Isolation and characterization of a Pseudomonas ...

Isolation and characterization of a Pseudomonas aemginosa bacteriophage with a very limited host range DIANNEBIGBYAND ANDREW M. B. KROPINSKI'

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by China University of Science and Technology on 06/04/13 For personal use only.

Department of Microbiology and Immunology, Queen's University, Kingston, Ont., Canada K7L 3N6 Received September 20, 1988 Accepted February 21, 1989 BIGBY,D., and KROPINSKI, A. M. B. 1989. Isolation and characterization of a Pseudomonas aeruginosa bacteriophage with a very limited host range. Can. J. Microbiol. 35: 630-635. A Pseudomonas aeruginosa bacteriophage, 4PLS743, with extremely limited host range has been isolated. It belongs to the virus family Podoviridae, morphological type C1, and possesses a head diameter of 45 nm. The phage has a buoyant density in CsCl of 1.516 g/cm3, and its mass is 45 x lo6 daltons. The phage particles are composed of double-stranded DNA (49.9 mol% G+C; 42.4 kilobase pairs) and 11 structural proteins (66% by weight). The major head protein, P5, has a M,of 34 500. The DNA is not cut by SalI or XhoI restriction endonucleases, but is cut by PvuII (1 site), KpnI and BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), and HindIII (12 sites). A restriction endonuclease map is presented. Key words: Pseudomonas, bacteriophage, DNA, restriction map, structural proteins, electron microscopy. BIGBY,D., et KROPINSKI, A. M. B. 1989. Isolation and characterization of a Pseudomonas aeruginosa bacteriophage with a very limited host range. Can. J. Microbiol. 35 : 630-635. Nous avons is016 un bactkriophage de Pseudomonas aeruginosa, 4PLS743, qui affiche une gamrne extr6mement restreinte de cellules-h6tes. Ce virus appartient ii la famille des Popoviridae, de type morphologique C1, et possMe une t6te dont le diamhtre est de 45 nm. Sa densit6 de flottaison dans le CsCl est de 1.516 g/cm3 et sa masse totale est de 45 x lo6 daltons. Les particules phagiques sont compos6es d'ADN ii double brin (G+C 49,9 mol% et 42 400 paires de bases) et de onze prot6ines structurales (66% du poids). La prot6ine principale de la t6te, P5, a un M, de 34 500. L'ADN n'est pas coup6 par les endonuclhses de restriction SalI ou XhoI, mais il peut 6tre coup6 par PvuII (1 site), KpnI et BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), et HindIII (12 sites). Une cartographie des endonucl6ases de restriction est pr6sentk. Mots elks : Pseudomonas, bactkriophage, ADN, cartographie des endonucl6ases de restriction, prot6ines structurales, microscopic 6lectronique. [Traduit par la revue]

Introduction Relatively few Pseudomonas bacteriophages have received the same in-depth study that has been directed at understanding the molecular biology of the coliphages. These include phages with long noncontractile tails (siphoviruses; Matthews 1982), such as the mutator phages D3112 (Krylov et al. 1980, 1982; Rehmat and Shapiro 1983) and PH2 (Akhverdian et al. 1984), the temperate polysaccharide-depolymerizingphage 2 (Castillo and Bartell 1974, 1976), and the Pseudomonas pseudojlava phages of the gd series (Auling 1978; Auling et al. 1980). Other well-characterized phages of this genus include the corticovirus, PM2 (Franklin 1978; Mindich 1978), the lipidcontaining cystovirus, 46 (Mindich 1978), and 4W-14 (Kropinski et al. 1973), a member of the Myovindae. Although members of the family Podoviridae represent the second most commonly characterized phages of Pseudomonas only five have been characterized to any extent. These are 9s-1 (Kelln and Warren 1977), 4PLS27 (Jarrell and Kropinski 1981), gh-1 (Lee and Boezi 1966), and the generalized transducing phage, F116 (Caruso and Shapiro 1982; Ackerman and Dubow 1987). Only in the latter case has the phage genome been characterized beyond the level of base composition. Korsten et al. (1979) have remarked on the amazing similarity of structure and intracellular replication strategy between virulent members of this viral group isolated against a variety of phylogenetically unrelated bacteria. There has been only one report in the scientific literature on the isolation and characterization of temperate members of the Podoviridae infectious for Pseudomonas aeruginosa (Yamamoto and Chow 1968; H.-W. Ackermann, personal communication). Enrich'Author to whom correspondence should be addressed. Rinted in Canada 1 Imprim6 au Canada

