FEMS MicrobiologyLetters 68 (1990)239-2,t0, Published by Elsevier
239
Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila S u s a n a Merino, Silvia C a m p r u b i a n d J u a n M. T~n::_~s Deparlamentodc Mwrohiologia. UnH'ersidadde Barcelona. Barcelona. Spare Received27 November 1989 Accepted 7 December 1989 Key words: Aeromonas; Bacteriophage PM2; Lipopolysaccharide
1. S U M M A R Y PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaecharide (LPS), specifically a low-molecular weight LPS fraction (LPS-¢ore oligosaecharides). Mutants resistant ro this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-~:ore. No other outer-membrane (OM) molecules appeared to be involved in phage binding.
2. I N T R O D U C T I O N r,lotile Aeromonas spp. are an ubiquitous component of the aquatic environment [1] and are also considered to be normal in habitants of the intestinal tract of fish [2]. Aeromonas hydrophda is an opportunistic, a~ well as primary pathogen of a variety of aquatic and terrestrial an'~mals as v.ell as humans [3]. The clinical manifestation of A. hydrophila infections range from gastroenteritis to Corresptmdence to: J.M. Tom/ts. Departamento de Mi~to~,ic~ Iogia. Universidad de Bercelona. Diagonal 645. 08071 Barcelona. Spain.
soft tissue infections, septicemia, and meningitis 14.51. The surface characteristics of A. hydrophila correlate with changes in virulence and can be used to classify strains 16,7], We have r~oently described a group of A ~;.g-c,phda strains belonging to ~ r o t y p e 034 with heterogeneous LPS Opolysaccharide chains, which previously were reported to be modest in their virulence for fish and mice 18.91. In this paper we report on the isolation and characterization of bacteriophage PM2. isolated on A. t~vdrophila AH-3 (serotype 034t which is able to form plaques on strains of this serot?.1~e and many other A. l~1"drophtta. A. sobrta and A. salmonicida strains. Spontaneous phage PM2 resistant mutants lack the LPS O-antigen polysaccharide chains and we demonstrate that LPS alone is the bacterial surface receptor for this bacteriophage. Furthermore. we show that a lowmolecular weight LPS (which is enriched in LPScore oligosaecharides) is involved in PM2 bacteriophage reception. 3. M A T E R I A L S A N D METHODS 3.1. Bacteria, bacteriophages and media The strains, their relevant properties, origirL~, and phage sensitivities are listed in Table 1.
0378-1097/qO/$03.50 q 1990 FederaTion of EuropeanMicrobiologicalSocieues
240 Bacteriophage Aeh-I was kindly provided by Dr. H.W. Ackermann [10l. Tryptone-soya-broth (TSB) was used for bacterial growth and phage propagation. TSB was supplemented with 1.5% agar (w/v) (TSA) and TSA soft-agar with 0.6% agar. For titration and inactivation assays, phage suspensions were diluted in phage buffer [11]. Spontaneous phage resistant mutants of A. hydrophila AH*3 or Ba5 were isolated, independently for each strain, by spreading a mixture containing l0 s bacteria and 109 pfu of the phages on TSA. After 48 h at 20oC individual colonies were purified by streaking and cross-streaked against the bacteriophage to confirm resistance. Resistance to other bacteriophages was assayed by spot test.
3.Z Bacteriophage isolation Freshwater samples were centriluged and the superuatant was incubated at 20~C :vith an exponential growth-phase culture of A. hydrophila AH-3. After overnight incubation, the bacteria were removed by centrifugation and filtration, and the supematant was plated en ,4. hydrophila AH-3 by using the double agar layered method of Adams [12]. Plaques which formed on the plates were stabbed with a needle and eluted with a small volume of phage buffer. Each phage suspension was serially propagated twice on the same strain.
3.3. General phage ~eLAn;q,es and production of phage lysates The methods of Adams [12] were use,d. The incubation temperature was initially 200C, and the plates were incubated 24 h. The bacteriophage host range was assayed by spot test. One-step growth experiments were performed using A. hydrophila AH-3 as host at a multiplicity of infection of 1. Solvent inactivation (ether and chloroform) of phages, as well a~ phage particle purification, determination of the buoyant density, determination of the nucleic acid type or the number of polypeptides were carried out as previously described [131.
deoxycholate (DOC)-solubilized OM proteins with or without treatment wit~ 1• , g of proteinase K [14]. 100/tg of purified I,PS. 100 ttg of purified LPS with equal volume of anti-LPS serum (diluted 1/10 in PBS), or 50 /zg of pooled high or lowmolecular weight LPS fractions. Chloroform (2-3 drops) was added and the suspension was mixed for 1 rain. The mixture was centrifuged at 12 bOO × g for !0 rain at 4°C. The supernatants were assayed for phage aclivity directly on A. hydraphila AH-3.
