FEMS Microbiology Letters 69 (1990) 277-282 277 Published by Elsevier

FEMSLE 04023

Isolation and characterization of bacteriophage PM3 from Aeromonas hydrophila the bacterial receptor

for which is the monopolar flagellum

Susana Merino, Silvia C a m p r u b i and Juan M. Tomfis

Departamento de Microbiologia, Universidad de Barcelona, Barcelona, Spain

Received 3 January 1990 Revision received 19 February 1990

Accepted 20 February 1990

Key words: Aeromonas hydrophila; Bacteriophage PM3; Flagellum

1. SUMMARY

PM3 is an Aeromonas-specific bacteriophage which was isolated and characterized on A. hydro- phila strain TF7. Spontaneous mutants resistant to PM3 were non-motile having lost their char- acteristic monopolar flagellum. In addition, puri- fied flagella inactivated PM3. PM3 is the first filamentous bacteriophage isolated on Aeromonas, the adsorption site for which is the monopolar flagellum.

2. INTRODUCTION

Some bacteriophages attack only motile strains, of either Gram-positive or Gram-negative bacteria. In spite of their probably widespread occurrence [1] only a few have been isolated. The be~t ~iudied

Correspondence to: Juan M. Tom;is, Departamento de Micro- biolngia, Universidad de Bmcelona, Avda. Diagonal 645.08071 Barcelona. Spain.

is X 1. isolated on motile strains of Salmonella [2]. x l has since been shown to possess a broad host range amongst the enteric bacteria [2-4]. Six other phages, designated X2 to X7 [1,4], have also been isolated and found tc iyse motile enteric bacteria. For Gram-positive bacteria two flagellotrophic phages have been reported: PBS1 which lyses motile strains of Bacillg~ subtilis [5] and PBP1 which is specific for motile strains of B. pumilus [6]. None of the filamentous phages isolated are able to plate on Aeromonas strains, for which motility is an important characteristic [7]. Motile Aeromonas spp. are an ubiquitous component of the aquatic environment [7] and are also consid- ered to be normal inhabitants of the intestinal tract of fish [8]. A. hydrophila is an opportunitic, as well as primary pathogen of a variety of aquatic and terrestrial animals including humans [9].

In this paper we report on the isolation and characterization of bacteriophage PM3, a fila- mentous phage, isolated on motile Aeromonas strains, the recepior for which is the monopolar flagellum.

0378-1097/90/$03.50 © 1990 Federation of European Microbiological Societies

278

3. M A T E R I A L S A N D M E T H O D S

3.1. Bacteria, bacteriophages and media The strains, their relevant proper t ies and

origins, as well as their phage sensitivity are listed in Table 1. Bacteriophage Aeh-1 was kindly pro- vided by Ackermann [10]. Tryp tone-soya-bro th (TSB) was used for bacterial growth and phage propagat ion. TSB was supplemented with 1.5~g agar ( w / v ) (TSA) and TSA soft-agar with 0 .6~ agar. For t i t rat ion and inact ivat ion assays, phage suspensions were di luted in phage buffer [11].

The selection of phage-resis tant clones of A. hydrophila TF7 was achieved by seeding on a top-layer of soft TSA (5 ml) 1 × 107cfu o f bacterial strain in the presence of 1 × 10 t° pfu phage PM3 before pour ing over the surface of a TSA plate.

Table 1

Bacterial strains used

Strain Relevant Sensitivity to Source characteristics PM3 Aeh-1

TF7 011, S-layer +, motile + - [22] AH-45 011, S-layer +,

nonmotile - - this work AH-57 011, S-layer +,

nonmotile - - this work LLI 011, S-layer +, motile + - [22 l ATC9071 011, S-layer +, motile - - ATCC AH-205 011, S-layer +, motile - - [23] AH-3 034, motile + + [24] AH-203 034, nonmotile - + this work Ba5 034, motile + + [24] AH-101 034, nonmotile - + [24] AH-10 034, nonmotile - + [24] AH-50 022, motile - + our lab. ATCC

7966 not serotyped, motile - + ATCC 43014 not serotyped, motile - + clinical 17577 not serotyped, motile - + clinical 24963 not serotyped, motile + - clinical 8973 not serotyped, motile + + clinical 2911 not scrotyped, motile - - clinical

A. sobria AS-28 011, S-layer +, motile + - [23] AS-76 011, S-layer +, motile - - [23]

+, sensitive; - , resistant, Note: all the Aeromonca' strains which have not been serotyped do not belong to serotypes 011 or 034.

