Copyright©1972 American Society for Microbiology PrintedinU.S.A.
Transcription After
Bacteriophage SPP1
Infection
in
Bacillus subtilis
G. MILANESI1 AND G. CASSANI
Laboratoric di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, 27100 Pavia, Italy, andIstitutodiGenetica, Universita diPavia,27100 Pavia, Italy
Received forpublication3April 1972
The role ofthe host polymerase in Bacillus subtilis infected with phage SPP1 was studied in vivo with regard to production of phage-specific and host-specific ribonucleic acid (RNA) and to phage yield. Evidence is presented that the sub-unit(s) of B. subtilis RNA polymerase which is sensitivetorifampin and streptoly-digin isnecessary atalltimes during infection for phage production. The synthesis ofphage RNA and the phage yield in strains resistant to either antibiotic were unaffected by the drug. Host RNA synthesis continued throughout infection; phage-specific RNA neveraccountedformorethan 20% of pulse-labeled RNA at
any time during infection.
A
number
of Escherichiacoli
bacteriophageshave been thoroughly investigated with
respect totranscription of their genomes during infection.
Someof
them
(3,
4,8) have been
found todirect
the synthesis
of a newribonucleic acid (RNA)
polymerase, which is then responsible for
tran-scription of
a majorportion of their
deoxyribo-nucleic acid (DNA).
Other phages are known todirect modifications
ofsome of thesubunits of
the host-cell RNA polymerase (6, 16, 20) in order
to
complete transcription of their
genome.SPP1 is
avirulent bacteriophage of
Bacillussubtilis which has
been isolated
andcharacterized
in this
laboratory (14).
Itssmall
chromosomewith
easily separable
strands and
high infectivity of
purified
DNAhave made it
auseful tool
forex-periments
onDNAreplication (12),
transcription
(13), and recombination (7,
17,18).
We
report here resultsconcerningthe
transcrip-tion
process in B. subtilis infected withbac-teriophage
SPP1. These results
showthat
atleast
a
portion of
thebacterial
enzymeis
necessary atall times
afterinfection
forphage
production.
Furthermore,
possible modifications
orsubstitu-tions of subunits in
the host-cell
RNApolymerase
during infection do
notalter
the effect
onthe
enzyme
ofrifampin
andstreptolydigin,
twodrugs
that interact
directly
with RNApolymerase (2,
19).
Asimilar
investigation has been reported
forB. subtilis infection with phage SPO1
(5).
MATERIALS
AND METHODSBacterial strains. The bacterial strains used are listed in Table 1. Spontaneous mutants resistant to
1Present address: Institut de Microbiologie, Faculte des Sciences, Bat.409, 91-Orsay, France.
rifampin orstreptolydigin were selected by spreading about 109 bacteria on nutrient broth plates con-taining various concentrations of antibiotic. The strains chosen were all resistant to either antibiotic at aconcentration of 100,g/ml.
Media. M40 medium (11) supplemented with 0.5% glucose,
1%
Norit-treated Casamino Acids (Difco), and 20 ug of tryptophan/ml was used for infection ofliquidcultures. Adsorption medium was M40 without carbon sources (glucose, asparagine, Casamino Acids, and tryptophan). Plates of TY (15) agar and TY soft agar were used for standardplatingtechniques.
Chemicals. Sodium dodecyl sulfate (SDS) was from The British Drug Houses Ltd.; rifampin (mo-lecularweight, 823)was agiftof G.Lancini, Lepetit
Corp.; streptolydigin (molecular weight, 610) was a
giftof G. B.Whitefield,TheUpjohnCo.
Phage.SPP1 waspurifiedfromlysateseitherwith a modification of thepolyethylene glycolsodium dex-tran sulfate method (10) or, more recently, with a modification of thepolyethylene glycol-NaClmethod
ofYmamoto et al. (21) and then by CsCl gradient
centrifugation.Titration ofphagewasdonebymixing
0.1 ml of the appropriate dilution in TY medium with 0.2 ml of B. subtilis PB 25 spores, leaving the mixtureat37 Cfor 30 min, and thenadding 2.5 ml ofsoft TY agar at 45 C and plating on TY agar. Morerecently,wefoundnochange in titers when the incubation step was omitted.
