JOURNAL OFVIROLOGY,Sept.1974,p.689-699 Copyright0 1974 AmericanSocietyforMicrobiology
Vol. 14, No. 3 Printed inU.S.A.
Characteristics
of
PRD1,
a
Plasmid-Dependent
Broad
Host
Range
DNA
Bacteriophage
RONALD H. OLSEN, JUNE-SANG SIAK, AND ROBERT H. GRAY
The University of Michigan, Department of Microbiology,The MedicalSchool,andDepartment of Environmental and IndustrialHealth, CellularChemistry Laboratory, The School of Public Health, Ann
Arbor,Michigan48104 Received forpublication 27 March 1974
Several distinctive properties of PRD1, an icosahedral plasmid-dependent phage, are described. The drug-resistance plasmid-dependent host range of
PRD1 extendsbeyond the P incompatibilitygroup and includes gram-negative bacteria containing plasmids of incompatibility groups N and W. PRD1 phage will infect pseudomonads and Enterobacteriaceae containing either a P or W incompatibilitygroupplasmid. PRD1 adsorbstothe cell wallofR+ bacteria and thus its infectivity indicates cell wall alterations by these drug-resistance plasmidgroups. PRD1 nucleic acid is duplexDNA withanestimated molecular weight of 24 x 106. The appearance of PRD1 in electron micrographs is suggestive of lipidcontentin additiontoitsbuoyant density of 1.348 in CsCl and its sensitivity tochloroform.The latent period ofPRD1 varies withtheR+ host bacterialstrain used forgrowth of thephage.
We have recently reported the isolation and characteristicsofPRR1,anRNAphage specific for the broad host range Pseudomonas drug-resistance plasmid R1822 (21, 23). Since that time,we havesentourstrains toothersworking with RP1, a plasmid whose origin is the same clinical isolate which servedasthesourceof our R1822 (15). When the percent guanine plus cytosine
(G+C),
molecularweight, and contour length ofR1822 and RP1DNA werecompared, identical results for RP1 and R1822 were ob-tained (M. Richmond, personal communica-tion). Accordingly, we are redesignating R1822 asRP1since it isindistinguishablefromRP1by any ofits known biologicalor physical proper-ties.Bacterial
drug-resistance
factors have been classified on the basis oftheir incompatibility (2, 19), i.e., their inability to coexist stably in thesamehost.This classification scheme previ-ously has beena convenient criterion, basedonbiological
assay, to determine the relationshipof a newplasmidtoknown groups. The efficacy of groupings based on the incompatibility of related plasmids is reinforced by thespecificity ofseveral phages for bacteria possessing plas-midsof a givencompatibilitygroup (13, 14, 20). The recent description of the RP1 plasmid specific phage, PRR1 (21), increases the num-ber of known plasmid compatibility groups having specific phages to include F, I, N, and P group plasmids. When PRR1 was isolated
from Kalamazoo, Mich., sewage, another ap-parently P
group-dependent phage
was ob-tained which wedesignated
PRD1. This phage differs significantly from other plasmid-de-pendentphages previously
described with respect to its receptor sites, morphology, host range, and composition. Some of the salient features of this phage and its purification are presented in this report. (A preliminary report of some of this work was presented at the Annual Meeting of the American Society for Microbiology, 23-28 April 1972, Philadelphia, Pa.)MATERIALS AND METHODS
Bacterial strains and growth medium. Most bacterial strains and growth media routinely used in this study have been described (21, 23). Additional strainsandtheir drug-resistance plasmids used in the presentstudy are described in the tables. PRD1 host range determinations on bacteria containing drug-resistanceplasmids considered representative of vari-ouscompatibility groups were done by using tryptone-glucose-yeast extract medium (TGE) or TGE contain-ing NaCl (8.5g/liter) (TGEN). Indicator bacteria for phage enumeration were grown overnight on TGE agar medium containing an antibiotic whose resist-ancewasspecified by the particular drug-resistance plasmid the bacteria contained (if any). Cells contain-ing RP1 were grown on medium withcarbenicillin at
500
,g/ml.
Theconcentration of other antibiotics usedwas at least four times the minimal inhibitory concen-tration observedforthe R- parent. A turbid suspen-sion was made from overnight growth on solid me-689
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dium and was inoculated into TGE broth medium.
These cultureswereincubated with agitation for2to3 h, or until approximately 108 cells/ml was achieved.
Allcultureswere grownat37C with theexception of Pseudomonas putidaPPO13.12and the psychrophile, P. fluorescens PFO15.4,whichwere grownat30C. All media usedforphagegrowthoradsorption contained
CaCl2at0.015% (wt/vol).
Preliminary studies had shown that Salmonella typhimurium LT2(RP1)wasthebest platinghost for
PRD1, and this strain was used for all estimates of
phagetiterunless otherwise indicated in thetext. Reference phage strains and growth of phage stocks. Several well-studied phages were used as a
point of reference for physical characterization studies
onPRD1. They included lambda and P22c received
from D. Friedman, University of Michigan, and
OX174
received from C. Kulpa, Universityof NotreDame. R- hostsforgrowth andassayofthesephages were Escherichia coli C80 grown on tryptone-yeast
extractagarmedium with maltose(0.1%)for lambda;
S. typhimurium LT2 for P22c; and E. colifor
OX174.
