Molec. Gen. Genetics 103, 105--115 (1968)
Genetic Analysis of lon Mutants of Strain K-12 of Escherichia coli * JOHN DONC~ a n d JosEP~ CxREENBERG** Palo Alto Medical Research Foundation, Palo Alto, California Received July 10, 1968 (and in revised form September 27, 1968) Summary. Following UV irradiation of ABl157 31 mucoid ultraviolet light UV sensitive mutants were isolated. These were all induced to form filaments by UV irradiation, i.e. they had all the phenotypic properties of Lon mutants. These lon mutants fell into two phenotypic classes based on their sensitivity to UV. The gene determining UV sensitivity and mucoidy in all mutants of both Class A and Class B was cotransducible with proC. Intra-class crosses by P1 transduction yielded no UV resistant recombinants. Inter-class crosses yielded UV resistant nonmucoid recombinants, the frequency depending on the direction of the cross. The data imply two adjacent blocks in the Ion region of E. coli and the order of markers in this region is probably proC tsx Ion Class A Ion purE Class B.
Introduction M u t a t i o n from L o n + (nonfilamentous after U V irradiation) to L o n (filamentous after U V irradiation) i n Escherichia coli K-12 is a c c o m p a n i e d b y overproduction of mucoid polysaccharide (HowARD-FLANDm~S, SIMSO~ a n d TttEI~IO~, 1964; MAI~KOVlTZa n d ROSEX]3AVM, 1965; DO~Clt a n d GI~V,EI~]~RG, 1968) a n d increased sensitivity to u l t r a v i o l e t (UV) irradiation. The high degree of association b e t w e e n L o n a n d m u c o i d y (Muc) makes i t possible to o b t a i n large n u m b e r s of U V sensitive m u t a n t s b y direct isolation of mucoid clones t h a t arise on m i n i m a l glucose m e d i u m i n c u b a t e d a t 37 ° C. I n this report we shall show t h a t L o n m u t a n t s fall i n t o two p h e n o t y p i c classes with respect to U V sensitivity whose genetic sites form two a d j a c e n t blocks both linked to proC. Materials and Methods Bacterial Strains. Strains used in these experiments are described in Table 1 and Fig. 1. Isolation o/ Mutants. Mutants were isolated from ABl157 as described by HOWAt~DFLA~)ERS, SIMSOI~and THERIOT(1964). Phage. The 1)1 vir mutant used in this laboratory has been previously described (DoI~cI~ and GREE~C~EI~G,1968). We shall refer to it as P1. Media. The minimal glucose-mineral salts medium (DE) has been described (DONeI~ and GRV.E~BEI~G, 1968). Complete (JN) broth consisted of tryptone 10 g, ~qaC1 5 g, yeast extract 5 g and glucose 1 g per liter of deionized water. Viable counts and filament formation studies were done on this medium without glucose, solidified with 1.5% Bacto Agar (Difco). Transductions, filament/ormation, U V survival curves and plating medium response ( P M R ) and host cell reactivation (HCR) were performed as previously described (GI~EENBEI~(~,1964, 1967; DONC~ and GREENBElCG,1968). UV treated bacteria were also plated on 0.75% pantoyl * This work was carried out under Public Health Service Grant CA 05687-08 from the lqational Cancer Institute. ** Recipient of a Public Health Service Career Development Award. 8
l~olec. Gen. Genetics 103
106
J.
D o ~ c ~ and J. GREENBERG:
Table 1. Characteristics o/ bacterial strains Strain
lacZ
pro
pure
tsx
str
Fil
Muc
Source
PAM 2011
~
-[-
+
+
--
+
+
Recombinant I4_frH × AB 1899
PAI~ 611 PAI~ 660 PAM 663 PAM 501
-----
-----
+ + +
-----
@ + +
+ @ -}-
+
-----
+
+
504
--
--
+
--
--
+
+
PAM 5 5 0 PAM 5 5 1 S77BM AB1899
PAM
--
--
+
--
--
+
÷
--
--
--
+
--
@
-
-
- -
+ + @
--
+
-
- -
+ + +
+ + -+-
A
-
-
-
B
I
I
5
7
W1895 a HfrH a Z478 W1485
-
-
-}-
.