Af te r incubat ion at 3 7 ° C , overgrowing colonies on the otherwise relatively clear surface were then picked off and puri f ied by repeated single colony isolat ion on TSA.

3.2. Bacteriophage isolation Sewage samples (ob ta ined front various sewage

works in the Barcelona area) were enr iched with b ro th cul tures of A. hydrophila TF7 according to the m e t h o d of Bradley [12]. Di lut ions of the super- na tan t s o f the lysates were then spot ted on to bacterial lawns growing on TSA. Plaques were puri f ied before phage purif icat ion. Phage lysates were ob ta ined by the m e t h o d o f A d a m s [13] af ter incubat ion at 37 ° C overnight .

3.3. General phage techniques The me thods of A d a m s [13] were used. The

bac te r iophage hos t range was assayed by spot test. Solvent inact ivat ion (ether and ch loroform) of phages were carr ied out as descr ibed by Acker- m a n n [14]. Phage part icle purif icat ion, de termina- t ion of the buoyan t density, de te rmina t ion o f the nucleic acid type or the n u m b e r o f polypcpt ides were pe r fo rmed as previously descr ibed [15].

3.4. Phage inactivation experiments Bacter iophages (103 pfu) were incubated with

ei ther 107 bacterial cells, 200 /~g of whole mem- branes (WM), 100 p g of pur i f ied flagella or 100 p g o f pur i f ied flagella wi th equal volume of anti- flagellar se rum (di luted 1 /10) . Af te r 20 rain at 3 7 ° C , ch lo fo rom ( 2 - 3 drops) was added and the suspens ion was mixed for 1 rain. The mixture was centr i fuged at 12000 × g for 10 rain at 4 ° C and the superna tan t s were assayed for phage activity directly on A. hydrophila TF7.

3.5. Cell surface isolation and analyses C¢11 envelopes were p repared as previously de-

scr ibed [16]. Purif icat ion of flagella was per fo rmed as briefly descr ibed. The bacterial overnight growth was vigorously vor texed for 2 rain to re- lease flagella f rom the cells. The coils were col- lected by centr i fugat ion (14100 × g for 20 rain) and the result ing superna tan t was sa tura ted to 30% with a m m o n i u m sulphate. The precipi tate

(which contained flagella) was suspended in 5 ml of TEAN (20 mM Tris, 1 mM EDTA, 0.1% sodium azide, and 0.25 M NaCI, pH 7.4) buffer, and then dialysed extensively against the same buffer. After dialysis, the preparation was centrifuged (14 100 x g for 20 min at 4 °C) and the supernatant was further centrifuged at 77300 × g for 2 h at 4 ° C to obtain purified flagella as pellets.

Membrane proteins were analyzed by sodium dodecyl sulphate-polyacrylamide gel electro- phoresis (SDS-PAGE) as previously described [161.

3.6. Electron microscopy Techniques for visualizing negatively stained

phage, whole cells, purified flagella and phage- flagellum mixtures were as previously described [151.

3. 7. Motility For the observation of motility, A. hydrophila

strains were grown overnight with shaking at 37 ° C in TSB. All strains were observed for motility by wet-mount preparations on a phase-contrast mi-

~:

2

• ,

Fig, i. Electron micrographs of purifid bacteriophage PM3 (l) and the bacteriophage plus purified flagella from strain TF7 (2 and 3) negatively stained with 1% phospbotungstic acid (bar = 100 nm).

280

croscope and by a semisolid culture technique on a broth with 0.4~ agar [17].