Phage stockswereprepared by harvesting confluentlylysed plates and clarificationofthe homogenized soft
agaroverlays by several4C centrifugationsat8,000x g.Theselysateswerestoredoverchloroformat3C in TGE brothmedium.
Electron microscopy. A bacterial culture growing inTGEN broth medium (approximately108cells/ml)
wasaddedtoaCaCl2-containing brothsuspensionof
phage resultinginamultiplicityofapproximately20
phageperbacterium. Phagewereallowedtoadsorb20 minatambienttemperaturefollowedbyimmergion of the mixture in an ice bath. From this, drops of the
suspensionweredepositedoncarbon-coated Formvar
grids. Excess fluid was removed from this grid with
filter paper and a drop of 1% phosphotungstic acid
(pH 6.8) was placed on thegrid for 1 min. Several dropsof1%aqueousammoniumacetatewereusedto wash excess phototungstic acid from the grid. The
ammonium acetate was removed with filter paper.
The grids were examined in an AEI Corinth 275 electronmicroscope.
Preparation of PRD1 high-titer lysates. P. aeruginosaPAO67(RP1) wasused asthe host
bacte-rium forthe growthofPRD1. AllTris-based buffers contained tris(hydroxymethyl)aminomethane. Other procedures used for PRD1growth, precipitation, and concentration were as reported previously for PRR1 (21),exceptthat the buffer(pH7.0) used for
suspen-sion of the concentrated phagecontained Tris (0.01 M, pH 7.0); NaCl (5%); CaCl2 (0.001 M); and tryptone (Difco) (0.5%) (TNCT). The phage suspen-sion from the ammonium sulfate precipitate was
storedovernightat4C. Thiswasfollowedby
centrifu-gationat 10,000 x gfor 20minat4C in the Sorvall
SS34 rotor (low-speed centrifugation). The
superna-tantwasthendecantedandcentrifugedat100,000xg for2hat4C in theSpincotype30rotor(high-speed centrifugation). The pellets from each tube were
suspended in 5 ml of TNCT and pooled. Twomore
cyclesof low- andhigh-speed centrifugationwerethen used to promote further purification. This phage lysate was further processed by centrifugation
through a 5 to 20% sucrose gradient in the Spinco SW27 rotor at 17,000 rpm for 150 min at 4 C. The sucrosegradientswerepreparedinTNCT buffer.The gradient tube showed a visible band containing phage particles confirmed by plating and which adsorbed stronglyat260 nm, indicating thepresence of nucleic acid. The phage band was collected through the bottom of the tube by a Beckman fraction recovery system,followedby dialysis against2liters ofTNCT buffer at 4C. The phage was then treated as de-scribed previously (22) with RNase and DNase, fol-lowed by another high-speed centrifugation and sus-pension of the phage pellet in TNCT. Table 1 indicates the phage recovery at each step. DNA and RNA contents of the purified phage lysate were determined by the diphenylamine and orcinol methods, respectively. RNA content was negligible and DNA content was 8 x 10-11 ,ug/PFU. When the composition and molecular weight of PRD1 DNA were subsequently estimated, we calculated that PRD1 lysates purified by this method had 50% noninfective DNA containing particles in the total phage popula-tion. All mediaorbuffersused were at pH 7.0 unless otherwise specified.
Phage DNA extraction. PRD1 and lambda phage DNA were extracted essentially as described by Sobieski and Olsen (26). Phage lysates were in SSC (0.15 M NaCl; 0.02 M sodium citrate, pH 7.0). The phage lysate was treated 2 h at 37 C with Pronase (10 mg/ml) before extraction with SSC-saturated phenol. PRD1 DNAwas extracted by using six serial phenol extractions; three wereused for lambda. After exhaus-tivedialysis against SSC at 4 C, PRD1 DNA usually had a 260/280 nm abosrption ratio of 1.4 to 1.5, and lambda DNA had an absorption ratio of 1.8 to 1.9.
RP1 DNA. Tritiated RP1 DNAwaspreparedfrom E. coliJC1553(RP1) by the method of Guerryet al. (8). DNA was labeled with [3H]thymine having a specific activityof15Ci/mmol (Schwarz/Mann).The supercoiled plasmid DNA prepared by the above procedure was nicked during dialysis against SSC overnight at 4C. The majority ofthe plasmid DNA became open circular molecules withasedimentation value of 43S, as previously reported for RP1 by Grinstedetal. (7).