-~-
-~
-+-
-~-
-}-
_
_
+
+
+
+
+
_ _
_ _
.
.
+
-~
.
.
-+-
.
.
.
+
.
.
@
.
--
--
UV irradiation of AB 1157
A. MARKOWTZ P. HOWARD-FLANDERS P. HOWARD-FLANDERS J.
LEDERBERG
J. LEDERBERG R. Cu~TIss I I I C. Y A N O F S K ¥
Abbreviations are those recommended b y DEMEREC, ADELBERG, CLARK and HART~AN (1966). Fil + means forms filaments following U V irradiation; Muc + means colonies produce large amounts of mucoid material at 37 ° C on minimal medium. For tsx and str + is sensitive is resistant. a Donor properties described in Fig. 1. - -
thr
leu
proA
proB
lacZ
proC
tsx
:
Class A
Class B
pure
W1895
: I-IfrH < . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fig. 1. Order of markers in the thr leu - - p u r e region on the E. coli chromosome. Class A and Class B lon m u t a n t s are placed between tsx and p u r e from data described in this paper. The origin and direction of donor strains W1895 and HfrI-I are shown b y arrow heads. The distances between markers are arbitrary lactone in J N agar (G~vLA and G~uLA, 1962). The results obtained by using this agent are identical to those obtained using D~¢[ agar. Recombination. Recombination experiments were performed b y mixing exponentially growing cells of donor and recipient in ratio of 1:10. The minority parent was always the donor. The mixture was allowed to form mating pairs for 5 minutes at 37 ° C, at which time pair formation was stopped b y gentle dilution of 1/100 into fresh J N broth containing 0.1% glucose prewarmed to 37 ° C. This dilution varied according to the fertility of the cross. I n t e r r u p t e d matings were performed b y removing 0.2 ml samples and diluting into cold 0.02 M phosphate buffered saline (PBS). Undiluted samples were blended for 1 minute on an eccentrically mounted rubber stopper as described b y RILEr and PAR~)EE (1962) in order to separate mating pairs. 0.1 ml blended samples were then spread onto selective medium. Direct Selection o/ U V Resistance. Direct selection of UV resistance was performed as described for transduetions (DoNcH and G~EENBE~G, 1968) except t h a t early ( 1 - - 3 × l0 s cells/ml) rather t h a n late log phase were used at an m.o.i, of 5 rather t h a n 0.1. After adsorption cells were centrifuged, resuspended in sterile water and appropriate dilations spread in 0.1 ml amounts onto supplemented DM glucose plates. Plates were then incubated for 3 0 - 4 5 minutes at 37°C t h e n irradiated with 616--1078 ergs/mm2/sec and rcincubated for 48 hours at 37 ° C.
Genetic Analysis of lon mutants of Strain K-12 of E. coli
107
Results and Discussion Classi/ication o/lon Mutants. Thirty-one mucoid, UV sensitive mutants were isolated from strain ABl157 following UV irradiation. The mutants fell into two classes, those placed into Class A (600 series) being less UV sensitive than those in Class B (500 series). Fig. 2 shows the fraction of survivors of seven representative mutants. The UV survival data of a derivative of AB1899 of HOWA~D-FLA~])v,RS, SI~SO~ and TH~IOT (1964), PAM 2011 is shown; it belongs
1,0 ~
>o
g <
Z
1
4
7
8
\
lr}-3L- II~'.-....~'-...~ ~ . . I:: I [ \ \
o
660
~2011
10-5l 0
