Salmonella bacteriophage glycanases: endorhamnosidases of Salmonella typhimurium bacteriophages.

0022-538X/79/11-0583/10$02.00/0

Salmonella Bacteriophage

Glycanases: Endorhamnosidases

of

Salmonella typhimurium Bacteriophages

STEFAN B.SVENSON,1* JORGEN LONNGREN,2 NILSCARLIN,' AND ALFA. LINDBERG'

Departmentof Bacteriology,NationalBacteriological Laboratory,S-10521Stockholm,'andDepartment of

Organic

Chemiistry,

Arrhenius

Laboratory,

University of Stockholm,

S-106

91

Stockholm,2

Sweden

Received forpublication15 May 1979

Twelve bacteriophages lysing only smooth Salmonella typhimurium strains wereshowntohave similar morphology-anicosahedric head to which a short,

noncontractile tail carrying sixspikes was attached. Allphages degraded their

lipopolysaccharide (LPS)receptors asshownby their ability to cleave off

['4C]-galactosyl-containingoligosaccharidesfrom S. typhimurium cells labeled in their

LPS. The oligosaccharidesinhibited the a-D-galactosyl-specific Bandeiraea

sim-plicifolia

lectinagglutination of humantype B erythrocytes, indicating thatall12

phageglycanaseswereof endorhamnosidase specificity, i.e.,hydrolyzedthe a-L-rhamnopyranosyl-(1 -- 3)-D-galactopyranosyllinkage in the S. typhimurium

0-polysaccharide chain.Twoof thephages,28Band 36, were studied in more detail.

Whereas the phage 28B

glycanase

hydrolyzed the S. typhimurium LPS into

dodeca- and octasaccharides, the phage 36 glycanase in addition cleaved off

tetrasaccharides. Both phageenzymeshydrolyzedthe0-polysaccharidechains of

LPS fromSalmonella belongingtoserogroups A, B, andDl, which arebuiltup

of tetrasaccharide-repeating units identical except for the nature of the

3,6-dideoxyhexopyranosylgroup(R). R

al

- 2)-a-D-Manp-(1-- 4)-a-L-Rhap-(1-+ 3)-a-D-Galp-(l

The phage 28Band36endorhamnosidases hydrolyzed also an LPS from which

the3,6-dideoxyhexosyl substituents had previouslybeen hydrolyzed off. However,

neither of theenzymes wasactiveonLPSpreparations in which the C2-C3 bond

of theL-rhamnopyranosyl ring had been opened by periodateoxidation.

Glucosyl-ationat0-6 of the D-galactopyranosyl residues in the S. typhimurium LPS was

foundtobeincompatible with hydrolysisby both enzymes. However, in an LPS

glucosylated at 0-4 of the D-galactopyranosyl residues, the adjacent

a-L-rham-nopyranosyllinkageswerefoundtobepreferentiallycleaved.

Receptors forseveral

bacteriophages specific

for smooth

gram-negative

bacteria have been shown to be located in the lipopolysaccharide (LPS) moiety of the bacterialoutermembrane (12). More

recently,

it has been revealed that

suchphage

adsorption

oftenis concomitantwith

an

enzymatic

hydrolysis

ofthe

polysaccharide

receptor.

Thus, adsorption

of

phages

P22 and

E'5totheirLPS receptors (seeFig. 1forgeneral

structureofSalmonellaLPS)ofsusceptible

Sal-monella species is accompanied by hydrolytic cleavage of a-L-rhamnosyl linkages within the LPS (7, 11). Likewise, one Escherichia coli

phage

Qi8

cleavesa-D-mannopyranosyl linkages

of the E. coli0-8LPS, and theShigella-phage

Sf6 cleaves the Shigella flexneriLPSatthe

a-L-rhamnopyranosyl linkages(13, 17).

The phage

glycanase

activities have so far been showntobe

exclusively

associatedwith the

phage tail. In some instances

(P22

and coli-phagesf18 and

29),

the

phage

enzymes have been

purified

andidentifiedastail structural

proteins

(2, 7, 17).

Theelucidation of

properties

and

specificities

of

phage

glycanases

is of interest for studiesof

phage-receptor interactions and host

specifici-ties.Furthermore,phage

glycanases

may alsobe

important

as tools for

specific

degradation

of

polysaccharides.

In a search for

phages

possessing

glycanase

activity, 12

0-antigen-specific

S.

typhimurium

phageswerefound tohaveLPSreceptor

cleav-ingproperties.In thepresent paperwereporton

the substrate

specificities

oftwoof these

phage

583

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enzymes (no. 28B and 36) and the isolation of phage 36subviralstructurescarrying glycanase activity.