3.5. Cell surface isolation and analyses Cell envelopes and outer membranes were prepared as previously described and analyzed by SDS-PAGE [14]. LPS was purified by the method of Westphal and Jann [15l as modified by Osbom [16]. Subfractionation of purified LPS by column chromatography was performed as we described previously [17]. Fractions were extensively dialyzed against distilled water, first at room temperature and then at 4°C, analyzed by SDS-PAGE and silver stained by the method of Tsai and Fraseh [18]. For chemical analysis, purified LPS was h y d r a a,ysed with 1 M HCI for 2 h at 105oC. Colorimettic a.-~lyses of the deoxy-D-mannooctulosonic acid (KDO) content of LPS was performed by the method of Karkhanis et al. [19]. Monosaccharides were also analysed as their alditol z_eetate derivatives by gas-liquid chromatography (GLC) on a 3% SP-~840 column (Supelco) at 225°C and 180oC. Alditol acetate _-'~rhohvdrate standards were either purchased from Supclco or prc},,t,e,d by us.
3.6. Anti-LPS serum Immune serum was obtained from adult New Zealand white rabbits injected with purified LPS from ,4 hydrot, hi!a AH-3 as previously describe~ I141. 4. RESULTS AND DISCUSSION
3.4. Phage inactivation experiments
4 I. Bacteriophage isolation and characterization
Bacteriophages (103 pfu) were incubated with either 107 bacterial cells. 200 pg of 1% (w/v)
Bacteriophage PM2 was isolated from one of three freshwater samples tested. Phage PM2 gave
241 Table 1 Bacterial strains used Strains of
,4. hydrophila AH-3 AH-35 Ba5 AH-54 AH-I01 AH-199 ALL50 AH-10 ATCC 7¢66 24830 24963 25616 TF7 AH-45 A|I-21
A ,'rd~rtJ AS-28 AH-,198 19975
A. salmt~mclda A 450 A 450-3
A 450-1
Relevant characteristics
Sensitivity to PM2
Aehl
0M,, S-layer isogenic mutant derived from AH-3. rough 034, S-layerisogenic mutant derived from Ba5, rough 034. S-layer isogenic mutant derived from AH-101, rough 022. S-layerrough, S-layernot serotyped. S-layer not serotyped, S-layernot serotyped. S.-laye,not ~roG',qped,S-layer 011, S-layer + isogenic mutant derived from TF7:011, S-layer isogenic mutant derived from TF7: rough, S-layer-
+
+
IS]
+
+
thi~ work 19]
+
thi~,v.t)rk [81
Sollrc~
-
, + + + * I + + +
-
+ + 4+ --
-
-
-
this ~ork our lab our lab ATCC clinical ~.iiail.al clinical 191 our
lab
our lab
0l 1. S-layer + isogenic mutant derived from AS-28; 01 l, S-layer rough, S-layer-
-
-
+ +
-
our lab clinical
smooth, A-layer + isogenic mutant derived from A 450; smooth. A-layer isogenic mutant derived from A 450: rough. A-layer -
+
+
1241
+
+
1241
+
+
i241
161
! . c,mshive: -,resistant. Note: All the Aeromona~ >,~;.~ ~'-t ~rotvped do not belong to serolypes 011 or 0.~4.
clear plaques, b o t h at 2 0 ° C a n d 3 7 0 C , w i t h a r a n g e o f sizes u p t o 3 m m with d i f f u s e edges. P h a g e P M 2 i n c o r p o r a t e d 3 H - t h y m i d i n e i n t o its nucleic acid, w h i c h is therefore D N A . F u r t h e r more, u s i n g Bradley's m e t h o d [20] we c o n f i r m e d t h a t p h a g e P M 2 h a s d o u b l e s : r a n d e d D N A . Also, certain restriction enzymes ( E c o R l , S a i l , H i n d l l l a n d B a m H l ) were able : o cleave P M 2 bacteriop h a g e D N A . B a c t e r i o p h a g e P M 2 p r o d u c e d 15 p o l y p e p t i d e s , there b e i n g three m a j o r p r o t e i n s o f
56.0. 42.0 a n d 18.> K ~ , . N~itFcr c h l o r o f o r m nor ether caused any loss o f F'+J2 inlecfi+~:7 Also, b a c t e r i o p h a g e P M 2 showed a b u o y a n t density o f 1.51 g / c m ~, a latent p e r i o d o f 30 rain, a rise p e r i o d o f 20 rain a n d a burst size o f 290 pfu. Bacteri¢ h~ge P M 2 s h o w s a polyhedric head with a collar, • .ntractile tail and a base plate with at least sL': spikes (Fig. 1 ). T h i s b a c t e r i o p h a g e seems very s i m i l a r t o the well characterized T-even E. coli bacteriophages. Bacteriophage P M 2 has a
242 Thus, PM2 is a bacteriophage that can be classifted in the Myoviridae family by Matthews [21] and in group A2 of A c k e r m a a n [22] according to their morphological physico-chemical and biological characteristics.
