-
JOURNAL OF BACTERIOLOGY, July 1969, p. 116-124Copyright ( 1969
American Society for Microbiology
Vol. 99, No. IPrinted In U.S.A.
Synthesis of Bacterial FlagellaII. PBS1 Transduction of
Flagella-specific Markers in Bacillus subtilis
GEOFFREY F. GRANT1 AND MELVIN I. SIMONDepartment of Biology,
Revelle College, University of California, San Diego, La Jolla,
California 92037
Received for publication 22 January 1969
The linkage relationship of mutants involved in the synthesis of
flagella was de-termined by PBS1 transduction. Mutants that affect
the structure of flagellin(hag) and temperature-sensitive mutants
(flaTS) that produce flagella when grownat 37 C but not when grown
at 46 C were examined. All of the mutants were found tobe linked to
the hisAl marker. The flaTS mutants fell into three clusters.
GroupA contained the majority of mutants which were loosely grouped
around the haglocus. Group B mutants were segregated from the hag
locus and appeared closelylinked to the phage adsorption site gene
(gtaA), and group C was only looselylinked to hisAI and thus far
contains only one mutant. A flagella locus (ifm) affect-ing both
the degree of motility and level of flagellation was shown to map
neargroup A. Mutants affecting motility (mot) were not linked to
hisAI by PBS1 trans-duction. Several markers previously shown to
link to hisAI were ordered withrespect to hisAI and the flagellar
genes.
Previous work on the genetic control of flagel-lation in
Bacillus subtilis (7) has defined theexistence of at least three
classes of mutationanalogous to those found in Salmonella
typhi-murium, hag, fla, and mot. The hag group involvesmodification
of the structure of the flagellar fila-ment subunit protein. The
wild-type W23 strain,for example, possesses immunologically
distinctflagellin (hag-2) which differs from wild-type168 (hag-i)
in both amino acid composition andpeptide sequence (S. Emerson,
personal com-munication).The fla mutants phenotypically lack
flagella
and are presumably defective in functions in-volved in the
synthesis and assembly of the or-ganelle. They are readily isolated
in B. subtilisbut are difficult to examine genetically. The
prob-lem stems from the fact that both transductionwith SPIO and
transformation establish linkagerelationships over only relatively
short, well-defined intervals of the map, whereas transduc-tion
with PBS1, which allows the transfer ofextensive fractions of the
genome, is mediated bya flagella-specific virus which does not
adsorb tofla recipient cells (5). To establish a system forboth
genetic and biochemical study of flagellasynthesis, we used the
known variants of thestructural gene (hag-i, hag-2, hag-3) and
alsoisolated a number of temperature-sensitive mu-
' Present address: Salk Institute for Biological Studies,
LaJolla, Calif. 92037.
tants (flaTS). The flaTS mutants allow the ad-sorption of PBS1
and hence transduction at37 C, but they do not possess flagella at
46 C andcan, therefore, be scored for recombination.A preliminary
report on the mapping of fla-
gella mutants in B. subtilis was presented at the68th Annual
Meeting of the American Society forMicrobiology, Detroit, Mich.,
5-10 May 1968.
MATERIALS AND METHODSMedia. Basal medium was a minimal salts
medium
(2) supplemented with either 0.1% Casamino Acidsand 30 lsg of
appropriate growth requirements per ml,or, when selective medium
was required, 20 ,g of allamino acids and requirements per ml
excepting theparticular growth factor used as a selective
agent.
Soft motility agar was composed of basal mediumsupplemented with
0.4% agar and 0.8% gelatin; whenappropriate, sufficient
flagella-specific antiserum wasadded to inhibit motility.
Antibodies and antigens. B. subtilis flagellar proteinwas
purified and antisera were prepared as previouslydescribed (6).