I N5041550"~,5 0 1 % 551 I 154 308 462 616 770 924 1078 u V DOSE,ergs/mm2 Fig. 2. Survival of the UV irradiation of Class A ton mutants (600 series) and Class B ton mutants (500 series). Both classes were derived from ABl157. 2011, ABl157 and Z478 are included for comparison to Class A. Twenty-four of the UV sensitive isolates fell into Class A and seven into Class B and there was no equivocation as to which category any particular m u t a n t fit. Mutants of both classes were found to be HCR+ for UV irradiated phages T1 and T3 and became filamentous following low doses of UV irradiation. The effect of the nature of the plating medium (ROBERTS and AL])o~;s, 1949) following UV irradiation as well as of pantoyl laetone (G~m~A and GRV-LA, 1962) are shown in Fig. 3. The mutants in both classes show a reduction in sensitivity to UV irradiation when plated on minimal medium or complex medium conraining pantoyl lactone. Location o/Lon Mutations by Conjugation. The Itfr donor strain W1895 was used to determine the approximate location of the lon gene of a representative m u t a n t in each class. Time of entry on the lon+ allele was determined by selecting proA+ recombinants at two minute intervals and examining them for nonmucoid, UV resistant clones, t~rom the results of several experiments (Fig. 4) it was clear that the lon+ allele was transferred to Class A and Class B mutants no later 8*
108
J. DO~TO]~a n d J. GREENBERG:
1,0
1,0
O
a
10-I ,~
~. ~ 663 ~~""[~"---~ 660 ,. 611
~"
U3 nO
2011
¢n
10-1
2011
>_ >
¢D U_ O Z
10-2
501 551 550
o 10-3 I,-,
[
5s0
o,_lO-3
U-
10-51 0
I
I
\
1.1_ 10-4
\\\
lff 4
iL -- ._
I
I
I
I
\
\
._.
~ " " ~ . ~ 504
\
10-5 |
154 308 /,62 616 770 924 1078 UV DOSE, e r g s / m m 2
154 308 462 616 770 924 1078 UV DOSE, e r g s / m m 2
0
a
b
Fig. 3 a a n d b. Effect of plating medium on t h e survival of Class A a n d Class B lon m u t a n t s after U V irradiation, a Minimal medium, b P a n t o y l laetone. I n b o t h instances upper a n d lower dash lines represent t h e survival of typical Class A and Class B mutants, respectively, when plated on complex medium following U V irradiation
O3 LLI
100 -
X
X _
E3 z
~X
x
6 7 < 75
_X~ >-~
x
so
0 {...9 WZ
o25
I
n
0
I
f
I
I
5 10 15 20 30 TIME OF INTERRUPTION IN MINUTES
Fig. 4. Kinetics of transfer of the wild type allele (nonmucoid a n d U V resistant) from W1895 a n d either a Class A or Class B recipient. Selection was made for Pro + S t r
Genetic Analysis of Ion Mutants of Strain K-12 of E. coli
109
t h a n proA + itself. A b o u t 60 t o 80% of proA + r e c o m b i n a n t s were U V r e s i s t a n t and nonmucoid. Transduction o] Ion Genes. W e n e x t i n q u i r e d into w h e t h e r t h e lon genes were c o t r a n s d u c i b l e p a r t i c u l a r l y w i t h proC. To do this Lae+ Pro+ T s x b u t still L o n r e c o m b i n a n t s d e r i v e d f r o m r e c o m b i n a t i o n of H f r W1895 w i t h Ion m u t a n t strains were u s e d as P1 donors in t r a n s d u c t i o n s . These r e c o m b i n a n t s were used because t h e lon m u t a n t s h a v i n g been i s o l a t e d f r o m A B l 1 5 7 carried m a r k e r s which w o u l d n o t h a v e allowed L a c + to be used as a selected a n d Tsx as a n unselected m a r k e r . S t r a i n Z478 was u s e d as recipient because i t h a d a p p r o p r i a t e m a r k e r s : i t is proC, lacZ a n d purE, a n d h a d been used p r e v i o u s l y in m a p p i n g studies of t h e Ion gene of E. coli s t r a i n B. AB1899 