[image:2.493.268.464.531.662.2]PRDI DNA. PRD1 DNA was readioactively la-beled usingP. aeruginosa PAO67(RP1) as the host bacterium. P. aeruginosa PA067(RP1) was grown
TABLE 1. Purification of PRD1 phage
Purif'icat ion step Vol(ml)PFU/ml ~~PFUTotal coverycover
Crude lvsate 7,200 2.7x 10'0 1.9x 1014 100
Ammonium sulf'ate 70 1.6 x 1012 1.1x 10" 57
precipitate
High-speed centrif'u- 15 7.4x 1012 1.1 x 1014 57 gation
5-20^7. sucrose gradi- 20 1.3x 10 2 2.6 1013 13
entanddialysis
DNase and RNase 4 6 x 102 2.4x 103 12.3
treatmentand
high-speed centrif-ugat ion
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CHARACTERISTICSOFPRD16 overnight at 37C onTGE agar medium containing
500jsgofcarbenicillin perml.This culturewasthen
inoculated into TGE brothmedium containing 20Ag ofuracil (non-radioactive) perml andgrown at37C to mid-logarithmic phase (A42. = 0.5). The culture wascentrifuged at13,000x gat25Cfor 10min. The pellets weresuspendedinTGEbroth mediumtoA42.
= 1.0 and warmed to 37C. A 2.5-ml suspension of
PRD1 phage (107 PFU/ml) at37 Cwasaddedto2.5 mlof cellsuspension, towhichCaCl2wasthen added
(final concentration of0.015%). [2-4C]uracil,having
aspecific activity of60mCi/mmol (Schwarz/Mann), wasadded20min afterinfection,and the culturewas
incubated statically at 37C for 6 h. After this preliminary infection, an additional 5 ml of P. aeruginosa PA067(RP1) (A426 = 1.0) growing
expo-nentially in TGE broth medium containing CaCl2, and more [2- 4C]uracil were added to the culture. Uracil labelingwasused sincepseudomonadsdonot takeupthymine. Phagegrowthwascontinuedforan
additional 16h.Cell debriswasremoved by
centrifu-gation at 13,000 x g for 10 min at 25C. The "IC-labeled phage was copurified with 10 ml of previously purified unlabeled PRD1 (6 x 1012 PFU/
ml) using three successive cycles of low- and high-speed centrifugations. The labeled high-titer lysate
was treated with RNase and DNase before DNA
extraction.
Lambda DNA.Ahigh-titer lysateoflambdaphage labeled with [3H]thymidine was prepared using the
method described by De Czekala et al. (5). It was
labeled with [3H]thymine havingaspecific activityof
15Ci/mmol(Schwarz/Mann).Thelambdalysatewas
purified by the method describedabove for PRD1. RESULTS
Hostrangeof PRD1. Not all RP1-containing
strains areable topropagatePRD1. This result resembles ourpreviousreportforRP1 plasmid-dependent RNA phage, PRR1(21). The plaque-forming ability of PRD1 on RP1-containing strains shown previously to act as conjugal donors (23) is shown in Table 2. These strains have beengrouped accordingto theirability to
show plaques when phagewas plated either by the overlay technique orthe spotting of phage dilutionsonsurface lawns of indicator bacteria. None of these strains plated PRD1 in the absence of the RP1 plasmid. Of the strains in group A, the plaque morphology was variable, ranging from 3- to 4-mm (diameter) clear plaquesonS.typhimurium LT2(RP1) to 1-mm
turbid plaques with poorly defined peripheries on Acinetobacter calcoaceticus ACJ1(RP1). The strains ingroup B, after repeated testing, nevershowed evidence of plaque formationeven whenspotting surface lawns. We have found the latter method to be a more sensitive test than either plating for PFU or the commonly used cross-streaktestonthesurface of solid medium. However, when any of the strains included in
TABLE 2. Host rangeof PRD1 onRPl-containing bacteria R+indicator R+straindesignationa Lyticreactionb Strains formingplaques
Pseudomonas aeruginosa PA067(RP1)FP- Plaque morphology variable depending upon
PA067(RP1) FP+ the R+ clonal isolate tested
PAT904(RP1)FP* PAT2(RP1) FP+
PT013(RP1)FP+ PAL1822
P.fluorescens PF015.4SmR(RP1)
P.putida PP013.12(RP1)
E. coli K12 W1177(RP1)
HfrH(RP1) HfrC(RP1) BWR3951(RP1)
C(RP1)
Salmonella typhimurium LT2(RP1)
Proteus mirabilis PM17(RP1)
Vibrio cholera VCF2(RP1)
Acinetobacter calcoaceticus ACJ1(RP1)
Strainsnotformingplaques
Azotobactervinelandii AVM(RP1) Noplaque-forming ability or growth inhibition
Neisseriaperflava NPM(RP1) byspotting surface lawns
Shigellaboydii SBM(RP1)
aForsourceand othercharacteristics see previous reports (21, 23).
bDeterminationswere done at 37 C with the exception of RP1containingPF015.4SmRandPP013.12,which wereperformed at 30 C.