Nomenclature. To assign consistent designations toflagellar
mutations in this study, we have adhered tothe conventions proposed
by Demerec et al. (3). Sincewe assume, on the basis of both our
data and the datapresented by Frankel and Joys (5) that the
flagellarantigens represent alternate alleles of a single hag
gene,the wild-type 168 antigen has been designated hag-1; the W23
antigen, hag-2; and the straight fila-ment mutation reported by
Martinez et al. (8), hag-3.Genetic analysis on B. subtilis is not
refined enough,at this stage, to completely rule out the
possibilityof multiple hag cistrons.
116
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/
-
TRANSDUCTION OF FLAGELLAR MARKERS
Strains. Tables 1 and 2 show the properties and in a number of
ways. The mutants described in thisorigin of strains of B. subtilis
used and prepared in paper were isolated following treatment
withthis investigation. All parent strains were selected for
N-methyl-N'-nitro-N-nitrosoguanidine at 100 &g/mlrapid motility
by passage through motility tubes. (approximately 50% survival)
according to the proce-The selection offlaTS mutants may be
accomplished dure of Adelberg et al. (1). They were selected by
TABLE 1. Strains of B. subtilis
GenotypeStrain Origin Derivation
Auxotrophic markers hag Other markers
W23MH-1BD71BR19Rog 1
Rog 3
BR19 (hag-2)
BR13BR13 (hag-2)
BR85SB-3FY'A'JH1057G-2
SO-49G-5
G-10
SC-3 and SC4
G-22
flaTS -I{o -10 +flaTS -32to -47+
flaTS -18 to -23
flaTS -51
G-25
G31
G26
trp-2 lyshisAl, ura, argC4hisAl, trp-2
hisAl, trp-2
hisAl, trp-2
ura-1, trp-2ura-1, trp-2
argC4, trp-2hisAl, trp-2, cysBtrp-2trp-2 met4hisAl, ura,
argC4
trp-2hisAl, argC4
argC4
trp-2
hisAl, ura
trp-2, lys
trp-2, ura
hisAl, ura, argC4
hisAl
hisAl
hisAl, ura
2
1
1
1
1
1
2
12
11
1
1
2
1
2
2
3
3
1
1
1
2
1
2
rou-l
rou-lspoCI
spoCl
rou-l
rou-lrou-l
rou-l
gtaA rou-luvr-l
motgtaA
gtaA
gtaA, uvr-1,ifm-l
gtaA, uvr-lifm-l
uvr-1, ifm-l
Wild type
Marburg wild typecured for sporula-tion marker withacridine
orange
BR19 transformedwith Rog 1 DNA
BR19 transformedwith W23 DNA
BR13 transformedwith W23 DNA
BD71 transformedwith W23 DNA
G2 transformed withexcess FY'A' DNAselection of gtaAby
congressioneliminating ura
G-5 transduced withPBS1 lysate ofMH-1
Nonmotile mutantpossessing straightflagella; antigen-ically
hag-i
BD71 transformedwith SC4 DNA
Nitrosoguanidinemutagenesis ofMH-1
Nitrosoguanidinemutagenesis ofBR13
Nitrosoguanidinemutagenesis ofBD71
G5 transformed withexcess JH1057 DNA
G25 transformedwith FY'A' DNA
G2 transformed withexcess JH1057 DNA
SpizizenSueokaDubnauReillyRogolsky
Rogolsky
Reilly
ReillyNesterYoungHoch
Joys (7)
Martinez
117VOL. 99, 1969
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/
-
GRANT AND SIMON
cycling cells through high (46 C) and low (37 C)temperatures and
transferring the fraction of theculture which agglutinated at 37 C
but not at 46 Cwith flagella-specific antibodies. The process
wasrepeated four or five times, and the cells were platedin soft
motility agar for single colonies at 46 C.Nonmotile clones were
picked, restreaked, and testedfor their ability to produce flagella
at both tempera-tures. The mutants used for mapping produce few
or
TABLE 2. Relative position of hag replacement locuson B.
subtilis chromosomea
SelectedRecipient strain auxotrophic
markers
BR-27........BR-5.........BR-62........BR-77........SB-8.........BR-19........BR-85........BR-51...BR-13........BR-123.......BR-44........BR-84........BR-76........BR-76........BR-50......