MATERIALS AND METHODS

Bacterial strains. S. typhimurium strainsSH4305

(0-antigens 4, 5, and 122) and SH4809 (0-antigens4, 5, and 12), S. enteritidis SH1262 (his- thr-thy-, 0-antigens9and 12)wereobtained from P. H. Miikela,

Central Public Health Laboratory, Helsinki, Finland. S.paratyphiAvar.durazzo(0-antigens2and12),S.

typhimurium LT2,and S.flexnerivar.Ywerefrom the collectionattheDepartmentofBacteriology, Na-tional Bacteriological Laboratory, Stockholn, Swe-den. StrainS. typhimurium SL3622 and the uridine

diphosphate-galactose epimerase-defective strain LT2-M1werefrom thecollection of B. A. D.Stocker,

Departmentof Medical Microbiology, Stanford Uni-versity,Stanford, Calif. S.typhimurium strains 1, 2, 3, 4, 5a, 6a,6b, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22,and23a werethephagetypingreference strains used at the Department of Bacteriology, National

Bacteriological Laboratory, Stockholm,Sweden.

Bacteriophages. The clearplaquemutant P22c2 was obtained from B. A. D. Stocker. Salmonella smoothspecificphages 2, 4, 8, 28B, 30, 31, 32, 33, 34, 36, 37, and 39 were those used for S. typhimurium phagetypingattheDepartment of Bacteriology, Na-tionalBacteriological Laboratory, Stockholm,Sweden

(15).

Preparation of bacteriophage stocks. All phages were grown in submerged culture on their corresponding S. typhimurium host strain (Table 1). Log-phase cells, approximately5x 108 cells per ml, grownat37°C in Marvin medium (16),wereinfected at amultiplicity of infection of1to5.The individual cultureswereheavilyaeratedfor5 to 6horuntillysis wasevident. Completelysisofcellswasimposed by theaddition of chloroform.Cell debriswassedimented bycentrifugationat2,500xgfor20min.Some of the phage stocks were further purified by polyethylene glycol (PEG) precipitation. PEG (average molecular weight, 6,000) and sodium chloridewereadded to the clear supernatantto givefinal concentrations of 8%

(wt/vol) and0.5M,respectively (23). After sedimen-tationfor 24 h at 4°C, aggregated phages were col-lectedbycentrifugationat4,000xgfor20minat4°C.

The phage pelletswere suspendedin M-9 base me-dium (1) toapproximately 1/100of the original vol-ume,and thePEGwasprecipitated by repeated chlo-roform additions and removedbylow-speed centrifu-gations. Theresulting phage stocks had titers of 5 x

1010to 5 x 1013plaque-forming units (PFU) per ml. Thephages28Band 36wereinadditionpurified by ultracentrifugation in CsCl gradients (Spinco SW 40 rotor at160,000xg for16h). Gradientswere fraction-ated from the bottom in about

250-Al

portions, and the densities of the phage bands were determined (for 28B,8=1.48; for 36, 8=1.49).The phage-containing fractions werefinally dialyzed against M-9base me-dium and titrated on their respective host strains (phage 28B,4.7x 10'3PFUml';phage 36,2.2 x1013

PFU*ml-').

Isolation ofphage36endoglycanase.Aheavily

aerated,5-literculture oflogarithmicallygrowingcells

of S. typhimurium strain 22 was infected at a multi-plicityofinfection of1.5.After5hof propagation at 37°C, the culture waslysed by the addition of a few

milliliters of chloroform and 0.5% (wt/vol) Sarkosyl. Cell debriswassedimentedat2,500xgfor 20 minat 4°C. Ammonium sulfatewasaddedto50%saturation of the clearsupernatant, andprecipitationwasallowed for24hat4°C.After sedimentation at 2,000xg,the precipitate was suspended in 500 ml of phosphate-buffered saline(PBS)anddialyzedagainst PBS over-night. The precipitation with 50% saturated ammo-niumsulfatewasrepeatedtwice. Afterfinaldissolution anddialysis againstPBS,thematerialwassubjected

to high-speed centrifugation to sediment remaining phageparticles.The enzymeactivityof the

superna-tantpreparationwasdeterminedby itsability to re-lease [14C]galactose-containing material from forma-linized S. typhimurium LT2-M1 cellslabeled in the LPS. Glycanase-active preparationswerealso exam-ined by electronmicroscopy.

PreparationofLPS. Bacteriaweregrownin sub-merged culture, and LPSwasextractedbythe

phenol-watermethod fromformaldehyde-killedbacteria(22).