Infection. Liquidcultureswere infected in the fol-lowing way. An overnight culture in M40 medium was centrifuged and resuspended in adsorption me-dium at
Aa40
= I and was shaken at 37 C for 60min. Attheend of this starvationperiod, RNA syn-thesis, as measured by short pulses of
'4C-uridine,
wasless than0.5% of the initialsynthesisimmediatelyafter resuspension of the cells in normal M40
me-dium. The cells were then infected with SPPI at
187
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TABLE 1.Bacillus subtilis strainis used
Strain Genotypea Origin
PB 25 his-2- try-2- M. Polsinelli PB 3366 his-2- try-2- Stlr From PB 25 PB 3367 his-2- try-2- stlr From PB 25 PB 3370 his-2- try-2- rifr From PB 25
aSymbols:
his-,
try- = requirement for histi-dine and tryptophan; stlr, rifr = resistant to streptolydigin andrifampin, respectively.a multiplicity of 10. After 5 min, the culture was
rapidly chilled and centrifuged to eliminate
unad-sorbed phages and was then resuspended in
com-plete M40mediumat 37C. Atthis time, all cells be-have as infected centers when plated with PB 25
spores as describedabove. A540was usually measured
atintervals, untillysis was complete.
Labeling RNA in infectedcells.RNAsynthesiswas
measured by1- or2-minpulses of'4C-uridine (Amer-sham Radiochemical Center, 55 mCi/mmole). Usu-ally, 0.9-ml portions of the culture were placed in
tubes at 37 C containing about 1 ,uCi of uridine
(-4.5 ,g) and the tubes were shaken. The pulses
werethen terminated byadding 5ml of5%'IX ice-cold trichloroacetic acidcontaining 100 ygof cold
uridine/
ml.Thesampleswereleftin ice foratleast 1 hrand were then filtered on WhatmanGF/C
glass filters;the filters were washed with 20 ml of the same tri-chloroacetic acid solution, then with 15 ml of cold 0.01 M HCl, and finally with 95%c ethanol. When RNA was to beextracted and purified, pulses
(usu-ally made on 25 ml ofculture) were terminated by
pouringthesample of the cultureon 20 gof iceplus
50 mg ofcold uridine and 2.5 mg ofstreptolydigin
(Nasalt), both dissolved in 1.5 ml of1.3 M NaCl.
RNA extraction. Thelabeled cellswereresuspended
in ice-cold 0.1 M sodium acetate (pH 5.2)-0.01 M
MgCl2, layered on ice-cold water-saturated phenol,
and treated at maximal output with an MSE ultra-sonic disintegrator for a total of 3 min with inter-ruptions every 30 sec, while cooling on ice. This homogenizes the mixture while disrupting the cells and, as judged by sedimentation profiles, is very effective inblocking nucleases. Aftersonictreatment,
SDS wasadded to
1%',
and three phenol extractionsat60 C werecarriedout (1). Finally, RNAwas pre-cipitated byaddingone-third volume of4M NaCl and two volumes ofethanol and leaving the mixture at
-20 Covernight. Aftercentrifugation, thepelletwas
dissolved in 0.01 M
tris(hydroxymethyl)aminometh-ane (Tris)-hydrochloride (pH 7.5) and stored at
-20 C. In thecaseof invitro-synthesized RNA, the
reaction mixtures were directly extracted at 60 C
withphenoland SDS, and the
sarre
procedure was followed.DNA extraction. DNA was extracted from
CsCl-purified phage diluted in 0.01 M Tris-hydrochloride (pH 7.5), 0.01 M ethylenediaminetetraacetic acid
(EDTA), and 0.05 M NaClby three extractions with
cold phenol saturated with 0.1 M Tris-hydrochloride
(pH 7.5) and 0.01 M EDTA, and was dialyzed first
against sterile 0.01 M Tris-hydrochloride (pH 7.5),
0.001 M EDTA, and 1.0 M NaCl and then against sterile0.01 MTris-hydrochloride, 0.001 MEDTA, and
0.1 M NaCl with repeated changes. Dialysis tubes
were boiled for 60 minwith 10% NaHCO3, washed withdistilled water,and stored in 50% ethanol-0.001 M EDTA.
