate the same amount of [32P]UMP into acid-precipitable material using the RNA polymerase assay (1). When the cloned fragments of DNA were used, it appeared that E. coli RNA polymerase recognized the vector promoters more efficiently than did the P. aeruginosa RNA polymerase. In addition, E. coli RNA polymerase appeared to produce the transcript of 145 bases which originated from nucleotide 615 more efficiently than did the RNA polymerase from P. aeruginosa. Overexposure of the autoradiogram was necessary to visualize the 145-base transcript when P. aeruginosa RNA polymerase was used. However, the overall patterns of transcription obtained with both RNA polymerase types were quite similar. It was observed previously with the xyl genes of Pseudomonas putida that the same initiation start sites are used in vivo in E. coli and P. putida despite the absence of a typical E. coli consensus sequence (23). Promoters recognized by the RNA polymerase of P. aeruginosa appear to have a different consensus sequence than those recognized by the RNA polymerase of E. coli, although a good consensus sequence for the promoters of P. aeruginosa is not yet available owing to the limited number of sequences analyzed. Preliminary data suggest that the promoter may have the general form GG (N)10,14 GC (5); however, not all promoters identified in P. aeruginosa conform to this pattern. Examination of the area where the transcripts initiated revealed at least two regions that resembled promoter sequences (Fig. 4). The sequence beginning at nucleotide 797 (P2) was particularly appealing, as it shared significant homology with the known promoter found in the TOL plasmid pNM74 (22) and was followed by a Shine-Dalgarno sequence (Fig. 7). The putative promoter (P1) between nucleotides 606 and 619 resembles the PAK pilin promoter sequence (8). However, no Shine-Dalgarno sequence was observed until nucleotide 874. In addition, stop codons occur in all three reading frames before this Shine-Dalgarno box. Perusal of (PLS27 DNA for E. coli-like promoters reveals two sequences, TATTGAC (N)18 TAGAGT (nucleotides 608 to 638) and TTGACA (N)16 TAGAGT (nucleotides 783 to 810), which bear considerable homology to the E. coli consensus sequence TTGACAT (N)7-17 TATAAT. Two other potential promoters are TTAGGCAT (N)16 TATAAC and TTGATAT (N)10 TATTGA, which begin at nucleotides 571 and 591, respectively. To determine the exact location of the promoter(s) and the site of initiation of transcription will require Si mapping and/or DNase footprinting with both P. aeruginosa and E. coli RNA polymerases. The promoters appear to be approximately 200 bp apart, which is slightly farther apart than the early promoters of T7, which are 125 bp apart (26), but significantly closer together than the early promoters of phage SP6, which are 1,000 bp apart (15). Both T7 and SP6 have a cluster of three early promoters. This may also be the case in 4PLS27, as the third promoter would be further along the genome than the length

of the fragments examined. ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council and by a Medical Research Council studentship (to B.J.A.).

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