4. RESULTS A N D DISCUSSION

4.1. Bacteriophage isolation and characterization Bacteriophage PM3 was isolated from one to

the five freshwater samples obtained from differ- ent sources and tested on A. hydrophila TF7. Phage PM3 gave turbid plaques, of approximately 2 mm, at both 2 0 ° C and 37°C. Phage PM3 incorporated [ 3H]thymidine into their nucleic acid, and therefore are DNA phages. Furthermore, Bradley's method [18] confirmed that PM3 con- tains double stranded DNA and certain restriction enzymes ( PstI, SalI, BglII, and BamHI) were able to cleave PM3 bacteriophage D N A .

Bacteriophage PM3 was found to express a total of 13 polypeptides with four major proteins of 43.0, 41.0, 39.0 and 15.5 kDa. Neither chloroform nor ether caused any loss of PM3 infectivity. Also, PM3 possessed a buoyant density of 1.50 g / c m 3, a polyhaedric head and a long ,loncontractile tail with a tapering end probably terminated with a tail fiber (Fig. 1).

PM3 is able to multiply on different serotypes of A. hydrophila strains but not on A. salmonicida, although some strains belonging to the same O- antigen serotype showed differential susceptibility. For example, A. hydrophila TF7 or LL1 (serotype 011) are sensitive to this phage while strains ATCCg071 or AH-205 (also 011) are resistant to PM3 (Table 1). Vibrio anguillarum (serotypes 01 and 02) strains and all the Enterobacteriaceae

: 1

: - . . " ," . , ' v ~ ..

°'" i

Fig. 2. Electron micrographs of A. hydrophila strains: l , strain TF7 (wild-type): 2, strain AH-45; and 3. strain AH-57, negatively stained with 1% phosphotungstic acid (bar ffi I p m).

tested were resistant to this bacteriophage (data not shown).

According to their morphological, physico- chemical and biological characteristics, PM3 can be classified in class B of Bradley [19] and in morphotype BI by Ackermann [20 l.

4.2. Bacteriophage surface receptor Mutants resistant to bacteriophage PM3 oc-

curred at a frequency of about 8 x 10 -7. We iso- lated aad characterized two independent mutants from strain TF7 resistant to PM3 (AH-45 and AH-57). These phage-resistant mutants showed similar LPS and OM protein profiles as the wild- type (data not shown). When we examined the motility of the mutant strains we found that un- like the parent strain they were nonmotile. All phage-resistant mutants obtained from a variety of motile A. hydrophila strains sensitive to the phage PM3 were nonmotile. Electron micrographs of strain TF7 and the mutant strains (AH-45 and AH-57) are shown in Fig. 2. As can be observed the mutant strains were devoid of the monopolar flagellum characteristic of the wild-type strain and showed alterations in flageilar number and mor- phology. Strain AH-45 showed multiple hair-like surface appendages. A similar phenomenon has been observed in some fla mutans of Salmonella t~phimurium [21]. These surface structures could either be multiple but inactive flagella (since AH- 45 is non-motile) or fimbriae. Further work is required to fully characterize these surface ap- pendages although they are unlikely to be fimbrlae since they are expressed even when AH-45 is grown under conditions known to repress fimbna- tion. Strain AH-57 showed two bipolar altered flagella and is also similar to some mutants of S. typhimurium [21]. Also, these PM3-resistant (S- layer + ) mutants, unlike strains belonging to sero- type 011 (S-layer+), were unable to autoag- glutinate (data not shown).

Phage PM3 adsorbed readily (more than 75% of phage inactivation) to A. hydrophila TF7 cells grown under motile conditions (shaking at 200 rpm at 370C) (Fig. 1), but was unable to adsorb (less than 2% of phage inactivation) to the same cells grown under restrictive motile conditions (static growth at 20 o C). This fact prompted us to isolate and purify flagella from strain TF7. Purl-

,!! ~L

,~N~.

.