Hybridization. Heat-denatured SPP1 DNA was hybridized to RNA insolution (0.4 ml), and hybrid formation was detectedon nitrocellulose filters (Sar-torius Membranfilter), according to the method of Nygaardand Hall (9). DNA excess conditions were
chosenfor thisset ofexperiments and werechecked
foreveryRNApreparation.
RNA polymerase assay. RNA polymerase was
ex-tractedfromB. subtilis PB 25 anditsderivatives and
waspartially purifiedup to acompleteDNA depend-ence by a modification of the procedure of
Geidu-schek and Sklar (5). In this procedure, cells were
resuspended in 0.01 M Tris-hydrochloride (pH 7.9),
0.01 M MgCl2,0.005 M 3-mercaptoethanol,and 0.001
M EDTA, sonically treatedfor 1 min with
interrup-tionsevery 20 secwhile coolingonice, andcentrifuged
at 120,000 X g for 90 min. The supernatant fluid
wasbrought to65%csaturationat0Cwith ammonium
sulfate and was centrifuged at 13,000 X g for 30 min. The precipitatewas resuspended in 0.01 M Tris-hydrochloride(pH7.9),0.01 MMgCl2,0.005MEDTA, 10-4M dithiothreitol, and 10% glycerol. The enzyme solution withanequal volume of87%-'/ glycerol added canbestoredat -20 Cforatleast6 monthswithout appreciable loss of activity. The assay mixture con-tained, in0.20ml, 0.1 MTris-hydrochloride (pH7.9)
at 20C, 0.01 M MgCl2, 0.05 M KCl, 10-4 M EDTA,
2 X 10-4 M dithiothreitol, 10-3 M adenosine,
guano-sine, and cytidine triphosphate, 104 M 3H-uridine triphosphate (20
gCi/',pmole),
and 0.1 mg of calf thymus DNA per ml. Assays wereincubated for 10min at 37 C, chilled inice, andprecipitated with0.5
ml of
10%/6
trichloroacetic acid containing 2% so-dium pyrophosphate. After5min,theprecipitatewas collectedonWhatmanGF/C glass filters and washed twicewith10 ml of2% trichloroacetic acidandthen with ethanol. Radioactivity was determined by scin-tillationcounting.RESULTS
Phage RNA synthesis in infected cells. Figure 1 shows a typical SPP1 infection of B. subtilis strain PB 25. Cells from an overnight culture
were resuspended in adsorption medium at a concentration of 108 to 3 X 108 cells/ml and infected at a multiplicity of infection of 10. After
5minhad been allowed for adsorption, the culture was cooled and rapidly centrifuged. Thepelleted bacteria were then resuspended in complete medium which had been prewarmed at 37 C, and this was taken as time zero of infection. Phage production, under these conditions, reached a plateau after more than 1 hr after time zero. RNA synthesis, as measured by 2-min pulses of
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[image:3.499.46.239.66.274.2]TIME ASTER INFECTION(MINuEs)
FIG. 1. Itnfectioni ofPB25withl SPPI. Antoverniighlt
culture of PB 25 in M40 supplemented with glucose, Casaminto Acids, anid tryptophan (see Materials anid Methods) was cenitrifuged anidresuspenidedin
adsorp-tion medium (carbonz-free) toA140 - I (about3 X 108
cells/ml). After 60 min, the cells were inifected at a
multiplicity of inifection ofJO. Timezeroisthetimeat
wliich the cenitrifuged intfected cells were resuspended
incomplete M40 mediumat37C. Symbols: A, A51o;
0, 2-mim incorporation of '4C-uridine (see Materials
andMethods); O, intracellularphage (plaque-forming units/ml) asmeasuredafter addinigsaturatintg CHCl3 tosamples oftheculture.
uridine, continued throughout the infection
period and paralleled, at late times, the
absorb-ance curve.