4.2. Bacteriophage surface zeceptor
Fig. l. Electron micrographyof purified bacteriophage PM2 negatively stained with 17o phosphotungstic acid (bar = 100 nm).
broad host range in that it is able to produce plaques on different Aeromonas strains. For example PM2 is able to produce plaques on different serotypes of A. hydJophila strains, on A. sobria and on A. salmonicida strains (Table 1). Vibrio anguillarum (serotypes 01 and 02) strains and all Enterobacteriaceae tested were resistant to this bacteriophage.
Mutants resistant to bacteriophage PM2 occuffed at a frequency of about 3 x 1 0 ~ and fell into a s i n ~ e class based on their phage sensitivities and LPS profiles. All mutants (20 tested for A. hydr~phila strains AH-3 and Ba5) lacked the O-antigen polysaccharide chains, showed a higher electrophoresis migration rate of their LPS-core oligosaccharides and they were resistant to bacteriophage Ach-1. Phage PM2 adsorbed readily to A. hydrophila AH-3 or Ba5 but not to the PM2-resistant mutants AH-53 and AH-54, respectively (Table 2) suggesting that phage resistance was due to an altered phage receptor. IX)C-solubilized O M fro~.a A. hydrophila AH-3 or Ba5 (wildqypes) and from PM2-resistant mutants showed similar phage adsorption, as did the corresponding whole cells (Table 2). Proteinase K digestion did not alter the ability of DOC-solubilized O M from the wild type strains to inactivate bacteriophage PM2 (Table 2). Furthermore, if we treated the whole cells of the wild t ) p e strains (AH-3 or Ba5) with s~ecific anti-LPS serum for 1 h at 3 7 ° C and remo,,ed the serum by centrifugation, the treated cells were no longer able to inactivate bacteriophage PM2.
Table 2 Inactivation of bac|criophage PM2 by wholecells and OM components
.4. hydrophila strains
%bacteriophageinactivationby Whole OM " OM with cells proteinase K treatment b
LPS c
HMW-LPS u
LMW-LPS a
AH-3 AH-53 Ba5 AH-54
98.3 < 1.0 97.8 < 1.0
77.2 < 1.0 78.3 < 1.0
< 2.0 NT < 2.0 NT
75.8 NT 76.9 NT
97.6 < 1.0 97.2 < 1.0
%.8 NT • 96.8 NT
* OM solubilized with 19~ DO(7 and 2 mM EDTA [14]. h Treatment with proteinase K (10/+g/rid) for 2 h at 45°C. ¢ Purified LPS (100 p.g)[15,16]. d Pooled high-molecularweight LPS fractions and pooled low-molecularweight LPS fracP.ons(50 pg) as it is shown in Fig. 2. e NT, not tested.
243
!'
A
B
123456
I
23
Fig. 2. SDS-PAGE of LPS from ,4 hydrophda strains. (A) Lanes: I, strain AH-3: 2, strain AH-53: 3, strain BaS: 4. strain AH-54: 5. strain AH-IOI: and 6, strain AH-199 (B) Lanes: ]. LPS from strain AH-3: 2. Iowmolecular weight LPS from strain AH-3 subfractionated according to materials and methods: and 3. high-molecular weight LPS from strain AH-3.
Purified LPS f r o m .4. hydrophila A H - 3 or Ba5 was able to inactivate bacteriophage PE'2, but purified LPS from A H - 5 3 or A H - 5 4 (spontaneous PM2-resistant mutants) was not. The degree of p h a g e inactivation was directly related to the LPS concentration in the incubation mixture (1.5 fig of LPS was required to reduce the p h a g e titer in 50%). LPS fractions c o n t a i n i n g low-molecular weight A H - 3 LPS (enriched on LPS-core oligosaccharide~XFig. 2) were able to inactivate bacteriop h a g e PM2, while LPS fractions from the wild type strains containing high-molecular ,-'eight LPS (enriched o n O-antigen chains) (Fig. 2) were not inhibitory (phage inactivation in all ca:,=.s < 1%).