ments, originally intended to isolate filamentous phages, have led to the isolation of a phage with a very limited host range, the characteristics of which are described in this manuscript.

Materials and methods Phage and bacterial strains The strains of P. aeruginosa used were PA01 (obtained from B. Holloway), AK1149 (flagella-; Jarrell and Kropinski 1981), AK1012 (LPS-defective; Jarrell and Kropinski 1981), AK1213 (pili-; Jarrell and Kropinski 1982), AK1380 (D3 lysogen; Kuzio and Kropinski 1983), and members of the Lanyi serogroups (Lanyi and Bergan 1978) obtained from B. Lanyi. Coliphage T7 and its host, Escherichia coli L21, were kindly provided by W. Studier. The phage lysates were stored at 4OC in sterile screw cap tubes containing 0.1 mL of chloroform. Bacterial strains were maintained at -70°C in broth containing 7.7% (v/v) dimethylsulfoxide. Phage titrations Phages were routinely titrated by the agar overlay technique (Adams 1959). The top and bottom layers consisted of tryptic soy broth (TSB; Difco Laboratories) containing 0.6 and 1.5 % (wlv) agar, respectively. The media were also supplemented with 1 mM CaCl,. Isolation of P . aeruginosa phage Ten millilitres of clarified raw sewage from the Kingston Township sewage treatment plant was mixed with 10 mL of double-strength TSB supplemented with 3 mM CaCl,, and 0.1 rnL of an overnight culture of PAO1. After a 1-day incubation at 37"C, samples were centrifuged (10 000 x g for 10 min) to remove cells and debris, and the supernatant was titrated. The center of a well-isolated turbid plaque was picked with a sterile Pasteur pipette and transferred to 1 mL of TSB in a centrifuge tube. The tube contents were vigorously mixed and then allowed to stand undisturbed for 30 min. After centrifugation to remove cells and

BIGBY AND KROPINSKI

agar, the supernatant was titrated. This procedure was repeated five times to yield a plaque-purified preparation of q5PLS743.