The fraction of newly synthesized RNA which was phage-specific was rather lowatall times of
infection. Table2 reportsthe percentage of pulse-labeled RNA that hybridized with denatured
SPP1 DNA in the linearportion ofa saturation curve(DNA excess). RNAwaslabeled for 3min
with 14C-uridine after
20,
40, and 60 min ofinfection.
Table2 also showsthe hybridization efficiency
of in vitro-synthesized SPP1 RNA. This was synthesized by use of B. subtilis PB 25 RNA
polymeraseand SPP1 native DNAas atemplate, and washighly asymmetric (unpublished data).
From a comparison of the hybridization effi-ciencies of the RNA from infected cells and of the RNA synthesized in vitro, a rough estimate canbe madethatnomorethan20%oofthe
pulse-labeled RNA after SPP1 infection is phage-specific. Therefore, evenatlate times after
infec-tion, a greatproportion oftheRNA synthesized
is stillhost-specific. The results ofhybridization
to B. subtilis DNA are also given in Table 2.
Although thehybridization efficiency in this case was much lower, the data suggest again that a large fraction of the RNA synthesized after in-fection is
complementary
to thehost chromosome. Effect of rifampin and streptolydigin on RNA synthesis and phage production. Addition of rifampin or streptolydigin to a growing culture of B. subtilis rapidly inhibited RNA synthesis (Fig. 2). Inhibition by streptolydigin was more prompt, as one should expect, considering that streptolydigin blocks both initiation and elonga-tion of RNA chains at the same time (2) whereasrifampin
inhibits initiation only.The effect of addition of either antibiotic on
phage
productionduring SPP1 infection is shownin Fig. 3. At the indicated times, three different
portions of
the infected cultures were mixed withrifampin
(20Ag/ml),
streptolydigin (50,g/ml),or CHC13. The portion to which CHC13 was added was immediately put on ice. The antibiotic-containing samples were further incubated at
TABLE2. Hybridizationtof pulse-labeled RNA from infected cells and ofSPPI RNA
syntthesized
in vitroa
Percentage Percentage
ofinputb ofinput
RNAprepn hybridizing hybridizing toSPPi toB. sub-DNA tilisDNA
14C-RNA
from infected cells Labeling timeFrom20to23min... 8
From40to43min... 8 5
From60to63 min... 10
'4C-RNA
from uninfectedcells (labeling time, 5 min). <0.1 7 In vitro-synthesized RNA... 48
a RNAfrom infected cellslabeled with
'4C-uri-dine and purified as described in Materials and Methods was used. Hybridizationwascarriedout in solution and was detected on nitrocellulose
filters. The values reported are the averages of three points obtained in the linear portion of saturation curves (DNA excess). The SPPl DNA concentration was 15,g/ml in the experiments
with RNA from infected cells and20,ug/ml with in vitro-synthesized RNA. The B. subtilis DNA concentration was 45,ug/ml. Blankvalues (assays
made in the absence of DNA) were always less than 0.2% ofinput radioactivity. In vitro RNA was synthesized in a mixture containing: 0.11 M
Tris-hydrochloride (pH 7.5),0.01 MMgCl2,0.05M NaCl,7X 10r M EDTA,2X 104Mdithiothreitol, 107 M adenosine, guanosine, and uridine
triphos-phate, 2 X 1-4 M
"4C-cytidine
triphosphate (5jACi/,umole),
120,ugofSPP1DNA/ml,andpartially
purified RNA polymerase from PB 25. bRibonucleaseresistant.