691
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GRAY
group B were used as RP1 donors for mating
with R- recipientstrainsshown ingroupA,the
resulting Rt exconjugants plated PRD1. Conse-quently, it wouldseem thatthegroupB strains
lack the abilitytoexpress PRD1susceptibility, although the genetic potential of the plasmid is maintained and subsequently expressed in ap-propriategroupAhosts. However, evenwith R+
strains in group A, purified exconjugants from mating experiments varied somewhat with
re-gardtoPRD1 plating. For example, when Vibrio cholera VCF2(RP1) clones derived froma
mat-ing experimentweretested, someoftheisolates showed no evidence of PRD1 susceptibility, although all the resistancedeterminantsaswell
asplasmid donor abilitywereexpressed. Aswas seen for group B isolates, when these PRD1-insensitive isolates were subsequently mated with other strains, PRD1 susceptibility again was expressed in these exconjugants. Thus, PRD1 susceptibility may relateto the particu-lar exconjugant tested among the group A isolates. However, S. typhimurium LT2(RP1) and most R+ Pseudomonas strains tested uni-formlydemonstrated phage susceptibility when exconjugant clones of these strainswerepurified and tested. When clones identified as PRD1 susceptiblewereserially restreaked and isolates tested, PRD1 susceptibility was maintained in all progeny, indicating the stability of the plasmid as well as the uniform expression of PRD1susceptibility.
Salt effect and PRDl plating on other
drug-resistance plasmid compatibility
groups. Several bacterial strains containing plasmids unrelated to RP1 were tested during the course ofour initial studies on PRD1 host
range. The susceptibility of some strains was
subsequently confirmed. This was followed by testing more representatives of previously de-scribed plasmid compatibility groupsforPRD1 susceptibility on salt- and non-salt-containing medium. The results of our testing these R+ bacteria on non-salt medium (TGE) were
uni-formly negative both by the criteria of plating for PFUs in soft-agar overlays and spotting on bacterial lawns. However, different results
ob-tainedwith asalt-containing medium (TGEN) and the results of this survey conducted with
salt-containing medium are shown in Table 3.
Ofthecompatibility groups tested,
representa-tives ofgroups N and Windicated susceptibil-ity. However, one plasmid-containing strain of
groupW, J53(R7K), showednoPRD1
suscepti-bility. This result has been confirmed by S.
Falkow (personal communication). Therefore, within a recognized compatibility group, the
expression or presence of plasmid dependent genetic information for the PRD1receptor may
TABLE 3. Drug-resistance plasmidhostrangeof PRD1
CornpatibiltvT PRDI E.coli R+straina b
suscepti-J53(R192-7) Fi+
J53(R538.1)
J53(R64-11) I
J53(R163-7)
J53(R15) N +
J53(RN3) +
J53(R46) +
J53(RsPa) W +
J53(R388) +
J53(R7K)
J53(RA1) A
J53(RA1-16)
J53(R391) J
J53(R402) T _
J53(R6K) X _
J53(R576.1) C _
J53(R64) C or 6
J5(R135) 7
J5(R11li) 8 _
J5(R71a) 9
aStrains receivedfromN.Datta, Royal Postgradu-ateMedicalSchool,London, England, and G.Jacoby, HarvardUniversity Medical School, Boston, Mass.
b Fordescription of compatibility groups see previ-ousreportsof Datta and Hedges (2) and others(19).
cPerformedby spotting surface inoculated lawns of
bacteriaonTGENagarmedium with a loop ofphage
suspension.Susceptibilityindicatedby the formation of cloudy plaques at appropriate phage concentra-tions.
vary. However, these results, when considered withthose in Table 2, show that the host range ofPRD1 extendsbeyond asinglecompatibility group and that those plasmids that confer PRD1 sensitivity contain functionally equiva-lent or identical genetic information for the specification of the PRD1 receptors, although differing inother respects.
MorphologyofPRDl.In viewof the specific-ityofPRR1 forpili (21), we nextdetermined if pili were the adsorption site for PRD1. The electron micrographs in Fig. 1 clearly indicate PRD1 adsorbed to the cell wall. Our survey of PRD1 phage RP1-containing bacteria never showed PRD1 adsorbed to RP1 pili clearly in view. Thus, among plasmid-dependent phages, PRD1 is unique since it apparently does not requirepiliforadsorption.PRD1was neverseen adsorbed to R- bacteria, and this confirms broth culture
adsorption
studies.During
the examination ofthese and other electronmicro-J.VIROL.
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[image:4.493.268.463.71.369.2]CHARACTERISTICSOF PRD16
graphs of RP1, RN3, and R388 plasmid-contain-ing strains, PRD1 phage was always seen ad-sorbed to the cell surface. In many instances,
pili associated with the plasmid were also evi-dent but they were free of adsorbed phage. Thus, weconclude on the basis ofourelectron microscopic observations that PRD1 is cell-wall specific. The morphological features of PRD1 resemble in several respects several other phages associated with Pseudomonas bacteria. For example, PM2, a lipid-containing phage specific fora marinepseudomonad, has similar icosahedral morphology and a well-defined membrane surrounding a central particle (6). Brushlike projections occurring at the vertices ofthe phagecoatarealsoseenwith PRD1 (Fig.