Some of the LPSpreparationsweremademorewater soluble bytreatmentwith0.15Msodiumhydroxide

for2hat1000C.Thistreatmenthydrolyzes phosphate

bonds and fatty acid ester linkages in the lipid A moiety.Preparationofabequose-deficient

polysaccha-ridewasperformed by heatingLPS fromS.

typhimu-riumSH4809 in50%acetic acidat90°Cfor8h.This treatment hydrolyzed the

2-keto-3-deoxy-D-manno-octulusonic acid linkages in thecoreregion of the LPS as well as all a-1,3-abequosyl linkages. Sugar and methylation analyses of the dialyzed preparation

showed the presence of theexpected methylethersof D-mannose, L-rhamnose, and D-galactose. Sodium

metaperiodate-sodium borohydridetreatmentof the S.typhimurium LPSwasperformedasdescribed(8).

LPS fromKlebsiella 012 and thecapsular

polysac-charidefrom KlebsiellaK47were thesame asused earlier(3,6).

Screening for phage glycanase activities. Screening for phage glycanase activities was per-formedbythemethod of Erikssonetal.(7).Inshort,

mid-log-phase cellsof the uridine diphosphate-galac-tose-4-epimeraseless mutant, S. typhimurium

LT2-Ml,werelabeled with

D-galactose-1-14C

(0.5,uCi/ml,

2mg/ml) in their LPS for3h. To measureglycanase

activities,the14C-labeledcells,twice washed inPBS, weremixed with dilutions of the phagetobe tested. After60min of incubationat 37°C, cellswere sedi-mented at 2,000 x g for 20 min in the cold, and portions of thesupernatantwerewithdrawn and

as-sayed for14Cactivity.

Screeningforendorhamnosidaseactivities. To testwhetheraspecific phage glycanase possessed the abilitytocleavethea-L-rhamnosyllinkages of the S.

typhimurium SH4809 LPS,the dialyzablecrude

oli-gosaccharide preparation(seebelow)wastestedforits

inhibitoryactivityonBandeiraeasimplicifolialectin

agglutination of human typeB erythrocytes. Inhibi-tion 50% or more of that obtained with melibiose

(calculatedonamolarbasisassuminga mean molec-ular weightof the crude oligosaccharide of -1,200) wasconsideredaspositive.

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585

Electron microscopy. Thepreparation tobe ex-aminedwasstainedwithuranylacetatebythe follow-ingprocedure:adropletof thepreparationwasplaced

on a grid (200-mesh copper with carbon-shadowed Parlodion film), andexcessfluidwassucked offwith filterpaper.Adrop offreshlyprepared 1% (wt/vol) uranylacetate wasthenappliedto the grid. Aftera

few minutes,excessfluidwasagainremoved by touch-ing the gridtothecornerofanadsorbent filterpaper.

Thepreparationswereexamined inaPhilips EM-200 electronmicroscope. The estimation ofphage dimen-sionswasmadeby comparisonwithacalibrationgrid. Methylation analyses.Methylation analyseswere performedasdescribed earlier(14). The oligosaccha-rideswerereduced with sodium borohydride,andafter methylation and subsequent work-up, the materials

werehydrolyzed. The resulting monosaccharideswere

transformedintoalditolacetatesandanalyzed by gas-liquidchromatography (GLC) andmassspectrometry. Forgas-liquid chromatography, a Perkin-Elmer 990 instrumentfitted witha3%OV-225colunmwasused. Gas-liquid chromatography-mass spectrometry was performedwithaVarianMAT311instrument.

Test for phage glycanase hydrolysis of

capsu-larorLPS. Thephagetobeassayedwasmixedata

ratio of 109to1010PFU/mg ofpolysaccharide in5mM ammoniumcarbonatebuffer, pH7.0,and incubatedat

37°Cwithinadialysis bag immersedin 10times the volume ofthesame buffer.After48hofincubation,

the surrounding dialysis fluid was concentrated to

drynessandheatedto50°C undervacuumtoremove

theremaining ammonium carbonate. Theamountof hexose inthe reaction mixture and the surrounding

dialysis fluid wasdetermined by the phenol-sulfuric acidmethod (5).

Miscellaneous methods. Gel chromatographyof oligosaccharides wasperformedon acolumn (170 by

2.5cm) ofBio-GelP-2 (200to400mesh)eluted with water,usingtrichlorobutanol (0.05%)asthe

bacterio-cidalagent.SeparationsweremonitoredbyaWaters

403refractometer,and the fractionswereassayed for

carbohydrate content by the phenol-sulfuric acid

method. For nuclearmagnetic resonancerecordings,

aJeol FX-100 instrument operatedin the PFT mode

wasused. Thespectrawererecorded for solutionsin

D20at850C, using extemaltetramethylsilaneas stan-dard.