ade-4ade-lade-5thr-1cysBhisAlargC4metA7ura-largOIleu-6phe-3lys-3trp-2met-6
Migrationthru minimalagar contain-ing antibody
+(+)+
Binding of1251-hag-2antibodyb
< 1,000
-
TRANSDUCTION OF FLAGELLAR MARKERS
Stock virus was prepared by infection of an early logculture of
B. licheniformis in Penassay Broth (Difco).After lysis, the phage
were purified and concentratedby differential centrifugation. The
preparation oftransducing lysates and the transduction were
carriedout according to the procedures of Reilly and
Spizizen(personal communication). Donor strains were inocu-lated
into Penassay Broth from overnight TryptoseBlood Agar Base (TBAB;
Difco) plates and weregrown to an optical density of approximately
150Klett units; they were then infected with stock PBS1at a
multiplicity of 5. The infected lysate was incubatedwith shaking
for 3 to 4 hr. The culture was thenallowed to undergo autolysis by
removing it from theshaker and incubating it overnight at 37 C. The
lysatewas treated with deoxyribonuclease (1 jug/ml) andcentrifuged
at 6,000 X g for 10 min. The supernatantfluid was sterilized by
passage through a 0.45-Mmsterile membrane filter.
Recipient strains were streaked on TBAB platesand grown
overnight; they were then heavily inocu-lated into Penassay Broth
and grown for 5 hr untilmaximal motility was obtained. The
recipient cultureand transducing lysate were mixed in equal
volumesand diluted 1: 2 into fresh Penassay Broth followed
byincubation for 20 min with shaking. The infected cellswere washed
twice in minimal salts by centrifugation,and were plated on
selective media in the presence ofsterile PBSI antiserum.
Recombinant clones werepicked and restreaked on selective agar. The
observedrecombination frequency was approximately 10-5/bac-terium.
Nutritional and phage (t25)-resistancemarkers (gtaA) were scored by
replica-plating by useof pads of velveteen; flagella markers were
scored by
replica-plating by use of an inverted flower holder(frog) which
was placed onto soft motility agar plateswith or without flagella
antiserum and incubated at37 or 46 C, or at both temperatures.
Figure 2 showshow flagella markers were identified.
Transformation. Transforming deoxyribonucleicacid (]DNA) was
isolated by the procedure of Massieand Zimm (9) with the use of
lysozyme and Pronase.Transformation was carried out according to
themethod of Anagnostopoulos and Spizizen (2). Flagellaantigenic
types were selected by inoculating trans-formed populations into
motility tubes containingantisera specific for the recipient
flagella and pickinga recombinant that passed rapidly through the
tube.
RESULTS
Linkage of hag locus to hisAl. The generallocation of the hag
gene on the B. subtilis chro-mosome was established by the use of
strainswhich have antigenically non-cross-reacting fla-gella (hag-i
and hag-2). A large number ofauxotrophic strains were used, all of
whichrequired indole (trp-2) and one other marker.The second marker
was chosen so that linkagewith various parts of the chromosome
could bedemonstrated (Table 2). Transducing lysateswere grown on
strains with hag-2 flagella andwere used to infect the hag-i
auxotrophs. Link-age of the hag locus was demonstrated (i)
bygrowing recombinants in selective media andmeasuring hag-2
flagellar antigen with radio-
2
4
370 C
hog-I Ab
I hog-I flogello2 hoq-2 flogello3 hog- I flI TS4 f/la
3460 C
hao-2 Ab
FIG. 2. Demonstration of the technique used to score flaTS and
hag recombinants. The flaTS recombinantswere scored by incubation
ofplates at 37 and 46 C (upper petri plates), and the hag
recombinants were identifiedby use ofagar containing flagellar
specific antibodies (lowerpetri plates).
119VOL. 99, 1969
1...