189
VOL. IO, 1972
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01 as2 5 67 89440
MINUTES
FIG.2. InhibitionofRNAsynthesis by rifampin and
streptolydigin. AnoverniglhtcultureofPB 25was
cen-trifuged, resuspendedinfive timesthe original volume offreshM40medium, and shakenat37Cforabout 40
min. At thistime, rifampini (SO0,g/ml)orstreptolydigin
(50
,Ag/ml)
wasadded(zerotime inthefigure). Pulses(I min) were giveni to 0.9-ml portions ofthe culture
(see Materials andMethods) anidterminatedby
addi-tionofcold5% trichloroaceticacidcontaining 100,ug
ofcold uridine/mI. The resultsare expressedas
per-centages ofa similarpulse given from 1.5 to0.5 min
before addition of the antibiotics. The time of the pulses, asplotted, represent the middles ofthe 1-min
intervals. Symbols: 0, +50 ug ofrifampin/ml; 0,
+50 jig ofstreptolydigin/ml.
37 C for 160min,atwhichtimeCHC13wasadded
toall ofthem andtotheoriginalinfected culture.
The data show (within the experimental error)
thatat notime after infectioncanthe number of
bacteriophages, already present in the infected cells, be increased after addition of either anti-biotic. In a similar experiment, the effect of
streptolydiginonRNAsynthesiswasmeasuredat various times after infection (Table 3). At the indicated times, two portions of the infected
culture were transferred to two tubes, one con-taining streptolydigin to a final concentration of 40,g/ml andthe other a corresponding volume ofbuffer. At 3 and 6 minafter thetransfer,
1.5-min pulses of '4C-uridine were given. Table 3
reports the per cent incorporation, measured 6
min after the addition of theantibiotic, relative
to corresponding controls without antibiotic. As a comparison, the data for an uninfected culture are shown. It can be seen that the
per-centageof inhibition of RNA synthesis was ofa
similar magnitude in infected and uninfected cells. To assure that the lowresidual RNA
syn-I T
0
* 1
a a
0 I
*
A A
E
9-10
-A
A
0
0
A
. ;
2
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!- Stl,5aAg/il
0 A s li, 0^U9/inl
*
a1 _1 1- 1 I _-I
20 40 60 80 100 420
[image:4.499.65.459.63.286.2]TIME AFTER INFECTION(MINUTES)
FIG. 3. Effect of rifampin and streptolydigin on phage production. Ani overnight culture ofPB 25 was centrifuged and infected(seeFig.1) withamultiplicity of infection of10. At the indicated times,sampleswere addedtorifampin (20 ,ug/ml),streptolydigin(50 ,ug/ml),
or CHCI3 (see text). Inicubation was continu(ed until 160minafterinfection, except for the CHCI3--contain-ingsamples, whichwereimmediately puton ice. Atthe
enid, CHCI3 was addedtoall samples and thephages
were titrated.
TABLE3. In;iibition ofRNA
synzthesis
bystreptoly-digin duriiig infectionia
Time of addition of Percentincorporation
streptolydigin (min)
Inzfected
cells15 3.1
30 5.6
45 6.9
60 4.3
75 4.1
90 4.6
105 5.6
120 7.7
Uninfected cells
30 3.8
60 3.0
aThe timesindicated are those at which
strep-tolydigin (40 ,ug/ml finalconcentration) in 0.1 M Tris-hydrochloride (pH 7.9) or a corresponding
amount of Tris buffer were addedto portions of
the infected culture. At6min after theaddition,
90-secpulses of
"4C-uridine
weregiven. The valuesreported represent percentincorporation relative to that observed in the absenceofstreptolydigin. Inthecaseofuninfectedcells, thesameprocedure was followed as for infection (see Materials and Methods), exceptthat no phagewasadded.
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[image:4.499.269.460.417.567.2]PHAGE SPP1 TRANSCRIPTION
thesis still present after the addition of the anti-biotic was notphage-specific, RNA labeled with '4C-uridine from70to73minwasextractedfrom infected cells to which streptolydigin had been
added at 60 min after infection. The extracted RNA was then hybridized with an excess of SPP1 denatured DNA; only 10% of the input label was found in hybrid, showing no increase over the values reported in Table 2. Thus, the low residual DNA synthesis after addition of streptolydigin isnot phage-specific.