We examined the protein composition of the OM~ ~f ,I. h)J, uyhila stratus A H - 3 a n d BaS. a n d their respective PM2-re~istant mutants. N o main difference were f o u n d in the O M protein profiles ( d a t a not shown). W e also examined purified LPS from the wild type strains a n d the phage resistant m u t a n t s by the method of Tsai a n d Frasch [18]. There was a n a p p a r e n t complete loss of the H M W - L P S (O-antigen) a n d a change on the relative electrophoretic mobility of the L M W - L P S (LPS-c.ore) on the purified LPS from the PM2-resistant m u t a n t s (Fig. 2), confirming the inactivation d a t a of Table 2. Chemical analyses of purified LPS from A. hydrophila A H - 3 a n d BaS, a n d their respective phage-resistant m u t a n t s is shown on Table 3. A reduction of glucose a n d a complete loss of hexosamines in the purified LPSs of the m u t a n t strains was observed, which c a n be correlated with the changes observed o n S D S - P A G E . This reduction in glucose m a y explain the alteration in the electrophoretic moiety of the L M W - L P S on the mutants, because these strains belong to type 2 a c c o r d i n g to Shaw a n d H o d d e r [23). a n d in this type glucose is a c o m p o n e n t of the LPS core. Also. it seems clear that hexosamines are c o m p o n e n t s of the O-antigen LPS in these strains because t h ~ were lost on the LPSs of the m u t a n t strains lacking H M W - L P S . T h u s it is a p p a r e n t that the I.~w-molecu!C.~ weight LPS (LPS core oligosa,~,:harides) alone is the receptor for bacteriophage P~'2. a n d spontaneous phage-resistant m u t a n t s unable to bind this p h a g e iack the O-antigen p o l y - s a c c h a n d e chains. As this bacteriophage ~howed a b r o a d host range
Table 3 Chemical compo.~iti~;,of purified LPS from A. hvdrophda strains LPS from strain
Amount ( #tool/rag of LPS) of: KDO a L-hepD-heptosa b rosa h
PenlOSe b
Gluco~ h
Galaclose b
Rhamno~ ~
Hexo~.,~mlne~ ~
AH-3 AH-53 Ba5 AH-54
0.018 0.051 0.019 0.050
0 0 0 0
1.57 132 1.55 1.29
0 0 0 0
0 0 0 0
041 0 043 0
A. [(vdrophda
0.31 1.25 0.31 1.26
a Assayed by colorimetric method [19]. b Assayed by ggs-liqaid chromatography.
0 0 0 0
244
o n Aeromonas spp., the isolation o f P M 2 - r e s i s t a n t m u t a n t s f r o m d i f f e r e n t Aeromonas s t r a i n s is a useful tool for o b t a i n i n g isogenic L P S m u t a n t s l a c k i n g a n O - a n t i g e n . T h e availability o f such isogenic m u t a n t s will allow u s to s t u d y the c o n t r i b u t i o n o f the O - a n t i g e n to the virulence o f .4 eromonas.
ACKNOWLEDGEMENTS W e t h a n k H . W . A c k e r m a n n for b a c t e r i o p h a g e • ' .A. a. k. . . . 1 ~o.,~,, J~ a Jtxipient of a student grant from C.I.R.I.T. (Generalitat de Catalunya).
REFERENCES |l] Neilsm. A.H. (1978) J. Appl. Bacteriol. 44, 259-264. [2] Trust, T.$. and Sparrow, R.A.H. (1974)Can. J. MicrobioL 20, 1219-1228. [3[ Howard, S.P- and Backley, J.T. (1985) J. Bacteriol. 161, 463-465. 14] Freij, BJ. 0984) Pediatr. Infect. Dis. 31. 164-175. [5] Ljungh, A, and Wadstrom. T. (1983) Toxicol. Toxin Rev. I, 257- 307. [6] Jartd& J.M., Clark, R.R and Brenden. R. (1985) Can'. Microbiol. 12, 163-168. [7] Lallicr, R., Bernard. F. and Lalo.qde, G. (1984) Can. J. Microbiol. 30. 900-904.
[8] Merino. S. and Tom,is. J.M. (1988) Microbiologla 4. 181-184. [9[ Minal. KR.. Lalonde, G.. LeBlanc. D.. Olivicr. G. and Lallier, R. (1980) Can. J. MicrobioL 26, 1501-1503. [10] Ackemmnn. H.W.. Dauguet. C.. Paterson, W.D.. Popoff, M,. RonL M.A. and Vien, J.F. (1985) Ann. Inst. Pasteur/ Microbiol. 136E, 175-199. Ill] Clowes, R.C. and Hayes, W. (1968) Experiments in Microbial genetics. BlackwelL Oxford. [12] Adams. M.H. (1959) Bacteriophages. lmerscience. New York. [13] Regu~, M.+ Tom~. J.M., Par6s+ R- and Jofre. J. (1981) Carr. Mierobiol. 5, 152-156. [14] Tom~, J.M. and Jofre, J. (1985) J. Bacteriol. 162, 1276- t +~'/% [15] WestphaL O. and Jann, K. (1965) Methods Carbohydr. Chem. 5. 83-91. [16] Osborn. M,J. (1966) Methods Enzyrnol. 8. 161-164. [17] Cimana. B and Tom~s. J.M. (1987) Infect. Immun. 55. 2741-2746. [181 Tsai. C.M..rod Frasch+ C,E. (i'~82) Anal. Biochem. 119, 115-119. []9] Karkb~;: s. Y.D.. Zehner. J.Y., Jackson. J.J., and Carlo, DJ. (1978) Anal. ,-~,ochem. 85, 595-601. [20] Bradley, D.E. (1966) J. Gan. Microbiol. 44, 383-391. [21[ MaUhews, R E.F. (1980) Cl&ssification et nomenclature des virus Troisi~me rapport du cc.~t~ international de taxononfie des virus. Paris, M ~ . [22] Ackermann. H.W. (1973) Handbook of Microbiology. CRC Press, Boca Raton. FL. [23] Shaw. D . H and Hodder. H.J. (1978) Can. J. Microbiol. 24, g64-868. 124] Munn. C.B., ]shiguro, E.E., Kay. W.W. and Trust. TJ. (1982) Infect. Immun. 36, 1069-107~.