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/
-
GRANT AND SIMON
active antibodies, and (ii) by inoculating thetransduced
population into motility tubes con-taining hag-i flagellar antibody
but lacking aspecific nutrient. Under these conditions, onlyhag-2
prototrophic recombinants could migratethrough the agar. The hag
locus was found to belinked only to the cysB and hisAl
markers(Table 2). Linkage was observed when the phagewere grown on
derivatives of 168 strains carryingthe hag-2 locus. When the phage
were prepared
directly on the W23 strain and used to infect168 derivatives, no
linkage was observed.
Relative map position of the hag locus. ThehisAl locus has been
shown to be linked to anumber of mutations. Dubnau et al. (4)
mappedhisAI between cysB and argC4, and found 20%linkage to each of
these markers. Other markersthat have been placed in this region of
the chro-mosome are the phage-resistance markers gtaA,B, and C
(12); a sporulation marker, spoCI
TABLE 3. Linkage relationships of markers to hisAl
Recipient Donor lysate marker Recombinant classes duction
BR19 BR13 (hag-2) his+ his+ hag-2, 350/686 51SB3 BR13 (hag-2)
his+ his+ hag-2, 128/260 49
his+ cysB+, 112/564 20his+ cysB+ hag-2, 14/260 5.4
BR85 BR13 (hag-2) arg+ arg+ his+, 0/120BD71 BR13 (hag-2) his+
his+ hag-2, 96/184 52
his+ arg+, 0/120arg+ arg+ his+, 0/120
BD71 FY'A' his+ his+ rou-J, 403/673 60his+ rou-i gtaA, 236/673
35his+ rou+ gtaA, 340/673 50.5
BR-19 (hag-2) FY'A' his+ his+ hag-i, 61/90 68his+ hag-i gtaA,
48/90 53.5his+ hag-2 gtaA, 2/90 2
Rog 3 BR13 (hag-2) his+ his+ spoCI hag-2, 96/192 50his+ spo+
hag-2, 28/192 14.5his+ spo+ hag-i, 8/192 4
BD71 Rog-1 his+ his+ spoCI, 24/150 16his+ arg+, 0/150
1H1057 G-10 his+ his+ uvr+, 108/140 77his+ uvr+ hag-2, 81/140
58his+ uvr+ hag-2 gtaA, 65/140 46.5
rou-l
40
hisAifm
uvr-I hog gztaA
A BfloTS gene
spoCI
Cclusters
gtaC orgC4. o1
9031
3742
5384
FIG. 3. PBSI transduction map of the hisAl linkage group in
Bacillus subtilis. Distances were determinedfromthe average of all
experiments carried out during the course of this work (Table 7).
Although the relative order ofifm and hag have been established
(Table 4), the relative position of the flaTS markers that fall in
this region hasnot as yet been completely determined.
r
120 J. BAcrERioL.
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/
-
TRANSDUCTION OF FLAGELLAR MARKERS
(10); a locus controlling radiation resistance,uvr-1; and a
morphological marker, rou-1. Theresults of crosses designed to
establish the rela-tive map position of these markers with
respectto hisAl and hag are shown in Table 3 and aresummarized in
Fig. 3 and Table 7.
Cotransduction of flagella-related markers.Mutations in the ifm
locus change the relativequantity of flagella per cell as well as
the motilityof the cells (see Materials and Methods); the
function that is affected in these mutants is notknown, and
crosses were designed to determine(i) whether ifm clearly
segregates from hag and(ii) its relative position on the map.