Adsorption of q5PLS743 to cells Adsorption of +PLS743 to strain PAOl was assayed as described previously (Jarrell and Kropinski 1976). Sensitivity of q5PLS743 to solvents, detergents, and deoxyribonuclease Two rnillilitres of chloroform, diethyl ether, or TSB was added to a stoppered 25-mL flask containing 2 mL of q5PLS743 (1.5 x 101° pfuImL). After incubation at 37°C and 200 rpm for 1 h, the solutions were briefly centrifuged, and the number of surviving phages in the aqueous layer was determined by titration. These phages, suspended in TSB, were also exposed to pepsin (2420 unitslmg; Sigma Chemical Co.), trypsin (11 250 unitslmg; Sigma), and deoxyribonuclease I (2000 unitslmg; Boehringer Mannheim Canada, Dorval, Que.) at 100 pglmL for 1 h at 37OC prior to titration. In addition, the stability of the phage at 37°C in the presence of 5 mM SDS was studied (Jarrell and Kropinski 1976). Purijication of q5PLS743 For isolation of nucleic acid and for PAGE analysis of the phage proteins, a large-scale purification of the phage was undertaken. PAOl grown overnight in 500 mL of TSB was subcultured into 4.5 L of TSB in a Labline S.M.S. Hi-Density fermentor (Labline, Melrose Park, IL) at 37OC, 200 rpm, at an aeration rate of 10 Llmin. When the culture had reached an A,,, of 0.2, q5PLS743 was added to a multiplicity of infection (MOI) of 0.1. After a further 8 -9 h of incubation, the phage lysate was centrifuged at 10 000 X g for 15 min. Phages in the supernatant were concentrated with 10% (wlv) PEG 6000 (Yamamoto et al. 1970) and the pellet obtained was suspended in 0.01 M Tris-HC1 buffer, pH 7.5, containing 0.01 M MgSO, (Tris-Mg2+). Because large amounts of contaminating cell debris were present, this phage preparation could not be purified further by CsCl isopycnic gradient centrifugation. Deoxyribonuclease I and sodicm deoxycholate were added to a concentration of 10 pg/mL and 5 mM, respectively. After incubation at 37°C for 4 h, the suspension was centrifuged at 10 000 X g for 10 min and the supernatant was layered onto a CsCl step gradient (Bachrach and Friedmann 1971). The tubes were centrifuged at 140 000 x g for 9 h (4°C). The phage band was collected and further purified by an isopycnic run. The twice-banded phage was dialyzed against Tris -Mg2+ buffer. Electron microscopy The gradient-purified phage was dialyzed against 1% ammonium bicarbonate (pH 8) and negatively stained on carbon-coated grids with neutralized 1% potassium phosphotungstate. The specimens were examined using a Zeiss EMlOCR transmission electron microscope. Side to side measurements of the phage particles were made with the magnification calibrated using a replica of the diffraction grating. Buoyant density The buoyant density of q5PLS743 in CsCl was determined by the methods of Bachrach and Friedmann (1971) and Bowes and Dowel1 (1974). q5PLS743 particle mass A mixture of q5PLS743 and coliphage T7 (lo8 pfu each) were layered over a sucrose gradient (10 -50 % , wlv) in 0.01 M Tris -HCl buffer, pH 7.1, and centrifuged at 80 000 x g for 45 min at 20°C using an SW41 Ti rotor. The sedimentation coefficient ($o,,) of q5PLS743 was determined by its position in the gradient relative to T7 using 453 as the s!,, of T7 (Dubin et al. 1970). The phage mass was calculated from the equation s,/s2 = (m11m2)213(Dubin et al. 1970), using 49.9 X 10, as the molecular weight of T7 (Bancroft and Freifelder 1970; Dubin et al. 1970).

,

Determination of protein and DNA content The DNA content of the purified phage was determined by the method of Ceriotti (1952) as modified by Weiner et al. (1976) with

63 1

calf thymus DNA (Boehringer Mannheim) as the standard. Protein was determined by the Coomassie blue procedure of Bradford (1976), modified by using a commercial reagent (Bio-Rad Protein Assay Dye Reagent Concentrate; Bio-Rad Laboratories, Richmond, CA). Isolation of phage DNA and % G +C determination Nucleic acid was extracted from purified q5PLS743 as outlined by Maniatis et al. (1982). The G + C content (mol%) was determined from the melting temperature (T,) in one-tenth strength standard saline citrate (SSC) using the equations of Mandel et al. (1970), De Ley (1970), and Kropinski (1974), assuming in the latter two cases that the T, in SSC was 163°C greater than in 0.1 X SSC (Mandel et al. 1970). SDS -PAGE The molecular weights of the structural polypeptides of the purified phage particles were estimated by SDS-PAGE using high and low molecular weight protein standards (Bio-Rad). Electrophoresis was carried out using 1.5 mm thick slab gels of polyacrylamide (7.5-15%, wlv) prepared in the manner described by Laemmli (1970). Following electrophoresis the gels were stained with Coomassie brilliant blue R250 prior to photography. Restriction endonuclease map of q5PLS743 DNA q5PLS743 DNA, dissolved in 6 mM Tris-HC1 buffer, pH 7.5, containing 6 mM MgC1, and supplemented with 6 (L), 60 (M), or 150 mM (H) NaCl (Schleif and Wensink 1981), was digested with endonucleases at 37OC. The restriction endonucleases BamHI (H), BglII (L), EcoRI (H), HindIII (M), HpaI (L), KpnI (L), PvuI (H), PvuII (M), SalI (H), B a I (H), and B o I (H) were purchased from Boehringer Mannheim. Electrophoresis was carried out using TEB buffer under the conditions described by Maniatis et al. (1982). A DNA fragments generated by digestion with EcoRI, HindIII, and a combination of these two enzymes were used as molecular weight markers (Maniatis et al. 1982). A restriction map was constructed using double- and triple-digests and through the analysis of fragments incompletely digested with the restriction endonucleases.