1A) aswith the PM2 phage envelope, although less well-defined here. Theaveragediameter of PRD1 is 62nmand thus issimilartothe 60-nm diameter reported for PM2 (6). PRD1 also resembles
06,
a P. phaseolicola-dependent lipid-containing phage which has been recently described (28). The head diameter reported for06
is 60 to 70 nm and it also possesses a membranous-like headstructure.However, un-like06,
noamorphous saclike headstructuresor adsorption topili has been observed for PRD1. Growth of PRD1. One-step growth kineticsof PRD1 areshown in Fig. 2. Phage growth on the four hosts shown here has beencorrected for unadsorbed phage. It is apparent from the differing profiles that the identity of the host influences the growth behavior of PRD1. For example, the phage latent periodwasobserved
to vary from 35 min for S. typhimurium
LT2(RP1) to 60 min for P. aeruginosa PAO67(RP1), and theorder of these differences was reproducible on repeated trials. However, as indicated here, the burst size of approxi-mately 100 is similar for the four hosts tested.
When growth (measured as plaque-forming ability) is tested on a psychrophilic P. fluorescens strain, PFO15.4(RP1), no plaques are produced at 3.5 C, although plaques are
clearly visible at 30 C. Thus, based on its growth at 37C, and not at 3.5 C, PRD1 is a
mesophilic phage capable of infecting a
psy-chrophile. Although we have previously
re-ported psychrophilic phages infecting meso-philic bacteria (22), this is the first instance ofa
mesophilic phage infecting a psychrophile. In
this case, as with psychrophilic phages, the
phage growth temperature range is phage de-pendent.
Phage adsorption to R+ bacteria. Data in Tables2 and3indicate variationsinthe
occur-rence of PRD1 phage susceptibility whichmay
reflect theoccurrence ofphagereceptorsand/or theexpression ofphage determinants during the
infective process.
Accordingly,
togain
informa-tion concerning the variance citedabove,
we determined therelationship
between cell lethal-ity and phage adsorption. This was done by using several RP1-containing bacterial strains (Table 4). We also compared differences in the adsorption process as shown by the profiles in Fig. 3.The data in Table 4 show that S.
typhimurium
LT2(RP1) adsorb more phageresulting ingreater
bacterial
lethality
fromthe infective process than observed for the R+ Pseudomonas strains. This conclusion is also supportedby
efficiency ofplating
studiestobediscussed
later. The datain Table 4,however,
do not
unequivocally
distinguish whether
thePseudomonas strains have fewer receptor sites per cell or malfunctioning phage receptors. However, the adsorption
profile
inFig.
3 pro-videssome information concerning thisconsid-eration. It is unusual that adsorption sites on
the R+
pseudomonads
should be saturated in viewof the low (1.0) multiplicity ofphage
input used here. This resultmayindicatepoor expres-sion ofPRD1 receptors in these strains.Thus,
the data here point to
quantitative
considera-tions concerning the incidence of PRD1recep-tors as,
alternatively,
poorly functioning
recep-tors would show less steep
adsorption profiles.
Therefore, the primary difference among the results for bacteria examined here seems to
reflect the density of functional
phage
recep-tors. Phage receptors may beconstantly
and uniformly present but notfully
oradequately
exposed.
This could reflect thepartial
presenceofcapsule orslimelayers associated with these
bacteria. Occlusion
ofphage
receptorsby
mu-coidsurface
layers
hasbeen demonstratedforE.coli
phages
(24).Restriction and
modification
of PRD1. The effect ofgrowth
hoston thesubsequent
plating efficiency ofPRD1 isshown in Table5.As will beshown later, PRD1 contains double-stranded DNA and hence we considered the possibility that the varianceinPRD1susceptibilityamongRP1-containing
bacteria (Tables 2 and 4) maypartially reflect the expression of modification and restriction mechanisms knowntofunction in E. coli (18) and Pseudomonas (12) species. The data inTable5 clearly indicate that some bacterial strains are better plating hosts than others, and this occurs independently of the host usedforgrowth of the phage. For example, E. coli B is a poorer plating host than E. coli K-12 for all phage lysates including E. coli B-grown phage. These data indicate the possi-bility that PRD1 lacks the appropriate nucleo-tidesequence(s) allowing restriction of its DNA. Thisisconsistent with a similar lack of
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OLSEN,
r..I
',.i*l:ea;,- -4
.. 4,
_
AFIG. 1. Morphology and adsorption of PRD1 toR+ bacteria. (A)PRD1 morphology, x160,000; (B) PRD1 adsorbed tothe cell wall of S. typhimurium LT2(RPl), x80,000; (C) PRD1 adsorbed to the cell wall of P. aeruginosa PA067(RP1), x 100,000; (D)PRD1 adsorbedtothe cellwallof E. coli J53(R388), x80,000. The bars are 100 nm.
cation
and/or
restrictionobservedforRP1 DNA (unpublished data) when transferred between hosts ofknown DNA modification and restric-tion specificity.Sedimentation constant ofPRD1. A
mix-tureoflambda, P22,
kX174,
and PRD1phages
was cosedimented on a 5to 20% sucrose
gradi-ent to characterize PRD1 in relation to some
otherwell-studiedphages (Fig.4).Byusingas a
reference the sedimentation coefficients
re-ported for lambda (30) and kX174 (10), a
sedimentation coefficient for PRD1 of 357 was J. VIROL.