In the first cross, a hisAl, uvr-1, hag-2, ifm-1strain was used
as recipient, and a hag-i, ifm+strain was used as donor. When
recombinantswere replicated onto 0.4% agar, the ifm recom-binants
could be clearly distinguished (Fig. 1).The data show that ifm
segregates from hag-i;
TABLE 4. Segregation of ifm, gta, and hag among his+
transductantsa
+ + + hag-i +(1) Donor MHI, genotype hisAl uvr-i ifm-l hag-2
gtaA
Recipient G25his uvr ifm hag gta No. in class
1 0 0 0 0 441 1 0 0 0 131 1 1 0 0 101 1 1 1 0 251 1 1 1 1 481 0
0 0 1 2
No. with donor allele 142 96 83 73 50 142
+ + + hag-3 +(2) Donor SC4 genotype hisAl uvr-i ifm-i hag-i
gtaA
Recipient G31his uvr ifm hag gSa No. in class
1 0 0 0 0 581 1 0 0 0 3
1 0 0 81 1 NDb 1 0 161 1 NDb 1 1 501 0 0 0 1 11 0 NDb 1 1 21 1 0
0 1 11 1 1 0 1 1
No. with donor allele 140 79 9b 68 55 140
+ + + hag-i gStaA(3) Donor FY'A' genotype hisAI urr-i irm-i
hag-2 +
Recipient G26his uvr ifm hag g8aA No. in class
1 0 0 0 0 381 1 0 0 0 81 1 1 0 0 61 1 1 1 0 161 1 1 1 1 681 0 0
0 1 11 1 0 0 1 21 1 1 0 1 2
No. with donor allele 141 102 92 84 73 141
a The frequencies of all observed recombinant classes are shown.
The donor allele is represented
determined. The total thereforeby a 1 and the recipient by a
0.
"The ifm character of the recombinants that were hag-3 was
notrepresents the recombinants that were ifm+, hag-l.
121VOL. 99, 1969
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/
-
GRANT AND SIMON
of the his+ recombinants tested, 10 of 140 wereifm+, hag-2
(Table 4). In a second cross, thedonor strain was hag-3, ifm+, and
the recipientwas hisAl, uvr-1, hag-i, gtaA, and ifm-i. Again,ifm
was found to segregate from hag, and 9 of140 recombinants were
hag-i, ifm+. In the thirdcross, the same pattern of segregation was
found.These data are consistent with the order uvr-ifm-hag-gta.
Table 5 shows the results of crosses performedto determine the
relationship of theflaTS markersto the hag locus. In all, 26
temperature-sensitivemutants were tested and approximately 150
his+recombinants were picked in each cross. Sinceeach cross
required lysates grown on the flaTSstrains, a fair amount of
variation in the fre-quency of recombination for the hag region
wasexpected. Table 5 includes the extremes in thevariation
obtained. In spite of these differences,the mutants clearly fall
into three distinct groups.Group A includes 21 mutants, all of
which mapnear hag. The ratio of the frequency of recombin-ants for
flaTS to that for hag-i is in the rangeof 1.1 to 0.92, and the
ratio of flaTS to gtaA is1.6 to 1.2. Group B contains three
mutants; theratio of flaTS to hag-i is 0.86 to 0.77, and theratio
of flaTS to gtaA is 1.0. There is thus faronly one mutant that maps
in Group C. It isclearly separable from the hag and the gtaA
loci.To check further the position of the group B
TABLE 5. Transduction offlaTS mutants
flaTSdonorlysate
9404328
2210377
204613
51
Percentage of hisAl recombinantsa
hag-i IflaTSI gtaA+
7268586566706574616855657058
8075566266656668627258565422
5557405152424048425047575544
hag-l,
fla0
1
866666442171939
kag-2, flaTSflaTS to hag
5
96460
715
860
72
1.111.100.970.961.000.931.020.921.021.061.050.860.770.38
Ratio
1.451.321.401.281.261.551.621.541.481.451.250.990.990.50
a The recipient in all these experiments was G-5(hisAI, hag-2,
gtaA). The donor lysates were pre-pared on the appropriate flaTS
strain. hisAI+recombinants were picked and tested for the
othermarkers. The results are presented as the per-centage of
hisAI+ recombinants that carry a givenmarker.
mutants, flaTS-I and flaTS-7 were put into astrain carrying the
gtaA marker and then crossedinto BD-71. In this cross, flaTS-I was
found tobe closely linked to gtaA; the ratio of recombin-ants for
flaTS to those for gtaA was 1.05, andthe ratio for flaTS-7 to gtaA
was 1.2.