was considered p h e n o t y p i c a l l y a lon m u t a n t of Class A. P A M 2011 is a d e r i v a t i v e of AB1899 [with t h e a p p r o p r i a t e m a r k e r s i n t r o d u c e d i n t o i t b y c o n j u g a t i o n (lae + tsx)]. D e t a i l s of e x p e r i m e n t s in which P A M 2011 was used as d o n o r for t r a n s d u c i n g proC + i n t o Z478 are given in T a b l e 2. The t r a n s d u c t a n t s were e x a m i n e d for t h e unselected donor m a r k e r s Tsx, U V sensitivity, a n d m u c o i d y . M u c o i d y a n d U V s e n s i t i v i t y always occurred together. T h e y were n o t t r a n s f e r r e d w i t h L a c + a n d o n l y r a r e l y w i t h PurE+. T h e y were t r a n s f e r r e d w i t h ProC +, t h e f r e q u e n c y of c o t r a n s d u c t i o n being given outside t h e p a r e n t h e s i s for m u c o i d colonies d e t e c t e d on t h e p r i m a r y selection plate. To this figure were a d d e d (inside t h e parentheses) t h e n u m b e r of n o n m u c o i d clones which segregated m u c o i d clones u p o n p u r i f i c a t i o n on selective m e d i u m . A p p r o x i m a t e l y 10% of t h e n o n m u c o i d s s e g r e g a t e d mucoids. This is i n t e r p r e t e d to m e a n t h a t t h e P1 p a r t i c l e c a r r y i n g t h e lon m a r k e r was able to persist as a n u n i n t e g r a t e d p a r t i c l e for four to five d a y s . F u r t h e r m o r e , i t is clear t h a t U V s e n s i t i v i t y - m u c o i d y is recessive to U V resistancen o n m u c o i d y . T h e f r e q u e n c y of c o t r a n s d u c t i o n of m u c o i d y w i t h proC + was 29 %. All m u c o i d s were i n d u c e d b y U V to f o r m filaments.
I t should be n o t e d t h a t all L a c + t r a n s d u c t a a t s were Pro+ b u t n o t all Pro+ were L a c +, a n a n o m a l y which was r e p o r t e d p r e v i o u s l y w i t h Z478 ( D o ~ c ~ a n d Table 2. Linkage relationship among lacZ+ proC+ purE+ and UV sensitivity and mucoidy a Selected marker
Number observed
lacZ+ nonmucoid
2031
proC+ nonmucoid mucoid purE+ nonmucoid mucoid
2981
Number tested
400 300 200 2762 300 20
Frequency (%) of unselected donor markers b lacZ+ proC+ tsx +
100 100 23 23 0 0 0 0
100 100 100 100 100 0 0 0
18 18 66 37 97 0 0 0
UV e
mucoid
0 0 0 0 21 (29) 21 (29) 0 0 100 100 0.1 0.1 0 0 0.1 0.1
purE+
0 0 0 0 0 100 100 100
a P1 donor was PA!VI2011 and the recipient was Z478. b Data is an average of four experiments. e UV means sensitive to ultraviolet irradiation. Data in parentheses represent the corrected value for segregation of hcterogenotes after purification of nonmucoid transduetants.
110
J. Do~c~ and J. GREE~ERG:
G~EENBWRG, 1968). F u r t h e r m o r e , T s x was t r a n s d u c e d w i t h L a c + a t a f r e q u e n c y of 18%, n o t a t all w i t h p u r E + a n d w i t h L o n a t a f r e q u e n c y of 97%. E x c e p t for t h e u n e x p l a i n e d c o t r a n s d u c t i o n of Proc+ w i t h Lae+ t h e order of m a r k e r s w o u l d a p p e a r t o be lac proC tsx Ion purE. This was t h e o r d e r of m a r k e r s e s t a b l i s h e d for t r a n s d u c t i o n of t h e lon m a r k e r of s t r a i n B ( D o ~ c ~ a n d G~E~NBW~G, 1968) i n t o Z478, t h e difference being t h a t t h e lon m a r k e r of B was c o