Results Isolation of P. aeruginosa bacteriophage In several enrichments for P. aeruginosa phages from raw sewage large (3 mm in diam.), uniform, very turbid plaques were observed in numbers that exceeded those of clear plaques. The fact that this phage produced turbid plaque and also failed to completely lyse the host cells even at high phage concentrations, suggested that it was temperate. Unfortunately, several attempts to isolate lysogenic cells failed. The phage responsible for this plaque morphology (q5PLS743) was plaque purified and its physicochemical properties are described below. Properties of dPLS743 Electron microscopy Phage q5PLS743 belongs to the virus family Podoviridae (Matthews 1982) and to Ackermann's morphological group C1 (Ackermann et al. 1978). It possesses an isometric head with a diameter of 45 nm and a short noncontractile tail approximately 15 nm in length by 6 nrn in width (Fig. 1). It is therefore somewhat smaller than coliphage T7 and about the same size as Klebsiella phage no. 11 (Rudolph et al. 1975) and P. putida phage gh-1 (Lee and Boezi 1966). Host range and receptor speciJicity The host range of this bacteriophage is extremely limited. Of the 12 strains of P. aeruginosa tested, q5PLS743 only lysed derivatives of strain PAO. Using mutants deficient in pili (AK1213), flagella (AK1149), and the LPS core polysaccharide (AK1012), it was observed that plaque develop-


632

CAN. J. MICROBIOL. VOL. 35, 1989

FIG. 1 . Electron micrographs of phage 4PLS743 negatively stained with neutralized 1 % phosphotungstic acid. (A) Bar, 50 nm. (B) Bar, 100 nm.

ment only occurred on strains deficient in cellular appendages and not on AK1012. This suggests that 4PLS743, like q5PLS27 (Jarrell and Kropinski 198I), is LPS-specific. The adsorption rate constant (k) of $PLS743 to PA01 was calculated to be 2.1 x 10-lo mL/min at 37°C in TSB. This is a very slow rate when compared with other C1-type LPSmL/min; specific phages, such as q5PLS27 (9.8 x

Jarrell and Kropinski 1981) or P22 (3.8 Bezdek and Amati 1967).

X

low9mL/min;

Buoyant density The buoyant densities of 4PLS743 and coliphage T7 were found to be 1.516 and 1.504 g/cm3, respectively. The value for coliphage T7 is identical to the most recent published

633

BIGBY AND KROPINSKl

values (1.50 g/cm3; Ackermann et al. 1978; Matthews 1982) but is somewhat less than other reported buoyant densities for this phage, namely 1.513 (Davis and Hyman 1971) and 1.515 g/cm3 (Bancroft and Freifelder 1970).


4PLS743 mass Velocity sedimentation analysis of GPLS743 in sucrose gradients showed that this phage was somewhat lighter than coliphage T7. The estimated particle mass of 4PLS743 was 45.0 X lo6 daltons, compared with 49.9 X lo6 for T7 (Bancroft and Freifelder 1970; Dubin et al. 1970).

Sensitivity to chemical agents Phage 4PLS743 was not sensitive to either diethyl ether or chloroform, suggesting that it does not contain lipid (Bamford et al. 1981). This rules out the possibility that 4PLS743 is a member of the family Tectiviridae (Matthews 1982). In addition, no decrease in titre was noted upon incubation of phage preparations in the presence of proteases, deoxyribonuclease I, or 5 mM detergents (SDS and sodium deoxycholate). With respect to its sensitivity to surface active agents, it resembles 4PLS27 (Jarrell and Kropinski 1981) rather than 4PLS-1 (Jarrell and Kropinski 1976).