1 2

Fig. 3. SDS-PAGE. Lanes: l, molecular weight standard from Pharmacia (14.0, 20.1, 30.0, 43.0. 67.0, and 94.0 kDa); 2.

purified flagella from A. hydrophila TF7.

lied flagella showed a unique polypeptide when analyzed by S D S - P A G E that is probabl~¢ a flagel- lin subunit of about 45 kDa (Fig. 3). When we incubated purified flagella from strain TF7 with bacteriophage PM3, the phage adsorbed readily to the flagella (Fig. 1) (more than 75% of phage inactivation). Also, if we preincubated for 1 h at 37 ° C purified flagella of strain TF7 or whole cells of :he same strain grown under motile conditions with anti-flagellar serum, there was a complete loss of PM3 bacteriophage adsorption. Thus, it seems clear that the PM3 bacteriophage receptor is the monopolar flagellum of A. hyd"ophila.

A C K N O W L E D G E M ENTS

S.M. is a recipient of a student grant from C.I.R.I.T. (Generalitat de Catalunya). We thank H.W. Ackermann for bacteriophage Aeh-1.

282

R E F E R E N C E S

[1] Edwards, S. and Meynell, G.G. (1967) J. Gen. Virol. 2, 443-444.

[2] Meynell, E.W. (1961) J. Gen. Micrubiol. 25, 253-290. [3] lino, T. and Mitani, M. (1967) J. Virol. 1,445-447. [4] Appelbaum, P.C., Hugo, N. and Coetzee, J.N. (1971) J.

Gen. Virol. 13,153-162. [5] Joys, T.M. (1965) J. Bacteriol. 90, 1575-1577. [6] Lovett, P.S. (1972) Virology 47, 743-752. [7] Neilson, A.H. (1978) J. Appl. Bacteriol. 44, 259-264. [8] Trust, T.J. and Sparrow, R.A.H. (1974) Can. J. Microbiol.

20. 1219-1228. [9] Janda, J.M., Clark, R.B. and Brenden, R. (1985) Curt.

Micrubiol. 12,163-168. [10] Ackermann, H.W., Dauguet, C., Paterson, W.D., Popoff,

M., Rouf, M.A. and Vien, J.F. (1985) Ann. Microbiol. (Inst. Pasteur) 136E, 175-199.

[11] Clowes, R.C. and Hayes, W. (1968) Experiments in Mi- crobial Genetics. Blackwell Scientific Publishing Co. Ltd., Oxford.

[12] Bradley, D.E. (1966) J. Gen. Microbiol. 44, 383-391. [13] Adams, M.H. (1959) Bacteriophages. Interscience, New

York.

[141 Ackermann. H.W., Audurier, A., Berthaume, L., Jones, L.A., Mayo, J.A. and Vidaver, A.K. (1978) Adv. Virus Res. 23, 1-24.

[15] Regu6, M, Tomfis~ J., Par6s. R. and Jofre, J. (1981) Curt. Microbiol. 5.153-156.

[16] Tomfis, J.M. and Jofre, J. (1985) J. Bacteriol. 162. 1276- 1279.

[17] Hanson, R.S. and Phillips, J.A. (1981) Manual of Methods for General Bacteriology. American Society for Microbi- ology, Washington, D.C.

[18] Bradley, D.E. (1966) J. Gen. Microbiol.. 44, 383-391. [19] Bradley, D.E. (1967) Bacteriol. Rev. 31,230-314. [20] Ackermann. H.W. and DuBow, M.S. (1987) Viruses of

prokaryotes. CRC Press. Florida. [21] Yamagughi, S.. Fujita. H., Aizawa, S.I., Macnab, R.M.

and Ishihara, A. (1986) J. Bacteriol. 166, 187-193. [22] Dooley, J.S.G., Lallier. R., Shaw. D.H. and Trust, T.J.

(1985) J. Bacteriol. 164, 263-269. [23] Janda, J.M., Oshiro, L.S., Abbott, S.L. and Duffey, P.S.

(1987) Infect. lmmun. 55, 3070-3077. [24] Merino, S., Benedi, V.J. and Tom:is, J.M. (1989) Micro-

bios 59. 165-173.