Table 5 also shows that in all the crossesrecombinants were
obtained that were eitherhag-i, fla+ or hag-2, flaTS, suggesting
that allthe flaTS markers mapped can segregate fromthe hag locus.
However, the data obtained thusfar do not allow us to assign the
precise positionsof all of theflaTS markers relative to one
anotherand to the hag locus.We have assumed thus far that there is
no
phase variation in B. subtilis. Our results couldbe complicated
if these strains carried two sepa-rable hag genes and only one was
phenotypicallyexpressed. To test this possibility, hag-3
mutantswere used. The hag-3 gene is derived from hag-iby mutation
and has been shown to differ inonly a single peptide (8). Strains
carrying hag-3are nonmotile and have flagella that are
anti-genically identical to the hag-i product but lackthe normal
long-period helix. In the crossesshown in Table 6, recombinants
that were non-motile were picked and tested for
antigenicspecificity. None of the nonmotile recombinantshad hag-2
antigen, and all of the nonmotilerecombinants had flagella.
Furthermore, norecombinants of the hag-i type were found. Infurther
crosses, over 1,000 recombinants havebeen picked and tested, and
thus far only asingle hag-i recombinant has been found. There-fore,
these data suggest that the strains do notcarry cryptic alternate
hag genes that are linkedto hisAI.We have also found that the
motility-negative
mutation (mot) reported by Joys and Frankel(7) does not
cotransduce with hisAI.
DISCUSSIONGenetic crosses done by use of phage PBS1
established the linkage of the hag locus to hisAI.The
relationship of flagella markers to othermarkers cotransferred with
hisAI is shown inFig. 3 and Table 7.
It was not found possible to demonstratelinkage of hisAI to
argC4, as was reported byDubnau et al. (4); in fact, the spoCI
marker ofRogolsky (10) was demonstrated to be lesstightly linked to
hisAI (16%) than the reportedlevel of cotransfer of the argC4
marker (24%).None of the hisAI linked markers could beshown to
cotransfer with argC4. However, F.E. Young (J. Bacteriol., in
press) and Grant andSimon (unpublished data) have shown that insome
specific strains hisAI and argC4 may be
122 J. BAcTEiuoL.
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/
-
TRANSDUCTION OF FLAGELLAR MARKERS
TABLE 6. Test for a cryptic hag gene
Recipient Donor Motility Recombinant
classesphenotypeReobnnclss
G5 SC-3 _ his+, hag-3, 94/192hisAl, hag-2, gtaA hag-3 _ his+,
hag-3, gtaA, 42/192
+ his+, hag-2, 56/192
G5 SC-4hisAl, hag-2, gtaA hag-3 _ his+, hag-3, 97/200
_ his+, hag-3, gtaA, 35/200+ his+, hag-2, 68/200
G22 G10hisAI, hag-3 hag-2, gtaA + his+, hag-2, gtaA, 79/140
+ his+, hag-2, 11/140his+, hag-3, 50/140
TABLE 7. Summary of cotransductioni frequencies ofmarkers with
hisA1a
Total fraction of Percentage ApproximateMarker his+ recombinants
of cotrans- linkage
for linked markers fer (Y) (100 - Y)
rou-l 403/673 60 40uvr-l 775/1,120 69 31ifm 444/700 63 37hag
899/1,540 58 42gtaA 863/1,820 47 53spoCi 65/412 16 84
a The relative order of these markers has beenestablished and
was consistently found in all ofthe transduction experiments. The
degree of link-age summarizes our data. The cysB marker wasfound to
map to the left of rou-i (Table 3).
cotransferred by PBS1 transduction. Young hasalso shown
transformation linkage between thephage-resistance markers gtaA and
gtaC whichdo not normally cotransfer in PBS1 transduction.This
behavior suggests the presence of a chromo-somal abnormality or an
unstable chromosomalelement that can be inserted in this region,
e.g.,a defective lysogenic phage or an unstable epi-some. However,
more experimentation is obvi-ously necessary to clarify this
problem.