RESULTS

Screening for phage glycanase activity. Twelve phageswereselected fromasetused for phage typing ofclinical isolates of S. typhimu-rium (15). The lytic spectraof these phageson the reference set of S. typhimurium strains is given in Table 1. The various phages were in-cubated withformaldehyde-treated S. typhimu-rium LT2-M1 cellswhich had been labeled with

['4C]galactose in theirLPS (7). After centrifu-gation, '4C-labeled material was found in all supernatants,indicating that all 12phages pos-sessed glycanase activities (Table 2). Thesame batch of ['4C]galactose-labeled LT2-M1 cells was also subjected to hydrolysis by the P22 endorhamnosidase byincubating the cellswith TABLE 1. Lyticspectra of S.typhimuriumphagesa

Bacteria 2 4 8 28B 30 31 32 33 34 36 37 39

1 + + + + + + + + +b +

2 + + + + + + + + +

3 + + i + + + + +

4 + + +b + + + + +

5a + +b + + + + + +

6a + + + + + + + +

6b +b + + + + + +

7 + + + + + +

8 + + + + + + +

9 + + + + + +

10 + + + + + + +

11 + +b+

12 + + +b + +

13 +b

14 +b + +

15 + + +

16 + +b

18 +b

19 + +

20 + + + +

21 + +

22 + +b +

23a + + + + + +

aPhagesinvestigated on the standard set of S. typhimurium strains used for phage typing of clinical isolates.

+,Sensitivity to phage indicated. bStrain was used forpropagation. VOL. 32,1979

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TABLE 2. Glycanase activitiesof S.typhimurium phages assayedby

["4C]galactose

release'

Phage Titer %Releaseb

2 5 x107 10 (4)

4 1.5 x

1010

49 (33)

8 3.2x1012 50 (48)

28B 3.1 x1012 55 (48)

30 6.7 x 1010 43 (48)

31 1 x109 46 (12)

32 1.6 x1010 45 (33)

33 1.4x 1012 64 (48)

34 1.3 x1010 65 (32)

36 1x 109 68 (12)

37 1x 1010 39 (30)

39 1x 109 54 (12)

a14C-labeled material was released from S. typhi-murium LT2-M1 cells.To 100-,ulportions of washed S. typhimurium LT2-M1 cells previously labeled in their LPS with [14C]galactose were added

900-pl

amounts ofthevarious phagepreparations. After in-cubation for 1 h at 37°C, cellswere sedimented by centrifugation, 500-,ul portions of the supernatants

were withdrawn, and the released 14C activity was

determined.The same batch of

[14C]galactose-labeled

S. typhimurium LT-2 M-1wasalso assayed against a

PEG-purified P22c2 phage stock. Maximal release (48% oftotal) was obtained at 5x 1010PFU per assay.

bValues in parentheses were obtained by using equal numbers of PFU of a purified phage P22c2 preparation.

differentamounts ofa

PEG-purified phage

P22

stock and the percentage ofrelease

compared

with those obtained

by

the 12

phages

under

investigation

(Table 2).In allinstances

(except

phage 30), the crude

phage

lysates

showed

higher release activities per PFU than did the

purified

phage

P22stock. To confirm that the

release of 14C-labeled material from the S.

ty-phimurium LT2-M1 cellswasdue to

phage-as-sociatedglycanase

activities,

crude

lysates

of the

respective propagation strains were tested for

hydrolytic properties. About 5 x 1011 cells of eachstrainwere

lysed by

sonication

(three

times 10 s at25W ina Labsonic 1510

sonicator),

and

the crude bacterial

lysates

wereincubatedwith

[14C]galactose-labeled

S.

typhimurium

LT2-M1

cells. Noneofthe bacterial

lysates

releasedany

14C-labeled

material. In a control

experiment,

the variousphage lysates were, after sonication asdescribed

above,

foundtobe

equally

activeas

before sonication.

The

PEG-purified phage

28B and 36 stocks

werefurtherpurified

by

density gradient

ultra-centrifugation.Inbothcases,the

phage-contain-ingfractions

displayed

the

highest

release activ-ities of

[14C]galactose-labeled

material from S.

typhimurium LT2-M1 cells. The 50% maximal release (45% of total label was

maximally

re-leased) values were, for

phage

28B and36, 8.8

J. VIROL.

x

109

and 1.1 x 108

PFU, respectively.

These

data indicated that the glycanase activities of

phages

28B and 36 were associated with the

phage

particles.

Screening

for endorhamnosidase

speci-ficities. In another series ofexperiments, the various

phages

were incubated with

partially

delipidated

LPS from S.typhimurium SH 4809

within

dialysis

sacksimmersedinbuffer.All 12

phages

degradedtheLPStodialyzable

oligosac-charides asevidenced byrecovery of 30to50%

of the total carbohydrate in the surrounding

dialysis

fluid

(data

not

shown).