239
Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila S u s a n a Merino, Silvia C a m p r u b i a n d J u a n M. T~n::_~s Deparlamentodc Mwrohiologia. UnH'ersidadde Barcelona. Barcelona. Spare Received27 November 1989 Accepted 7 December 1989 Key words: Aeromonas; Bacteriophage PM2; Lipopolysaccharide
1. S U M M A R Y PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaecharide (LPS), specifically a low-molecular weight LPS fraction (LPS-¢ore oligosaecharides). Mutants resistant ro this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-~:ore. No other outer-membrane (OM) molecules appeared to be involved in phage binding.
2. I N T R O D U C T I O N r,lotile Aeromonas spp. are an ubiquitous component of the aquatic environment [1] and are also considered to be normal in habitants of the intestinal tract of fish [2]. Aeromonas hydrophda is an opportunistic, a~ well as primary pathogen of a variety of aquatic and terrestrial an'~mals as v.ell as humans [3]. The clinical manifestation of A. hydrophila infections range from gastroenteritis to Corresptmdence to: J.M. Tom/ts. Departamento de Mi~to~,ic~ Iogia. Universidad de Bercelona. Diagonal 645. 08071 Barcelona. Spain.
soft tissue infections, septicemia, and meningitis 14.51. The surface characteristics of A. hydrophila correlate with changes in virulence and can be used to classify strains 16,7], We have r~oently described a group of A ~;.g-c,phda strains belonging to ~ r o t y p e 034 with heterogeneous LPS Opolysaccharide chains, which previously were reported to be modest in their virulence for fish and mice 18.91. In this paper we report on the isolation and characterization of bacteriophage PM2. isolated on A. t~vdrophila AH-3 (serotype 034t which is able to form plaques on strains of this serot?.1~e and many other A. l~1"drophtta. A. sobrta and A. salmonicida strains. Spontaneous phage PM2 resistant mutants lack the LPS O-antigen polysaccharide chains and we demonstrate that LPS alone is the bacterial surface receptor for this bacteriophage. Furthermore. we show that a lowmolecular weight LPS (which is enriched in LPScore oligosaecharides) is involved in PM2 bacteriophage reception. 3. M A T E R I A L S A N D METHODS 3.1. Bacteria, bacteriophages and media The strains, their relevant properties, origirL~, and phage sensitivities are listed in Table 1.
0378-1097/qO/$03.50 q 1990 FederaTion of EuropeanMicrobiologicalSocieues
240 Bacteriophage Aeh-I was kindly provided by Dr. H.W. Ackermann [10l. Tryptone-soya-broth (TSB) was used for bacterial growth and phage propagation. TSB was supplemented with 1.5% agar (w/v) (TSA) and TSA soft-agar with 0.6% agar. For titration and inactivation assays, phage suspensions were diluted in phage buffer [11]. Spontaneous phage resistant mutants of A. hydrophila AH*3 or Ba5 were isolated, independently for each strain, by spreading a mixture containing l0 s bacteria and 109 pfu of the phages on TSA. After 48 h at 20oC individual colonies were purified by streaking and cross-streaked against the bacteriophage to confirm resistance. Resistance to other bacteriophages was assayed by spot test.
3.Z Bacteriophage isolation Freshwater samples were centriluged and the superuatant was incubated at 20~C :vith an exponential growth-phase culture of A. hydrophila AH-3. After overnight incubation, the bacteria were removed by centrifugation and filtration, and the supematant was plated en ,4. hydrophila AH-3 by using the double agar layered method of Adams [12]. Plaques which formed on the plates were stabbed with a needle and eluted with a small volume of phage buffer. Each phage suspension was serially propagated twice on the same strain.
3.3. General phage ~eLAn;q,es and production of phage lysates The methods of Adams [12] were use,d. The incubation temperature was initially 200C, and the plates were incubated 24 h. The bacteriophage host range was assayed by spot test. One-step growth experiments were performed using A. hydrophila AH-3 as host at a multiplicity of infection of 1. Solvent inactivation (ether and chloroform) of phages, as well a~ phage particle purification, determination of the buoyant density, determination of the nucleic acid type or the number of polypeptides were carried out as previously described [131.
deoxycholate (DOC)-solubilized OM proteins with or without treatment wit~ 1• , g of proteinase K [14]. 100/tg of purified I,PS. 100 ttg of purified LPS with equal volume of anti-LPS serum (diluted 1/10 in PBS), or 50 /zg of pooled high or lowmolecular weight LPS fractions. Chloroform (2-3 drops) was added and the suspension was mixed for 1 rain. The mixture was centrifuged at 12 bOO × g for !0 rain at 4°C. The supernatants were assayed for phage aclivity directly on A. hydraphila AH-3.