Chemical analysis of purijied dPLS743 Analysis of 1012 pfu showed the presence of 1.93 mg of protein (66% by weight) and 1.00 mg of DNA (34% by weight). These values closely correspond to the weight percentages found in purified P. putida phage gh-1 (Lee and Boezi 1966). Purified phage was examined by SDS-PAGE, using gels of various percentages (7.5, 10, 12.5, and 15%, w/v). The best resolution of the phage polypeptides was obtained with the 10 and 12.5% gels (Fig. 2) where a total of eleven bands were observed. The most abundant phage protein (P5) has a molecular weight of 34 500 and probably corresponds to the major head protein. The molecular weights of the other phage structural proteins are presented in Table 1. The phage DNA, in one-tenth strength SSC, exhibited a T, of 74.8 "C (average of six determinations), while the control DNA (E. coli) melted at 75.3 "C (average of three determinations). The experimental value for E. coli DNA corresponds to the reported value of 75.55"C (Mandel et al. 1970). Using the linear regression equations of Mandel et al. (1970), de Ley (1970), and Kropinski (1974), the mol% G +C in the phage DNA are 49.9, 52.9, and 55.0, respectively. Sensitivity of 4PLS743 DNA to restriction endonucleases and construction of a restriction map With the exception of SalI and XhoI which failed to cut 4PLS743 DNA, the other nucleases cut the DNA as follows: PvuII (1 site), KpnI and BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), and HindIII (12 sites). The sizes of the fragments generated with individual nucleases are listed in Table 2. By summing the sizes of the EcoRI, BamHI, and HindIII fragments, the average size of 4PLS743 DNA was calculated to be 42.4 kilobase pairs (kbp). Using PvuI, PvuII, KpnI, and BglII, singly and in combination (Table 2), a restriction map (Fig. 3) was constructed. This represents the second physical map for a Pseudomonas podovirus (see Caruso and Shapiro 1982).

Discussion 4PLS743 is an interesting bacteriophage in that it superficially resembles a temperate phage in failing to lyse cells at

FIG.2. Separation of the structural proteins of phage 4PLS743 on 10 (A) and 12.5% (B) polyacrylamide gels in the presence of SDS. (A) Lanes a and b, molecular weight markers (myosin, 200000; P-galactosidase, 116 250; phosphorylase B, 92 500; bovine serum albumin, 66200; ovalbumin, 45 000; carbonic anhydrase, 31 000; soybean trypsin inhibitor, 21 500; lysozyme, 14 400); lanes c and d, phage 4PLS743. (B) Lane a, molecular weight markers (14 400 92 500); lane b, phage structural proteins. The molecular weight values for phage proteins P1 to PI1 are given in Table 1.

TABLE1. Molecular weights of the structural polypeptides of phage 4PLS743 Polypeptide no.

Molecular weight

high MOI. Yet, numerous colony isolates from plaques and confluently infected areas on plates were sensitive to superinfection by 4PLS743 and did not spontaneously produce phage. This phenomenon could be due to the phage being poorly infectious (low k; small burst size; infectious for a specific subpopulation of cells; high frequency of abortive infections) or due to the existence of a high frequency of pseudolysogenic cells. It is of interest that in spite of its low k and extremely limited host range, this phage was the predominant isolate in several enrichments from sewage. One possibility, which would explain both its presence in sewage and the limited host range, is that many of the P. aeruginosa

CAN. J. MICROBIOL. VOL. 35, 1989

TABLE 2. Size (kbp) of DNA fragments generated by digestion of 4PLS743 DNA with restriction endonucleases Restriction endonuclease