Glycanases

cleaving the a-L-rhamnosyl

link-agesin the S.

typhimurium

SH4809 LPS(Fig. 1, Table

4)

should

give

rise to

oligosaccharides

possessingaterminal, nonreducing

ca-D-galacto-syl

group. The agglutination ofhumantype B

erythrocyteswith the a-D-galactosyl-specificB.

simplicifolia

lectin was inhibited by the crude

oligosaccharide

preparations(obtained fromthe

dialysis

experiments described above),thus

sug-gesting

that all 12phages exhibited

endorham-nosidase activity.

Electron microscopic examination of phages.InFig. 2,themorphology typical forall

12

phages

isshown. Theyall havea headwith

hexagonal symmetry about50 nmindiameterto

which a short, noncontractile tail carrying six

spikesisattached. Thisappearanceistypicalfor type C phages, based on the classification of

Bradley (4).

Two ofthe phages, 28B and 36, were chosen

foramoredetailed study. Phage28B waschosen

for itsnarrowlyticspectrum (Table1) and ease ofpropagation. Phage36,however,had abroad lyticspectrum(Table 1) alsolysingtheS. typhi-murium SL3622 (Fig. 1 and Table 4).

Further-more,

phage

36showeda

high

'4C

release

activ-R D-Glcp

a

a3i

4/6

[a-D-Ma1nP-(l1- 4)-a-L-RhaP-(l1- 3)-D-Gaip]n

-+2)-R

1J1

al 3

a-D-Manp-(1

4)-a-L-Rhap-(l

3)-a-D-Galp-(1 -*CORE LipA

FIG. 1. Generalstructureof LPS fromSalmonella serogroups A, B, andDl. Rindicates abequose (3,6-dideoxy-D-xylo-hexose) in Salmonella B, ortyvelose (3,6-dideoxy-D-arabino-hexose)andparatose (3,6-di-deoxy-D-ribo-hexose) in Salmonella Dl andA, re-spectively. Broken arrow indicates substitution oc-curring onlyin some strains. n,Numberof repeating units in0-chain,varyingfrom4to30.

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.--; i-'! - * < =, *S t In%

*

4;'r

''1'v'" '.

.''

'" ' -,;,.,

--.''

'"

FIG. 2. Electron micrographs of(A)phage 28B; (B)phage 36; and (C) isolated baseplate-like

struc-tures of phage 36. Each line shown represents 100

nm.

ity as compared with the phage P22 standard (Table 2); i.e., it could be overproducing the glycanase.

Isolation ofphage 36 subviral elements possessing glycanase activity. Subviral

structuresexhibitingglycanase activitywere

iso-lated by ammonium sulfate precipitation of a

crude lysateofphage36. More than 60%ofthe total glycanase activity was recovered in the precipitate fraction. To remove contaminating intact phages and ghosts, the dialyzed

precipi-tate fraction was subjected to high-speed

cen-trifugations. The final preparation contained <105PFU ofproteinper mg.

Electron microscopic examination of the su-pernatantfraction revealed thepresence oflarge numbers ofdoughnut-shaped structures (15 to 20 nm in diameter), each with a central hole (Fig. 2c). Thesestructuresresemblewhole phage tails or baseplates. No ghosts or intact phage particles were seen. The glycanase activity of the supernatant fractionwas about 1,000 times higher (per milligram ofprotein) than that of

PEG-purified phage36particles.

Phage 36- and 28B-mediated hydrolysis of S. typhimurium SH4809 LPS. Purified phage36and28B wereincubated withpartially

delipidated LPS from S. typhimurium SH4809. The crude dialyzable oligosaccharide

prepara-tions obtainedweresubjectedtogel chromatog-raphy (Fig. 3a andb). The phage 28B-cleaved material showed peaks corresponding to a do-deca- (A) and an octasaccharide (B),

respec-tively. Phage 36-cleavedmaterialshowed

essen-tially the same elutionpattern, but in addition to the dodeca-

(A')

and octasaccharide

(B1),

a

tetrasaccharide

(C')

wasalso isolated.

Toconfirm the estimated sizes ofthedifferent oligosaccharides, the individual peaks were

pooled,

andafteranadditional

chromatography

on the same column the saccharides were

re-duced and subjected to methylation analysis (Table 3). The only difference compared with

the

analyses

oforiginal LPSwasthepresence of

2,3,4,6-tetra-O-methyl-D-galactose,

demonstrat-ing that the

a-L-rhamnosyl

linkages

had been

selectively

cleaved in the S.

typhimurium

SH4809 LPS.Fromtheratio of tetra-O-methyl-D-galactosetotri-O-methyl-D-galactose,the size

of theoligosaccharidescanbeestimated.Thus, fractionsAandBfrom thephage28B

hydroly-satecorrespondtododeca-and

octasaccharides,

respectively.