3.5. Cell surface isolation and analyses Cell envelopes and outer membranes were prepared as previously described and analyzed by SDS-PAGE [14]. LPS was purified by the method of Westphal and Jann [15l as modified by Osbom [16]. Subfractionation of purified LPS by column chromatography was performed as we described previously [17]. Fractions were extensively dialyzed against distilled water, first at room temperature and then at 4°C, analyzed by SDS-PAGE and silver stained by the method of Tsai and Fraseh [18]. For chemical analysis, purified LPS was h y d r a a,ysed with 1 M HCI for 2 h at 105oC. Colorimettic a.-~lyses of the deoxy-D-mannooctulosonic acid (KDO) content of LPS was performed by the method of Karkhanis et al. [19]. Monosaccharides were also analysed as their alditol z_eetate derivatives by gas-liquid chromatography (GLC) on a 3% SP-~840 column (Supelco) at 225°C and 180oC. Alditol acetate _-'~rhohvdrate standards were either purchased from Supclco or prc},,t,e,d by us.
3.6. Anti-LPS serum Immune serum was obtained from adult New Zealand white rabbits injected with purified LPS from ,4 hydrot, hi!a AH-3 as previously describe~ I141. 4. RESULTS AND DISCUSSION
3.4. Phage inactivation experiments
4 I. Bacteriophage isolation and characterization
Bacteriophages (103 pfu) were incubated with either 107 bacterial cells. 200 pg of 1% (w/v)
Bacteriophage PM2 was isolated from one of three freshwater samples tested. Phage PM2 gave
241 Table 1 Bacterial strains used Strains of
,4. hydrophila AH-3 AH-35 Ba5 AH-54 AH-I01 AH-199 ALL50 AH-10 ATCC 7¢66 24830 24963 25616 TF7 AH-45 A|I-21
A ,'rd~rtJ AS-28 AH-,198 19975
A. salmt~mclda A 450 A 450-3
A 450-1
Relevant characteristics
Sensitivity to PM2
Aehl
0M,, S-layer isogenic mutant derived from AH-3. rough 034, S-layerisogenic mutant derived from Ba5, rough 034. S-layer isogenic mutant derived from AH-101, rough 022. S-layerrough, S-layernot serotyped. S-layer not serotyped, S-layernot serotyped. S.-laye,not ~roG',qped,S-layer 011, S-layer + isogenic mutant derived from TF7:011, S-layer isogenic mutant derived from TF7: rough, S-layer-
+
+
IS]
+
+
thi~ work 19]
+
thi~,v.t)rk [81
Sollrc~
-
, + + + * I + + +
-
+ + 4+ --
-
-
-
this ~ork our lab our lab ATCC clinical ~.iiail.al clinical 191 our
lab
our lab
0l 1. S-layer + isogenic mutant derived from AS-28; 01 l, S-layer rough, S-layer-
-
-
+ +
-
our lab clinical
smooth, A-layer + isogenic mutant derived from A 450; smooth. A-layer isogenic mutant derived from A 450: rough. A-layer -
+
+
1241
+
+
1241
+
+
i241
161
! . c,mshive: -,resistant. Note: All the Aeromona~ >,~;.~ ~'-t ~rotvped do not belong to serolypes 011 or 0.~4.
clear plaques, b o t h at 2 0 ° C a n d 3 7 0 C , w i t h a r a n g e o f sizes u p t o 3 m m with d i f f u s e edges. P h a g e P M 2 i n c o r p o r a t e d 3 H - t h y m i d i n e i n t o its nucleic acid, w h i c h is therefore D N A . F u r t h e r more, u s i n g Bradley's m e t h o d [20] we c o n f i r m e d t h a t p h a g e P M 2 h a s d o u b l e s : r a n d e d D N A . Also, certain restriction enzymes ( E c o R l , S a i l , H i n d l l l a n d B a m H l ) were able : o cleave P M 2 bacteriop h a g e D N A . B a c t e r i o p h a g e P M 2 p r o d u c e d 15 p o l y p e p t i d e s , there b e i n g three m a j o r p r o t e i n s o f
56.0. 42.0 a n d 18.> K ~ , . N~itFcr c h l o r o f o r m nor ether caused any loss o f F'+J2 inlecfi+~:7 Also, b a c t e r i o p h a g e P M 2 showed a b u o y a n t density o f 1.51 g / c m ~, a latent p e r i o d o f 30 rain, a rise p e r i o d o f 20 rain a n d a burst size o f 290 pfu. Bacteri¢ h~ge P M 2 s h o w s a polyhedric head with a collar, • .ntractile tail and a base plate with at least sL': spikes (Fig. 1 ). T h i s b a c t e r i o p h a g e seems very s i m i l a r t o the well characterized T-even E. coli bacteriophages. Bacteriophage P M 2 has a
242 Thus, PM2 is a bacteriophage that can be classifted in the Myoviridae family by Matthews [21] and in group A2 of A c k e r m a a n [22] according to their morphological physico-chemical and biological characteristics.