From these results we can conclude that (i) there is no phage-specific RNA polymerase,
synthesized
after SPP1 infection, which isre-sistant to rifampin or streptolydigin and which can by itselfsupport phage production, and (ii) transcription is necessaryat alltimes, before the phage production reaches its maximal value, for the number of phagestoincrease.
Infectionof PB 25mutantsresistanttorifampin andstreptolydigin.Wenextlooked for the possible synthesis of a phage-specific RNA polymerase sensitivetothetwoantibiotics. Forthispurpose, a number ofB. subtilis mutants resistant to the
two
antibiotics
were selected.Table 4 shows the characterization of some resistantmutantsselected from B. subtilisPB 25. RNA polymerase activity of partially purified
extracts of these mutants was strongly resistant to either rifampin or streptolydigin. These
mu-tantsweretheninfected with SPPl in thepresence
or absence of the antibiotic to which they were resistant. The lysis of the infected cultures was
followed by monitoring absorbance at 540 nm. After lysis had occurred, CHC13 was added and thephageweretitrated. From theresults in Table
5, it can be seen that in none of the mutants
analyzed was there any reduction of the phage
yield
in the presence of the antibiotic. In otherwords, no
phage-specific
RNA polymerase,sensitivetoeitherrifampinorstreptolydigin, and necessaryfor phage production, was synthesized after SPP1 infection.
DISCUSSION
Several conclusions can be drawn from our results. After infection ofB. subtilis PB 25 with
SPP1, no new RNA polymerase is synthesized which, alone, continues the infection process.
More specifically, thetwo followingpossibilities
canbe excluded: (i) thesynthesisofa newRNA
polymerase, sensitive to rifampin and
strep-tolydigin and necessary for phage production;
and (ii) the synthesis atany time after infection
of a new RNA polymerase resistant to either
rifampin
orstreptolydigin andsufficient forphageproduction. Other possibilities cannot be
ex-cluded, but they seem rather unlikely. For
ex-TABLE 4. RNA polymerase activity in partially purified extracts of PB 25 and resistant mutants
Per-Molarityin cent-Strain Drug theassay ageof
trol" PB 25stls, rif8 Rifampin 0 100
6 X 10-8 22 6X 1-7 6.5 6 X 106 0 1.2 X 10-5 0
PB 3370stls,rifr Rifampin 0 100 6 X 10- 100 6 X 10-7 100 6 X 10-6 100 1.2 X 10-i 100
PB 25
stl',
rifs Streptolydi- 1.2 X 10-6 100gin 1.2 X 10-5 38
1.2 X 104 0 1.2X 10-3 0
PB3366stlr,rifs Streptolydi- 1 .2 X 10-6 100
gin 1.2 X 10- 100
1.2 X 10-4 100 1.2 X 10-3 51
PB 3367
stls,
rif8 Streptolydi- 1.2 X 10-6 100gin 1.2 X 10-5 100 1.2 X 1Ir 100 1.2 X 10-3 53
a Values represent the percentage of controls
without inhibitors. Control assays correspond to
[image:5.499.248.445.468.640.2]approximately 0.5 nmole of 3H-uridine triphos-phateincorporatedinto RNA in 10 min at 37 C.
TABLE 5.
Effect
ofrifampin andstreptolydiginz
onphageproduction in strainssensitive or resistant to the inhibitorsa
Strain Antibiotic PFU/ml PFU(%) PB 25 sti', rifs None 3.3 X 1011 100 PB25stls,
rift3
Rif, 10 1.5 X 108 0.04yg/ml
PB25
stli, rif'
Stl, 50 1.1 X 108 0.03fig/ml
PB 3366 stlr None 5.0 X 1010 100 PB3366 Stlr Stl, 40 5.0 X 1010 100
yg/ml
PB3367 stlr None 5.0 X 1010 100 PB3367 stlr Stl,40 5.5 X 1010 100
/Ag/ml
PB3370 rifr None 6.3 X 1010 100
PB 3370rifr Rif, 50 7.5 X 101" 100
.Ug/ml
aRif = rifampin; Stl = streptolydigin; stl, stlr = sensitive or resistant to streptolydigin;
rifs, rifr = sensitive or resistant to rifampin; PFU = plaque-forming units.