t r a n s d u c e d w i t h proC + a t a f r e q u e n c y of o n l y 7 %. T h e p r o c e d u r e used w i t h PAY[ 2011 was u s e d w i t h Class A a n d Class B U V sensitive m u t a n t s . The results of these t r a n s d u c t i o n s are given in T a b l e 3. T h e f r e q u e n c y of c o t r a n s d u c t i o n of Class A m u t a n t s i n c l u d i n g P A M 2011 w i t h proC + v a r i e d f r o m 24 t o 2 9 % . Those in Class B v a r i e d f r o m 12 to 17%. Table 3. Transduction o/ UV sensitivity and mucoidy from P1 grown on UV sensitive mucoid mutants to )~478 P1 donors
Number observed
Class
Frequency of UV s mucoid among proC+ lacZ+ and pure + ~ransduetants proC+
lacZ+ pure +
PAM 611 PAM 660 PAM 663 PAM 2011
1089 1231 1074 2981
A A A A
17 19 12 21
(24)a (26) (24) (29)
0 0 0 0
0.1 0.1 0.1 0.1
PAM 501 P A ~ 504 PAM 550 PAM 551
1437 1332 1030 1253
B B B B
11 9 14 8
(12) (13) (17) (16)
0 0 0 0
0.1 0.1 0.1 0.2
a The value within the brackets represents the corrected value obtained by adding to the observed mucoid clones on the primary selection the per cent mucoids obtained by purifying 200 nonmucoids on the medium used for primary transductions. Thus a percentage of the nonmucoid clones on the original selection plates were heterogenotes. All mucoid transduetants were fotmd to be UV sensitive. The values shown represent an average of 5 experiments. I n Fig. 5 are shown s u r v i v a l curves of two t r a n s d u c t a n t s of )/478 i n t o which the Ion m a r k e r of a Class A or a Class B m u t a n t was t r a n s d u c e d . I n b o t h cases t h e r e was a n 8 to 10-fold decrease in s e n s i t i v i t y to U V i r r a d i a t i o n c o m p a r e d t o t h a t of t h e d o n o r of t h e lon gene. T h i s was earlier o b s e r v e d w h e n t r a n s d u c t i o n s were p e r f o r m e d using E. coli B a s a d o n o r a n d Z478 as a r e c i p i e n t (DoNcH a n d G ~ E ~ B ~ R G , 1968). This decrease in U V s e n s i t i v i t y of g478 t r a n s d u c t a n t s is n o t clearly u n d e r s t o o d . I t m a y be t h a t t h e genetic b a c k g r o u n d of Z478, w h i c h has a n e x t r e m e l y high intrinsic resistance to U V i r r a d i a t i o n , p r o d u c e s a s u b t r a c t i v e effect on t h e U V s e n s i t i v i t y of t h e L o n p h e n o t y p e . Direct Selection o/ U V Resistance. P r e v i o u s workers ( H o w ~ D - F L A ~ D ] ~ S , SIMSO~ a n d THERIOT, 1964) h a v e shown t h a t i t was possible t o select d i r e c t l y for U V resistance in c o n j u g a t i o n e x p e r i m e n t s . I t E ~ T M ~ a n d L y d I A (1967) a n d CLARK (1967) h a v e shown t h a t d i r e c t selection for U V resistance was also possible in t r a n s d u c t i o n s . I t occurred t o us t h a t this t e c h n i q u e could p o s s i b l y be a p p l i e d to our m u c o i d strains. I n e x p e r i m e n t s w i t h t h e lon+ s t r a i n W1485 as d o n o r
Genetic Analysis of lon Mutants of Strain K-12 of E. coli
111
i t was f o u n d t h a t t h e a d d i t i o n of P1.W1485 t o either Class A or Class B lon recipients increased t h e n u m b e r of s u r v i v o r s af t er U V i r r a d i a t i o n r e l a t i v e to recipients in selling controls in which recipients were i n f e c t e d w i t h P1 g r o w n on themselves.