Similarly,

A', B',

and

C'

fromthe

phage-36

hydrolysate

correspond

to

dodeca-,

octa-, and tetrasaccharides, respectively. All analyses showed the presence of significant amountsof

3,4,6-tri-O-methyl-D-mannose.

This was most likelydue to acidichydrolysis of the

abequosyllinkage duringthework-up.

Oligosaccharides B and Bl and the

octasac-charide obtained by phage P22 endorhamnosi-dase-mediated hydrolysis of S. typhimurium

SH4809 LPSwere shown tobeidenticalby

'H

nuclear magnetic resonance (8). Signals were

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E z

-4

-I

o*0C

0'

Fraction number

FIG. 3. Gelchromatography ofcrude oligosaccha-ride preparationsobtained after phage 28B (upper panel)andphage36(lower panel) hydrolysis of par-tially delipidatedLPSfromS.typhimuriumSH4809. The crudeoligosaccharideswereappliedtoacolumn (170 by2.5cm) ofBio-GelP-2(200to400mesh)and eluted withwater(8.5 ml/h).Thefractionvolumewas

5ml,and the total hexosecontentwasestimatedby the phenol-sulfuric acid method. For molecular

weightcalibration ofthecolumn,thefollowing

sac-charideswereused:melibiose, stachyose, cellohexose,

S. typhimurium dodecasaccharide, and fluorescein isothiocyanate-labeleddextran ofmolecular weight 2,900.

obtained, inter alia, at 8 4.9 to 5.4 (anomeric protons), 8 2.0 to2.2 (H-3 of abequosylgroup),

and 8 1.2 to1.5 (H-6 of abequosylgroupand L-rhamnosyl residue).

Under the conditionsused, both phageswere foundtoproducemoreoctasaccharide than do-decasaccharide. However, with the phage 36 en-dorhamnosidase, tetrasaccharides were also formed. The approximative molar proportions ofdodeca-, octa-, and tetrasaccharide were for phage 28B, 2:8:0,and forphage 36, 2:6:2.

Substrate specificities of phage 36 and

28B endorhamnosidases.In additiontothe S.

typhimurium (SH4809,serogroup B)LPS,both phages cleaved the S. enteritidis (SH1262,

se-rogroupD1) and S.paratyphi A(serogroup A)

LPS (Table 4). Since Salmonella bacteria be-longingtoserogroupsA, B,and Dlallshare the same trisaccharide backbone in their tetrasac-charide-repeating unit (Fig. 1) and differ only in the type of3,6-dideoxyhexosyl group linked to

[image:6.507.64.256.56.268.2]

0-3 of the D-mannosyl residue, these results show that different 3,6-dideoxyhexosyl groups linkedtothispositionareaccepted by the phage 36and28B endorhamnosidases.

TABLE 3. Methylation analyses ofoligosaccharide fractions obtained by phage-mediatedhydrolysis of

S. typhimurium SH4809 LPS

Methylated Mol%C

Hydrolysis sugar (major Tb

components)a A B A' B' C'

Phage 28 2,4-Abed 0.28 7 5 medi- 2,3-Rha 0.92 24 18 ated 2,3,4,6-Gal 1.19 10 16 3,4,6-Man 1.82 15 19 2,4,6-Gal 2.03 24 19 4,6-Man 2.92 19 22

Phage 36 2,4-Abed 0.28 5 5 7

medi- 2,3-Rha 0.92 23 23 5

ated 2,3,4,6-Gal 1.19 10 15 48 3,4,6-Man 1.82 12 4 18 2,4,6-Gal 2.03 24 19 2

4,6-Man 2.92 26 34 19

a2,4-Abe indicates

2,4-di-O-methyl-abequose;

2,3-Rha indicates2,3-di-0-methyl-L-rhamnose, etc.

bRetention time of the corresponding alditol ace-tates relative to

1,5-di-O-acetyl-2,3,4,6-tetra-0-methyl-D-glucitolon anOV-225 column. cFractionsaccordingtoFig.2.See text.

d

Most

ofthis volatile compound and derivatives

werelost during evaporations.

LPS from S. typhimurium SH4809in which

the

3,6-dideoxyhexosyl

groups had been

re-movedby mild acidic

hydrolysis

wasalso hydro-lyzed by theenzymes,

although

less efficiently. However, a

preparation

of S.

typhimurium

SH4809 LPSinwhich the

L-rhamnosyl

residues had beenmodified

by periodate

oxidation-boro-hydride reduction was found resistant to both

enzymes.