4.2. Bacteriophage surface zeceptor
Fig. l. Electron micrographyof purified bacteriophage PM2 negatively stained with 17o phosphotungstic acid (bar = 100 nm).
broad host range in that it is able to produce plaques on different Aeromonas strains. For example PM2 is able to produce plaques on different serotypes of A. hydJophila strains, on A. sobria and on A. salmonicida strains (Table 1). Vibrio anguillarum (serotypes 01 and 02) strains and all Enterobacteriaceae tested were resistant to this bacteriophage.
Mutants resistant to bacteriophage PM2 occuffed at a frequency of about 3 x 1 0 ~ and fell into a s i n ~ e class based on their phage sensitivities and LPS profiles. All mutants (20 tested for A. hydr~phila strains AH-3 and Ba5) lacked the O-antigen polysaccharide chains, showed a higher electrophoresis migration rate of their LPS-core oligosaccharides and they were resistant to bacteriophage Ach-1. Phage PM2 adsorbed readily to A. hydrophila AH-3 or Ba5 but not to the PM2-resistant mutants AH-53 and AH-54, respectively (Table 2) suggesting that phage resistance was due to an altered phage receptor. IX)C-solubilized O M fro~.a A. hydrophila AH-3 or Ba5 (wildqypes) and from PM2-resistant mutants showed similar phage adsorption, as did the corresponding whole cells (Table 2). Proteinase K digestion did not alter the ability of DOC-solubilized O M from the wild type strains to inactivate bacteriophage PM2 (Table 2). Furthermore, if we treated the whole cells of the wild t ) p e strains (AH-3 or Ba5) with s~ecific anti-LPS serum for 1 h at 3 7 ° C and remo,,ed the serum by centrifugation, the treated cells were no longer able to inactivate bacteriophage PM2.
Table 2 Inactivation of bac|criophage PM2 by wholecells and OM components
.4. hydrophila strains
%bacteriophageinactivationby Whole OM " OM with cells proteinase K treatment b
LPS c
HMW-LPS u
LMW-LPS a
AH-3 AH-53 Ba5 AH-54
98.3 < 1.0 97.8 < 1.0
77.2 < 1.0 78.3 < 1.0
< 2.0 NT < 2.0 NT
75.8 NT 76.9 NT
97.6 < 1.0 97.2 < 1.0
%.8 NT • 96.8 NT
* OM solubilized with 19~ DO(7 and 2 mM EDTA [14]. h Treatment with proteinase K (10/+g/rid) for 2 h at 45°C. ¢ Purified LPS (100 p.g)[15,16]. d Pooled high-molecularweight LPS fractions and pooled low-molecularweight LPS fracP.ons(50 pg) as it is shown in Fig. 2. e NT, not tested.
243
!'
A
B
123456
I
23
Fig. 2. SDS-PAGE of LPS from ,4 hydrophda strains. (A) Lanes: I, strain AH-3: 2, strain AH-53: 3, strain BaS: 4. strain AH-54: 5. strain AH-IOI: and 6, strain AH-199 (B) Lanes: ]. LPS from strain AH-3: 2. Iowmolecular weight LPS from strain AH-3 subfractionated according to materials and methods: and 3. high-molecular weight LPS from strain AH-3.
Purified LPS f r o m .4. hydrophila A H - 3 or Ba5 was able to inactivate bacteriophage PE'2, but purified LPS from A H - 5 3 or A H - 5 4 (spontaneous PM2-resistant mutants) was not. The degree of p h a g e inactivation was directly related to the LPS concentration in the incubation mixture (1.5 fig of LPS was required to reduce the p h a g e titer in 50%). LPS fractions c o n t a i n i n g low-molecular weight A H - 3 LPS (enriched on LPS-core oligosaccharide~XFig. 2) were able to inactivate bacteriop h a g e PM2, while LPS fractions from the wild type strains containing high-molecular ,-'eight LPS (enriched o n O-antigen chains) (Fig. 2) were not inhibitory (phage inactivation in all ca:,=.s < 1%).