~
1,0
8
"-, 0
15/* 308 /*62 616 770 92/, 1078 UV DOSE, ergs/mm 2
Fig. 5. Survival after UV irradiation of E. coli •478, a typical Class J lon mutant upper dash line and its transductant 657, and a typical Class B lon mutant lower dash line and its transductant 533. Both transductants obtained using X478 as a recipient Table 4. Direct selection of U V resistance P1 donor
Recipient Class A
A) W1485
Phenotype a Class B
PAM 2011 P A M 504
B) PAM 2011
PAM 611 PAM 504
PAM 504
PAM 2011 PAM 550
C) PAM 2 0 1 1 PAM 611 PAM 504 PAM 550
PAM 2011 PAM 611 PAM 504 PAM 550
nonmucoid mueoid 289 384
9 15
0 0 0 0
18 15 11 17
0 0
17 16 23 11
0
0
a As expressed on supplemented DM glucose medium after UV irradiation and incubation at 37 ° C for 48 hours. D e f i n i t i v e e x p e r i m e n t s were done in which P l . W 1 4 8 5 was a d d e d to recipients in Class A a n d Class B. T a b le 4 p a r t A shows t h e results of such e x p e r i m e n t s . T h e baselines for s u r v i v a l of re'eipient strains are shown in p a r t C, selling controls,
112
J. Do~c~ and J. GREElgBERG:
in which no nonmucoid, and only a few mucoid clones were found. These were as sensitive to UV as the recipient strains. We considered these to be the rare survivors of UV treatment of the recipient strains. A comparable number of mucoid clones were found on the plates in which P1.W1485 was used to infect lon mutants of Class A and B. From the experimental plates 200 nonmueoid survivors were restreaked on minimal glucose medium and incubated at 37 ° C. Between 80 and 90% of these nonmucoid clones segregated mucoid colonies. Approximately 10--20% of the nonmueoid clones did not segregate mucoid colonies, indicating that they were reeombinants at the lea+ gene. Further purification of nonmucoid clones (from the original 80--90%) revealed that 10--15% continued to segregate mucoid colonies. There were two features of the direct selection for UV resistance that are striking: first, there were nonmueoid survivors, indicating the inheritance of the donor property, second, most of these nonmucoid survivors behaved as persistent heterogenotes, since a small number of colonies continued to segregate mucoid colonies even after two purifications. I t had already been observed in experiments summarized in Table 3 that persistent heterogenotes for mueoidy occurred in the cotransduction with ProC + of Ion from Class A and B mutants to Z478. The increase in the heterogenotic state following selection for UV resistance (from about 10% in Table 4 to 90%) was not unexpected in view of the observations that UV irradiation increases synapses and subsequent recombination (JAcoB and WOLLMA~, 1955, 1961 ; CU~TlSS, 1968). On the other hand, persistent heterogenotes m a y result from the effect of UV on the Pt particle or to the fact that virulent mutants of P1 stop host DNA synthesis (IKEDAand TOMIZAWA, 1965) or to a combination of these factors. Complementation with Pl Phage. The results of the direct selection of UV resistance afforded us an opportunity to test for complementation between mutants of each class and thus demonstrate the existance of two cistrons. P1 grown on mutants in each class was added to recipients in each class. Table 4 part B shows the results using Class A and B as donors and Class A and Class B as recipients. I t is evident that no complementation occurred when the results are compared to both part A in which a ~zvr+ donor was used and part C which describes the results of selfing experiments. These results indicate that Class A and Class B mutants are probably mutations in the same cistron although similar results would be obtained if the Class B mutants were polarity mutations. While these experiments gave negative results, further experiments are in progress using the episome F'13 labelled with lea mutants of each lea class. Intraclass Recombinations. As can be seen from Table 2 the Ion marker can be transduced with 1pure at low frequencies (0.1 to 0.2%). This fact made it possible to conserve the 19roC marker in transducing lon to Z478, a necessary step for the experiments to be described in this and the next section. Transduetion by P1 among members within each class was first attempted, selection being made for proC+. All combinations within each class were tested. I n no instance were typically nonmucoid transductants obtained. On occasion,