Different glucosylations of the D-galactosyl residues in the S. typhimurium 0-side chain occur. Partial

glucosylation (about 40%)

at 0-6 of the

D-galactosyl

residuesinthe LPSof theS.

typhimurium

P22

lysogen

strain SL3622 lowered

bothenzymeactivities asmeasuredby the deg-radation of LPS intodialyzable oligosaccharides.

Upon

methylation analysis

of these

oligosaccha-rides,

only

the

2,3,4,6-tetra-0-methyl-galactose

etherwasfound.Hence,the absence of

2,3,4-tri-0-methyl-galactose

indicated that none of the

phageenzymes could cleavea-L-rhamnosyl link-agesadjacentto0-6-glucosylated galactosyl

res-idues.

Also, LPS from the S. typhimurium strain SH4305 which is

glucosylated

(about

70%)

at

0-4 of its D-galactosyl residues was subjected to

degradationbythe

phage

enzymes. Portions of theLPSwere mixed withphage 28B and36in

dialysis bags.. After 48 h ofhydrolysis with si-multaneousdialysis,boththedialyzable saccha-rides aswellasthose retained within thedialysis

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590 SVENSON ET AL.

bag weresubjected to methylation analysis.

Ta-ble 5shows the molar percentage of the various

methyl ethers ofD-galactosein these fractions. The presence of 2,3,6-tri-O-methyl-D-galactose

in thedialyzable fractions showed that the phage enzymescleaved linkages adjacent to galactosyl residues glucosylated at 0-4. Furthermore, in both cases thesmallincrease in 2,3,4,6-tetra-0-methyl-D-galactosecompared with that of

2,3,6-tri-0-methyl-D-galactoseshowed that

a-L-rham-nosyllinkages adjacent to 0-4-substituted D-ga-lactosyl residues were preferentially cleaved. The same pattern was also seen in similar

ex-periments in which the enzymes were given

equalamountsof unglucosylatedLPS (S.

typhi-murium SH4809) andLPS glucosylated at 0-4

(S. typhimurium SH4305) as substrates (data notshown).

The high specificities of the present endo-rhamnosidases were also evidenced by their

in-ability to degrade three other polysaccharides

(S. flexneri var.Y,Klebsiella 012, and

Klebsi-ella K47) containing a-L-rhamnosyl residues

(Table4).

DISCUSSION

It has been shown that phages infecting

en-capsulatedorsmoothbacteria likeSalmonella, Klebsiella,E.coli, andShigellahaveglycanase

activities associated with their tail structures

(forareview, see reference 12). These glycanases seem toassist in the penetration of the outer-most layer of carbohydrates surrounding the

bacterial cell,thus allowing the phage to bind to

postulatedsecondary receptors close to the cy-toplasmic membrane from where injection of the phage nucleic acid then occurs. Because

cleav-TABLE 5. Methylation analyses ofsaccharides after phage-mediatedhydrolysisof S.typhimurium

SH4305LPS

Mol % of total

galac-Methyl tose content

Hydrolysis ethersof Tb

Undi-D-galac- alyzable Dialyza-tose' saccha- ble sac-rides charides Phage 28 me- 2,3,4,6-Gal 1.19 13 19

diated 2,4,6-Gal 2.03 53 0

2,3,6-Gal 2.22 0 28

2,6-Gal 3.14 34 53

Phage 36 me- 2,3,4,6-Gal 1.19 20 22

diated 2,4,6-Gal 2.03 43 0

2,3,6-Gal 2.22 0 38

2,6-Gal 3.14 37 40

See footnote a to Table 3.

bRetention time of thecorrespondingalditol acetates rel-ative to1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitolon an OV-225 column.

age of the impeding

carbohydrate

layer

is a

necessity forsuccessful

infection,

the substrate

specificities of these

phage

glycanases

set the

limits to their host range.

Phage

P22 causes

glycosylation at 0-6 of

D-galactosyl

residues. A

strainwhich is

lysogenic

with this

phage

is

re-sistant to the

endorhamnosidase

and

conse-quentlyto

multiple

infection

by

this

phage.

Inthisstudy, 12S.

typhimurium

smooth

spe-cific phages,

originally

isolated

by

Lilleengen

(15) and used in the routine

phage

typing

of

clinical S.

typhimurium

isolatesin

Sweden,

were

investigated.

Thephageswerescreenedfor

glycanase

activ-ity by their

ability

to release

1"C-labeled

LPS

fragments fromwhole S.

typhimurium

LT2-M1 cells (7). All were shown to

degrade

the

LPS

(Table2). When

partially

delipidated

LPS

from

S.

typhimurium

SH4809 wasmixed with crude

lysates ofthe various

phages,

dialyzable

oligo-saccharides

couldbe

isolated,

giving

further

con-firmation to the

hydrolytic

capacity

of the phages.