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[image:6.493.74.457.76.558.2]CHARACTERISTICS OFPRD16 perfractionasdeterminedby weighing samples intermediate to the two phage peaks. The buoyant density calculated for PRD1rununder
these conditionswas1.348. Thisdensity and the membranous morphology of PRD1 are
reminis-centof similar observationson phages PM2 (6)
and
06,
which areknowntocontain lipid. Nucleic acidcomposition of PRDl. Nucleic acid extracted from PRD1 lysates asdescribedpreviously (26) was subjected to
[image:7.493.43.230.58.248.2]chromatogra-10 20 30 40 50 60 70 80 90 100 110
MINUTES
FIG. 2. One-step growth ofPRD1onPseudomonas,
Salmonella, and Escherichia hosts. Approximately 108 cells/ml logarithmically growing in TGE broth mediumat37Cwereinfectedatamultiplicity of0.01
phage/cell and incubated under static conditionsfor 20min. Following phage adsorption, theculturewas
diluted into prewarmed broth to approximately 100 infective centers/ml and incubation continued with aeration at 37C. All samplings were plated on S. typhimurium LT2(RPl) indicator bacteria with
ap-propriatedilution of samplesat the times indicated. Unadsorbed phage at 20 min was determined by
filtration and subtractedfrom subsequent phage ti-ters.
calculated. The sucrose gradient waslinear, as evidenced by comparing the positions in the gradient of the other phages, and the sedimen-tation behaviorforPRD1wasconsistent withits morphology and size.
Buoyant density of PRD1. Our initial at-tempts to determine the buoyant density of PRD1 yielded unusual results. Using a CsCl solution having a mean density of 1.6 g/cm3, which is usually used for the purification of DNA phages, resulted in greater than 90% inactivation ofPRD1, and the surviving PFUs
were recovered from the top (light) portion of the gradient. Wewere ableto preventthe CsCl inactivation ofPRD1 by including 0.5% tryp-tone (Difco) intheCsCl solution. However, the unexpected buoyancy of PRD1 and the electron micrographs of phage resembling those forthe lipid-containing PM2 phage prompted us to
consider that PRD1 may contain lipid. We therefore ran CsCl equilibrium density
gradi-ents starting with a mean density of approxi-mately 1.38, and included in thesame tube for
calibration checks an RNA phage, f2, havinga buoyant density of 1.43 (11). The results of this determination areshown inFig. 5.The pitch of
thegradientwas adensity change of0.004g/cm3
100
90
80
0
u 70
60
0 260
a
~4
Z.5
-030
0-20
10
PA067(RP1)
A
PA
AT904Revl(RP1) T2R(RP1I) [image:7.493.249.440.193.469.2]10
20
30
MINUTES
FIG. 3. Adsorptionkinetics of PRD1. Unadsorbed phagewasdeterminedasdescribed in Table4except thata multiplicity ofonephageper bacterium was used.
TABLE 4. PRD1 phageadsorption and cell killa Hostcells %PRD1 %cells Host cells adsorbed
killed
S.typhimuriumLT2(RP1) 60 95 P.aeruginosaPAT904(RP1) 40 47 P.aeruginosaPA067(RP1) 17 56aForthis,aculture growing logarithmically at 37 C
andcontaining 108 bacteria/ml was mixed with 5 x
106 PFU/ml phage suspension in TGE broth and mixed. CaCl2 wasthen added to 0.015%, and phage wasadsorbed at 37C under static conditions for 20 min followed by dilution in prewarmed TGE broth and plating for surviving bacteria or unadsorbed phageonS. typhimurium LT2(RP1).
7w a
I
m
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[image:7.493.246.443.521.593.2]TABLE 5. Effect of growth host on efficiency of plating (EOP)
EOP" on RPl-containing indicator Phage growth host
strains"
LT2 CR34 B PAT904 PA067 S. typhimurium 1 0.3 0.03 0.4 0.8
LT2(RP1)
E. coli K-12 1 0.6 0.08 0.6 1.2 CR34(RP1)
E.coli B(RP1) 1 0.1 0.02 0.6 0.9 P.aeruginosa 1 0.2 0.03 0.4 0.8
PAT904(RP1)
P.aeruginosa 1 0.3 0.07 0.6 0.8 PA067(RP1)
aFor the various phage suspensions, the EOP on
LT2(RP1)wastaken as unityregardless of the growth host.
bSee text for genus-species designations
corre-spondingtoabbreviated nomenclature used here.
dard. PX4hasbeen shown previouslytocontain DNA with44.4% G+C (22). When PRD1 DNA wassubjectedtothermal denaturation (Fig. 6), thepercentG+C, calculatedfrom inspection of the Tm profile, was50.3.Thusthe percent G+C determined by chromatography and Tm deter-minations are in close agreement. The Tm profile is also typical of that observed for double-stranded DNA since these DNA prepa-rations were resistant to alkali hydrolysis and sensitive to thermal denaturation.