All of the hag and flaTS mutants that we havetested thus far are
linked to hisAl by cotrans-duction. Although the data do not allow
us toestablish unequivocally the position of all ofthese markers,
it is clear that most of them clusteraround the hag locus. Some
mutants (flaTS-Iand -3) appear to be more closely associatedwith
the gta locus and segregate from hag. Onemutant (flaTS-51) is
clearly separated from theothers.The lack of more markers in this
region and
the absence of a reliable complementation systemin B. subtilis
has prevented us from establishingdiscrete functional classes for
the flaTS mutants.The data available, however, suggest that mostof
them do not directly affect the structural genefor flagellin. This
is certainly clear for the groupB and C mutants, which can be
readily separatedfrom the hag locus. However, even the group
Amutants, in almost all of the crosses, were foundto segregate from
hag and give hag-2, flaTS orhag-i, fla+ recombinants.
Furthermore, tests of the flagellin proteins ofthese mutants
also indicate that they do notdiffer from the wild-type protein
(Dimmitt andSimon, unpublished data). These data suggestthat the
flaTS mutants are defective in ancillaryfunctions that are required
for the formation ofbacterial flagella. Further work is being
directedtoward elucidating these functions, and towarddetermining
the gene order in the group Aregion.
ACKNOWLEDGMENTS
We thank J. Spizizen, J. Hoch, and especially B. Reilly andF. E.
Young, of Scripps Clinic and Research Foundation for pro-viding,
not only many of the strains used, but also many ideasand
criticisms of the work.
This work was supported by grant GB-6980 from the
NationalScience Foundation.
LITERATURE CITED
1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965.Optimal
conditions for mutagenesis by N-Methyl-N'-N'Nitrosoguanidine in
Escherichia coll. Biochem. Biophys.Res. Commun. 18:788-795.
2. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements
fortransformation in Bacillus subtills. J. Bacteriol.
81:741-746.
3. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hart-man.
1966. A proposal for a uniform nomenclature in bac-terial genetics.
Genetics 51:61-76.
4. Dubnau, D., C. Goldthwaite, I. Smith, and J. Marmur.1967.
Genetic mapping in Bacillus subtilis. J. Mol. Biol.27:163-188.
123VOL. 99, 1969
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/
-
124 GRANT A
5. Frankel, R. W., and T. M. Joys. 1966. Adsorption
specificityof bacteriophage PBSI. J. Bacteriol. 92:388-389.
6. Grant, G. F., and M. Simon. 1968. Use of radioactive
anti-bodies for characterizing antigens and application to thestudy
of flagella synthesis. J. Bacteriol. 95:81-86.
7. Joys, T. M. 1965. Correlation between susceptibility to
bac-teriophage PBSI and motility in Bacillus subtills. J.
Bac-teriol. 90:1575-1577.
8. Martinez, R. J., A. T. Ichiki, N. P. Lundh, and S. R.
Tronick.1968. A single amino acid substitution responsible
foraltered fagellar morphology. J. Mol. Biol. 34:559-564.
N[D SIMON J. BACTERIOL.
9. Massie, H. R., and B. H. Zimm. 1965. Molecular weight ofthe
DNA in the chromosomes of E. coil and B. subtilts.Proc. Nat. Acad.
Sci. U.S.A. 54:1636-1641.
10. Rogolsky, M., and R. Slepecky. 1968. The response of
sporo-genesis in B. subtilis to acriflavine. Can. J.
Microbiol.14:61-70.
11. Takahashi, I. 1963. Transducing phages for Bacillus
subtilis.J. Gen. Microbiol. 31:211-217.
12. Young, F. E. 1967. Requirement of glucosylated teichoicacid
for adsorption of phage in Bacillus subtilis 168. Proc.Nat. Acad.
Sci. U.S.A. 58:2377-2384.
on April 5, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
http://jb.asm.org/