191
VOL. 10, 1972on November 10, 2019 by guest
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ample, a new RNA polymerase could be made after infection, resistant to either rifampin or streptolydigin and necessary for infection, but such that it would be able to support phage pro-duction only in conjunction (at the same time) with the host enzyme.
On the basis of our results,
modifications
or substitutions of some of the subunits of the bac-terial enzyme cannot be ruled out. It is important in this respect to observe that, if the subunit(s) responsible for the interaction with rifampin and streptolydiginis modified after infection, this does not alter its interaction with either antibiotic, both in the case of a sensitive and of a resistant strain. The most likely interpretation of our results is that the subunit(s) that confers resist-anceorsensitivity to the hostRNApolymeraseis
notmodified after infection. Itssubstitution with completely new, phage-specific subunits is sub-jected to the same criticism as the case
discussed
above (i) of a completely new enzyme.
We
have
established that host RNAsynthesis
continues throughout infection, but we do not know whether this is necessary for infection to proceed.
What
wedo knowis
that RNAsynthesis
(host
orphage
orboth) is always
necessaryfor
the phage yield to reach
its
maximum.Secondary
effects of
the antibiotics,
distinctfrom inhibition
of RNA
synthesis
(e.g.,
onassembly)
areruled
out by the experiments with resistant mutants. Thissubject is currently under investigation.From all of the experiments reported here,it
is
probably safe to conclude that no dramatic
alteration
of thetranscription machinery
occursafter
SPP1infection
of B. subtilis. Mostlikely,the
host RNA polymerase itself can completely
transcribe
DNAduring infection. This hypothesis
is also supported by some preliminary in vitro evidence.
ACKNOWLEDGMENTS
Wegratefully thank M.Polsinielliand P.Schaeffer forletting us knowtheirresults prior to publicationand F. Melgara for skillful technical assistance.
LITERATURE CITED
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2. Cassani, G., R. R. Burgess, H. M. Goodman,andL.Gold. 1971. Inhibition of RNA polymerase by streptolydigin. NatureN. Biol. 230:197-200.
3. Chamberlin,M., J. McGrath, and L. Waskell. 1970. New RNApolymerase from E. coliinfectedwithbacteriophage T7. Nature(London) 228:227-231.
4. Dunn, J. J., F. A. Bautz, and E. K. F. Bautz. 1971. Different templatespecificitiesofphageT3 and T7 RNA polymer-ases.Nature N.Biol.230:94-96.
5. Geiduschek, E.P., and J. Sklar. 1969. Role of host RNA polymerase inphage development. Nature (London) 221: 833-836.
6.Hager,G., B. D.Hall,and K.L.Fields.1970.Transcription factors from T4 infected E. coli. Cold Spring Harbor Symp.Quant.Biol. 35:233-239.
7. KLOTZ, G.,AND H. C. SPATZ. 1971. Abiologicalassayfor intracellular SPPI DNA. Mol. Gen. Genet. 110:367-373. 8. Maitra, U. 1971. Induction of a new RNA polymerase in
E.coliinfected withbacteriophageT3.Biochem.Biophys. Res. Commun. 43:443-450.
9. Nygaard, A. P.,and B. D. Hall. 1963. A method for the detection of RNA-DNA complexes. Biochem. Biophys. Res.Commun. 12:98-104.
10. Philipson, L., P. A. Albertson, and G.Frick. 1960. The purifi-cation andconcentration ofviruses byaqueous polymer phasesystem. Virology11:553-571.
11. Polsinelli,M., and M. Baretta. 1966.Geneticrecombination incrosses betweenStreptomnyces aureofaciens and Strepto-mycesrianosus. J. Bacteriol.91:63-69.
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