We examined the protein composition of the OM~ ~f ,I. h)J, uyhila stratus A H - 3 a n d BaS. a n d their respective PM2-re~istant mutants. N o main difference were f o u n d in the O M protein profiles ( d a t a not shown). W e also examined purified LPS from the wild type strains a n d the phage resistant m u t a n t s by the method of Tsai a n d Frasch [18]. There was a n a p p a r e n t complete loss of the H M W - L P S (O-antigen) a n d a change on the relative electrophoretic mobility of the L M W - L P S (LPS-c.ore) on the purified LPS from the PM2-resistant m u t a n t s (Fig. 2), confirming the inactivation d a t a of Table 2. Chemical analyses of purified LPS from A. hydrophila A H - 3 a n d BaS, a n d their respective phage-resistant m u t a n t s is shown on Table 3. A reduction of glucose a n d a complete loss of hexosamines in the purified LPSs of the m u t a n t strains was observed, which c a n be correlated with the changes observed o n S D S - P A G E . This reduction in glucose m a y explain the alteration in the electrophoretic moiety of the L M W - L P S on the mutants, because these strains belong to type 2 a c c o r d i n g to Shaw a n d H o d d e r [23). a n d in this type glucose is a c o m p o n e n t of the LPS core. Also. it seems clear that hexosamines are c o m p o n e n t s of the O-antigen LPS in these strains because t h ~ were lost on the LPSs of the m u t a n t strains lacking H M W - L P S . T h u s it is a p p a r e n t that the I.~w-molecu!C.~ weight LPS (LPS core oligosa,~,:harides) alone is the receptor for bacteriophage P~'2. a n d spontaneous phage-resistant m u t a n t s unable to bind this p h a g e iack the O-antigen p o l y - s a c c h a n d e chains. As this bacteriophage ~howed a b r o a d host range
Table 3 Chemical compo.~iti~;,of purified LPS from A. hvdrophda strains LPS from strain
Amount ( #tool/rag of LPS) of: KDO a L-hepD-heptosa b rosa h
PenlOSe b
Gluco~ h
Galaclose b
Rhamno~ ~
Hexo~.,~mlne~ ~
AH-3 AH-53 Ba5 AH-54
0.018 0.051 0.019 0.050
0 0 0 0
1.57 132 1.55 1.29
0 0 0 0
0 0 0 0
041 0 043 0
A. [(vdrophda
0.31 1.25 0.31 1.26
a Assayed by colorimetric method [19]. b Assayed by ggs-liqaid chromatography.
0 0 0 0
244
o n Aeromonas spp., the isolation o f P M 2 - r e s i s t a n t m u t a n t s f r o m d i f f e r e n t Aeromonas s t r a i n s is a useful tool for o b t a i n i n g isogenic L P S m u t a n t s l a c k i n g a n O - a n t i g e n . T h e availability o f such isogenic m u t a n t s will allow u s to s t u d y the c o n t r i b u t i o n o f the O - a n t i g e n to the virulence o f .4 eromonas.
ACKNOWLEDGEMENTS W e t h a n k H . W . A c k e r m a n n for b a c t e r i o p h a g e • ' .A. a. k. . . . 1 ~o.,~,, J~ a Jtxipient of a student grant from C.I.R.I.T. (Generalitat de Catalunya).
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[8] Merino. S. and Tom,is. J.M. (1988) Microbiologla 4. 181-184. [9[ Minal. KR.. Lalonde, G.. LeBlanc. D.. Olivicr. G. and Lallier, R. (1980) Can. J. MicrobioL 26, 1501-1503. [10] Ackemmnn. H.W.. Dauguet. C.. Paterson, W.D.. Popoff, M,. RonL M.A. and Vien, J.F. (1985) Ann. Inst. Pasteur/ Microbiol. 136E, 175-199. Ill] Clowes, R.C. and Hayes, W. (1968) Experiments in Microbial genetics. BlackwelL Oxford. [12] Adams. M.H. (1959) Bacteriophages. lmerscience. New York. [13] Regu~, M.+ Tom~. J.M., Par6s+ R- and Jofre. J. (1981) Carr. Mierobiol. 5, 152-156. [14] Tom~, J.M. and Jofre, J. (1985) J. Bacteriol. 162, 1276- t +~'/% [15] WestphaL O. and Jann, K. (1965) Methods Carbohydr. Chem. 5. 83-91. [16] Osborn. M,J. (1966) Methods Enzyrnol. 8. 161-164. [17] Cimana. B and Tom~s. J.M. (1987) Infect. Immun. 55. 2741-2746. [181 Tsai. C.M..rod Frasch+ C,E. (i'~82) Anal. Biochem. 119, 115-119. []9] Karkb~;: s. Y.D.. Zehner. J.Y., Jackson. J.J., and Carlo, DJ. (1978) Anal. ,-~,ochem. 85, 595-601. [20] Bradley, D.E. (1966) J. Gan. Microbiol. 44, 383-391. [21[ MaUhews, R E.F. (1980) Cl&ssification et nomenclature des virus Troisi~me rapport du cc.~t~ international de taxononfie des virus. Paris, M ~ . [22] Ackermann. H.W. (1973) Handbook of Microbiology. CRC Press, Boca Raton. FL. [23] Shaw. D . H and Hodder. H.J. (1978) Can. J. Microbiol. 24, g64-868. 124] Munn. C.B., ]shiguro, E.E., Kay. W.W. and Trust. TJ. (1982) Infect. Immun. 36, 1069-107~.