Sedimentation of PRD1 DNA. The sedi-mentation properties of PRD1 DNA are shown inFig. 7A.The molecular weightofPRD1 DNA isestimated tobe24 x 106 asdetermined from theposition ofPRD1 DNA (Fig. 7A) relative to that for PR1 DNA (39 x 106 daltons, M.
Richmond,
personal communication), orlinearlambda DNA (34 x 106 daltons [27]). Further analysis of PRD1 DNA in alkaline sucrose gradients(Fig. 7B) indicates that PRD1 DNAis linearbasedonthe expected change inS value (25). Thisestimate ofthe sizeofPRD1 DNAis also compatible with the dimensions of the intact phage particle when this relationship is compared with that for the Salmonella phage P22.
10 20 30 40 S0 60
FRACTION NO.
FIG. 4. Sedimentation of PRDI in a 5 to 20% sucrose gradient. Gradients were produced using a Beckmangradient-forming device and contained, in addition to sucrose, 0.01 M Tris (pH 7.0) and 0.5% tryptone (Difco). A mixture of 0.2 ml of PRD1, lambda, P22, and<bX174phageswasaddedatthe top and the gradient was centrifuged at 22,000 rpm at 25C for45minin theSpincoSW50.1rotor.Samples
werecollecteddropwise fromthebottom,diluted,and platedonappropriate indicator bacteria.
phy usingtheproceduresofBendich(1).The
R,
values for PRD1 nucleic acid bases corre-spondedtothoseexpectedforadenine,guanine, cytosine, and thymine. PRD1 DNA was49.2%
G+C calculated from several experiments. For
this work we used DNA from a psychrophilic Pseudomonas phage, PX4, as areference
stan-10 20 30 40 50
FRACTION NO.
FIG. 5. Cosedimentation to equilibrium of PRDI
andf2 phagesinCsCI. CsCIwas added to a mixtureof PRDI and f2 in 0.05M Tris (pH 7.0)- 0.5% tryptone (Difco), to result in a mean densityof1.38 g/cm'.A portion of this mixture was centrifuged at 35,000 rpm for48h at 4CintheSpinco SW50.1 rotor.Fractions werecollecteddropwise from the bottom of thetube, diluted,andplatedonappropriate hosts.
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[image:8.493.63.254.293.491.2] [image:8.493.260.455.373.590.2]CHARACTERISTICSOFPRD16
1.3F
f..
1.2
F
0
1n 4(
1.1
50 60 70 80 90
TEMPERATURE (C)
FIG. 6. Meltingprofile ofPRD1 DNA.PhageDNA was prepared from a purified lysate as reported previouslyforPseudomonasphageCB3(26). TheTm determination was done asreported by Mandeland Marmur(16) in0.1SSC. CalculationofpercentG+C
wasaccordingtoMarmur andDoty (17).
Solvent
sensitivity
of PRD1. Our initial difficulties with thepreparation
ofhigh-titer
lysates and
subsequent
observations onphage
morphology indicatedPRD1 maycontain
lipid.
[image:9.493.39.229.52.340.2]Accordingly, we tested the sensitivity of PRD1 to agents known to inactivate lipid-containing phage (6, 29) and these results are shown in Table 6. Salmonella phage P22 was
generally
resistant to agents known to
disrupt
lipid-con-taining phage. PRD1 was sensitive to chloro-form when exposed in broth, and this chloro-form sensitivity was significantly enhanced when PRD1 was exposed to Tris-chloroform. Ethyl ether hadno effect on PRD1. This result differs from observations on the
lipid-contain-ing phage
06
(29), but duplicates observations onthelipid-containingcoliphage Sd summarized by Tikhonenko (28). Phage Sd also is disrupted by chloroform treatment but not by ether. Ac-cordingly, ourobservations leadus tothe tenta-tive view that PRD1 phage may contain lipid. However, analternative view is possible. PRD1 may have a structural protein required for in-fectivity that is uniquely chloroform sensitive. We consider this possibility less likely since, on the basis of morphology, buoyant density, and chloroform sensitivity, PRD1 clearly re-sembles three other phages known to contain lipid. Furthermore, one of these phages,Sd,
is inactivated by chloroform but not other solvents aswe also observe for
PRD1.
a
WA
at
m
ul
0 u
0U
E
G.
10 20 30 40 IS 25 35 45
FRACTION NO.
FIG.7. Sucrosegradient centrifugationof lambda, PRD1, and RP1 plasmid DNA. Experimental techniques are aspreviously described (21, 26). Samples were centrifuged at 39,000 rpm for 100 min at 20 C in the Spinco SW50.1rotor.Sedimentation is from right to left. (A) Neutral sucrosegradient of lambda, PRD1 andRPJDNA. (B) Alkaline gradient ofPRD1 DNA.