Theinabilityof crudelysatesof the

propaga-tion strains to cleave off

oligosaccharides

from

14C-labeled

S.

typhimurium

LT2-M1 cells

sug-gested that the

glycanase

activities were of

phage and not of bacterial

origin.

In the in-stanceswhere the phageswere

purified by

den-sity gradient

ultracentrifugation,

the

glycanase

activities werefoundassociatedwith the

phage

particles, indicatingthat the enzymes are

inte-gralpartsof thephage

particles

rather than viral

exitenzymes.

All phages (except phage 30) showed

higher

14C release values (per PFU) when

phage

P22 glycanase activity wasusedas standard

(Table

2). The higher activity could be accounted for

bythe presence of

enzymatically

active butnot

viableghostsor

overproduction

of

enzymatically

active phage

protein

or both. The isolation of subviral

baseplate-like

structures of

phage

36

(Fig. 2c) exhibitinga

1,000-fold higher

glycanase

activity(permilligram of

protein)

than the PEG-purified wholephage 36

particles,

showed that

thehigh release value obtained with the crude lysate of thisphage mostlikely was causedby overproduction of thephageenzyme.

Inhibition of the

a-D-galactosyl-specific

B.

simplicifolialectinagglutinationof humantype

B erythrocytes with the crude

oligosaccharide

preparations obtained after phage

degradation

of S. typhimuriumLPS

suggested

thatthe

gly-canasesof

all

12phageshadendorhamnosidase specificity.

Phages 28B and 36, which wereexamined in moredetail, had

essentially

equal substrate pro-files(Table4). Hydrolysisoccurredirrespective

of thetypeof3,6-dideoxyhexosylsubstituentin

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[image:8.507.66.259.499.640.2]

the polysaccharide. Bothenzymes were shown to preferentially cleave at galactosyl residues

glucosylated

at 0-4

(S. typhimurium SH4305)

(Table5). Thiswas evidentby the preferential

appearance of2,3,6-tri-0-methyl-D-galactosein

the methylation analysis of the oligosaccharide

preparations obtained after degradation of the

S.

typhimurium

SH4305 LPS

(Table

5). Also,

when theS. typhimurium 4305LPS wasmixed

atequal ratio with the unglucosylated S.

typhi-muriumSH4809, thesame methyl ether

(2,3,6-tri-0-methyl-D-galactose)

wasdominating (data

notshown). In contrast, glucosylation at 0-6 of

theD-galactosyl residueswas anobstacle to the enzymes, as demonstrated by the rather low

degradation of the LPSfromtheP22lysogen S.

typhimurium

strainSL3622

(Table 4).

No 2,3,4-tri-0-methyl-D-galactose could be

detected in the

methylation

analysis of the oli-gosaccharides prepared by degradation of this LPS, indicating that

glucosylation

at0-6 of

ga-lactosyl residues makes such

a-L-rhamnosyl

linkages resistantto

hydrolysis.

Abequose-deficient LPS from S.

typhimurium

SH4809 is cleaved

by

both

phage

28Band36. In

thisrespect,these

phages

differ fromphage P22, which does not

hydrolyze

LPS preparations lacking 3,6-dideoxyhexosyl groups (8). Modifi-cationof the

L-rhamnosyl

residues

by

periodate-borohydride treatment

made,

as

expected,

the S.

typhimurium

substrate inerttobothenzymes.

In parallel with

phage

P22,

phage

28B and36

endorhamnosidases were unable to cleave the

a-L-rhamnosyl

linkages occurring

in theS.

flex-neri var. Y and the Klebsiella 012 LPS or in

the

capsular

polysaccharide

of

Klebsiella

K47

(Table 4). Although the

phage

28B endorham-nosidase, like

phage P22,

mainly yields

octasac-charides, phage 36inaddition alsoproduced20

mol %tetrasaccharides.

Although the enzymes of the 28B and 36

phageshavesubstrate

profiles

similartothatof

phage P22 (8), differences are discernible. This

setof

phage

enzymescould be of value for

prep-aration of

0-antigen-specific oligosaccharides

from

Salmonella.

Such

oligosaccharides

have

been used for the elucidation of the size of 0-antigen determinants and also for the

prepara-tion ofartificial Salmonella vaccines

(9, 18-21;

H. J.

Jorbeck,

S.B.

Svenson,

andA. A.

Lindberg,

J.Immunol., inpress).

ACKNOWLEDGMENTS

Thisworkwasinpartsupported by grantsfrom the Swed-ishMedicalResearch Council (no.B77-16X-00656) andthe Swedish Board of TechnicalDevelopment(no. 75-5809).

Weareindebted to BertLarsson, Birgitta Sundberg,and Viveka Eriksson for technical assistance. Ulla Eriksson is gratefullyacknowledgedfor valuablediscussions.

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