Fragment no. A B

C D E

F G H I

J K

EcoRI

Hindm

BamHI

PvuII

KpnI

PVuI

BglU

8.6 6.3 5.9 5.3 5.3 4.4 3.3 1.7 1.35 1.2

9.8 6.0 4.2 4.05 3.7 3.0 2.8 1.55 1.4 1.3 1.25 1.15 0.95

14.5 9.0 4.6 4.2 3.9 3.8 1.95 0.72

37.0 7.8

23.0 19.0 3.0

25.0 8.5 5.9 3.4 1.3

31.0 13.7 0.5

L

M A B

A

10

Kpn I

c

1

I

A I

a

I C I

A

I E I O I

IC Bgl lf

8

B

I

I

eo

I 30

Pvu I Pvun

I 40

KILOBASE PAIRS

FIG. 3. Restriction endonuclease map of the genome of phage +PLS743. strains in nature may be stably lysogenic for dPLS743 or homoimrnune phages. The host range studies further suggest that LPS functions as the cellular receptor for this phage. Furthermore, the lysis of PA01 and the D3-convertant, AK1380, suggests that the 2,3-(1-acetyl-2-methyl-2-imidazolino-5 ,4)-2,3-dideoxy-D-mannuronyl (1 4) 2,3-diacetamido-2,3-dideoxy-D-mannuronyl portion of the 0-antigenic trimer may be important in the receptor activity. It is also interesting to note that strains serologically identical to PA0 from the Lanyi serotyping set were insensitive to 4PLS743. In possessing 11 structural proteins this phage resembles coliphage T7 (Studier 1969) and 4PLS27 (Jarrell and Kropinski 1981), but in size it is somewhat smaller than either. The results of the SDS-PAGE of the 4PLS743 particles suggest that the major head protein has a molecular weight of 34 500. This compares favourably with the values found for Citrobacter ViIII phage protein (35 000; Kwiatkowski et al. 1973), for coliphages 29 (36 000; Rieger et al. 1975) and T7 (38 000; Studier 1969), and for P. aeruginosa phage 4PLS27 (39 000; Jarrell and Kropinski 1981). The sum of the molecular weights of the structural polypeptides, 507 050, is closer to the value for the Citrobacter phage (429 000) than T7 (653 000) (Studier 1969). Considering the size of the genome (42.4 kbp) this indicates that approximately 32 % of the genome is concerned with structural proteins, a value quite similar to P22 (25 % ; Bezdek and Amati 1967). The viral family Podoviridae includes those phages that possess isometric or elongated heads, short noncontractile tails, and a linear double-stranded DNA genome. Within this morphogroup one finds several distinct "genera." One such genus contains virulent phages with the following additional characteristics: (i) the genome contains short terminally redundant sequences; (ii) the DNA contains fewer than

-

expected hexameric pallindromic nucleotide sequences and, as a result, exhibits resistance to certain common restriction endonucleases such as BamHI, EcoRI, and HindIII; and (iii) a common infection strategy is employed which includes the synthesis of a rifampicin-resistant RNA polymerase which is responsible for the transcription of late genes. This assemblage includes coliphages T7 and T3, and the Pseudomonas phages 4PLS27 (Allan et al. 1989) and gh-1 (Lee and Boezi 1966; Kortsen et al. 1979). Other possible genera would include the phylogenetically unrelated temperate phages, F 116 and P22, and the large virulent phage N4 (Kiino and RothrnanDenes 1988). GPLS743 superficially resembles the T7 group in possessing similar morphology, genome size, and a limited number of structural proteins, but it differs from this group in a number of respects. The major head protein is smaller and the sum of the molecular weights of the structural proteins is less. Furthermore, its DNA is very sensitive to the abovementioned restriction endonucleases which do not cleave DNA from T7-type phages. Furthermore, the lytic pattern appears more like that of a temperate than a virulent phage. Obviously further studies are required to define the characteristics of the different genera within the family Podoviridae. The phage has been deposited with the American Type Culture Collection and the FClix d'Herelle Reference Centre for Bacterial Viruses at Laval University (Quebec, Canada).

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