697
VOL.14,1974
I
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[image:9.493.94.384.407.620.2]TABLE 6. Inactivation ofPRD1 by solvents and sodiumdeoxycholate
%SurvivalP/Phage Treatmenta
P22 PRD1
T-chloroform 93 43
T-ethyl ether 101 109
T-toluene 98 79
T-sodiumdeoxycholate 92 78
Tris 109 80
Tris-chloroform 88 0.4
Tris-ethyl ether 77 92
Tris-toluene 76 68
Tris-sodium deoxycholate 92 64
a0.5 ml ofeach phage stock suspension was added
to a flaskcontainingTGE broth (T) or Tris, 0.05 M (pH 7.0). To a flask containing both phages the indicated additions were made as follows: chloroform (10 ml); ethyl ether (10 ml); toluene (10 ml); and sodium deoxycholate to 0.5%. The resulting 50-ml mixtures were swirled in a water bath shaker at 80 rpm at 7C for 24 h.
bCalculated as the percent surviving phage in relation to a Tbroth control phage suspension. Phase separation for solvent-containing mixtures occurred prior to removal ofsamples for dilution and titering usingPA067(RP1) in this instance for PRD1 titer and LT2 forP22 titer.
DISCUSSION
PRD1 isclearly unlike other
phages
described to date when itsplasmid-dependent
cell-wall adsorption,possible lipid
content, DNAcon-tent, and broad host range are considered
to-gether. The
ubiquity
of RP1 and relatedplas-mid-dependent
phages
is alsoapparent in viewof the facility with which these phages have been isolated from sewage
samples
obtainedfrom
geographically
distantareas(unpublished
data).
Previously
describedplasmid-dependent
phages have had their host range confined to a
particular
plasmid
group. Forexample,
bacteriacontaining the E. coli sex factor, F, orrelated
plasmids plate
themale-specific
E. coliphages
(20); the I compatibility group phage, Ifl,
at-tacks bacteria containing I-like
plasmids (14);
the N
compatibility
groupspecific
phage,
Ike,
attacks bacteria containing N group
plasmids
(13). In addition, we have described an RNA phage, PRR1, whose host range is confined to
theP-group
plasmids (21, 23).
Thesephages,
in mostinstances, have been eitherpilus-specific
RNA phages or filamentous
single-stranded
DNA phages. The icosahedral
morphology
of PRD1 represents anexample
of a new type ofplasmid-dependent phage.
It differssignifi-cantly
from the small RNAphages
orsingle-stranded DNA
containing
filamentousphages
bothrequiring the expression of plasmid genes fortheir adsorption.
Of particularinterest to us is theobservation that plasmidsof threedistinctly different com-patibility groups have in common the genetic information forPRD1receptors, although the P group (RP1and RP4), W group
(R-,,
andR388), and N group (R15, R46, and N3) plasmids do not share many otherproperties. For example, the hostrange ofthePgroup is broad (4, 7, 23), whereas the N group plasmids are confined to somegeneraoftheEnterobacteriaceae (3).The plasmids we used here of the P, W, and N groups also vary widely with regard to their molecular weight and DNAcomposition.Interestingly, a further degree of complexity with regard to criteria for relatedness of plas-mids is introduced when takinginto considera-tion the exception that a W group plasmid, R7K, will notallow plating of the phage. From thisobservationwe draw theinference that the specification of the PRD1 receptor has not necessarily been uniformly conserved (with R7K) throughout the evolutionofthe P,W, and N compatibility group plasmids. It is possible, however, that plasmids specifying the PRD1 receptor may have evolved from a common ancestral plasmid or, alternatively, that the plasmids allowing plating of PRD1 evolved independently through a common host from which was obtained the genetic information requisite for PRD1 adsorption. In addition,new phage
type-compatibility-type
plasmids may havebeengenerated by recombination between unrelatedplasmids
in agiven host (9).The observations ofothers (P. Guerry, R. W. Hedges, N. Datta, and S. Falkow, manuscript in preparation) also suggest homology between plasmids ofdifferingcompatibility groups con-sidered here. For example, RsPa and N3, group WandN,respectively, show8 to10%homology, RP1 and
Rs-a,
group P and W,respectively,
show 5% homology, and RP1 and N3, group P
and N,
respectively,
show 2 to 4%homology.
However, it should be pointed out that the homologous regions oftheseplasmids need not necessarily represent receptor site information. An alternate hypothesis is
possible,
namely, thatthe receptor siteconfiguration requirement of PRD 1 is not as stringent as presumed for other cell wall adsorbing phages.Insummary, it is clearthat PRD1represents anewand unique class ofphageswhose
growth
isplasmid-dependent. In this case, PRD1
sus-ceptibility iscontingenton a
plasmid
modifica-tionofthecellsurface.Additionally,
the critical host contributions to phage growth and host bacterium adaptability to aplasmid-specified
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CHARACTERISTICS OF
PRD16
cell wall modification have been genetically conserved. This has occurred
throughout
the evolution of hostshaving
disparate
DNA com-position andphysiological
properties.
ACKNOWLEDGMENTS
We are grateful to Deanna Thomas for her technical assistance.
This investigation wassupportedbyPublic Health Service Research grant AI07533 to Ronald H. Olsen from the National Institute ofAllergyandInfectious Diseases and the American Cancer Society Institutional Research grant IN-40N toTheUniversityofMichigan.
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