Genetic Analysis of Ion Mutants of Strain K-12 of E. coli
113
however, small a p p a r e n t l y n o n m u c o i d clones a p p e a r e d , b u t when purified p r o d u c e d all m u e o i d clones. Interelass Recombinations. R e c o m b i n a t i o n s were also p e r f o r m e d b e t w e e n classes of Ion m u t a n t s (Table 5). N o n m u c o i d t r a n s d u c t a n t s were observed. E a c h a p p a r e n t n o n m u c o i d t r a n s d u c t a n t was p i c k e d a n d purified twice on p r i m a r y selection m e d i u m . Those which p e r i s t e d as n o n m u c o i d s u n d e r conditions (37 ° C on m i n i m a l agar) where m u e o i d could be expressed were t e s t e d for addit i o n a l lon+ c r i t e r i a : 1) a b i l i t y t o t r a n s d u c e proC lon (mucoid) to proC + lon+ (nonmueoid). 2) I n a b i l i t y to t r a n s d u c e a proC lon+ (nonmucoid) to proC +lon (mueoid). 3) U V resistance. O n l y those m u t a n t s which m e t all these criteria are shown in T a b l e 5. Table 5. Interclass recombinations o/Lon mutants Phenotypic class
Recipients (ProC+ Lon+ × 104) 501
504 550
551 611
611 660 663
A A A
5 2 4
6 5 4
5 5 3
4 5 6
501 504 550 551
B B B B
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
P1 donors
660
663
0 0 0
0 0 0
0 0 0
77 86 73 79
87 70 70 84
85 67 79 81
Selection was made for ProC + transductants. Recipients were prepared by transducing the
Ion marker to Z 478 and selecting for PurE+ (Table 2) thereby conserving the proC locus. Each value is the total of two to three experiments in which a total of 1,900---4,700 recombinants were examined for Lon+ on the primary selection plate. Lon+ were further tested for adherence to the Lon+ criteria.
Ordering o/ Class A and Class B Mutants at proC. F r o m t h e c o t r a n s d u e t i o n frequencies s h o w n in T a b l e 2 Class A m u t a n t s were m o r e closely l i n k e d t o proC t h a n were Class B. I n addition, t h e response to U V r a d i a t i o n (Fig. 2) is characteristic of a given class. F u r t h e r m o r e , intraclass r e c o m b i n a t i o n s l e a d i n g to L o n + were n o t o b s e r v e d w i t h i n t h e resolving p o w e r of our system. I n t e r c l a s s r e c o m b i n a tions leading to L o n + s u p p o r t t h e i d e a of t w o cistrons even t h o u g h complemenr a t i o n t e s t s were negative. The l a c k of r e c i p r o c i t y shown in T a b l e 4 can be explained, b y t h e p o s i t i o n of m u t a n t s w i t h r e s p e c t to proC s h o w n b y T a b l e 2. W h e n Class A m u t a n t s were used as donors a n d Class B m u t a n t s as recipients a n a d d i t i o n a l crossover w o u l d be necessary (see Fig. 6a) to p r o d u c e a Lon+ r e c o m b i n a n t a n d such a n e v e n t w o u l d occur a t low frequency. This was n o t t r u e w h e n Class B m u t a n t s were used as donors a n d Class A m u t a n t s as r e c i p i e n t s (Fig. 6 b). Fig. 1 r e p r e s e n t s t h e genetic m a p of t h e thr fen p u r e region of t h e E. coli K-12 c h r o m o s o m e b a s e d u p o n this i n t e r p r e t a t i o n of t h e results o b t a i n e d . T h e lon m u t a n t s are b o u n d e d b y proC a n d purE. T h e tsx m a r k e r is to t h e left of t h e Ion m u t a n t s . This is b a s e d u p o n t h e r e s u l t o b t a i n e d with E. coli B ( D o ~ c H a n d G~EE~B]~C, 1968) a n d those shown in T a b l e 3.
114
J. Do~c~ and J. GREEI~BERG: Crossover region
Crossover region
A a Donor Class A lon
B
+
Class A
+
Recipient Class B Ion
b Donor Class B lon
proC
~-
Class B
+
+
Class B
Recipient Class A lon
i proC
Class A
+
Fig. 6 a and b. Crossover regions in the proC -- Class B segment on the E. coli chromosome M~RKOVITZ and his collaborators have isolated several mucoid m u t a n t s which has been m a p p e d a t proC (MARKOVITZ, 1964 ; MARKOVITZ and ROSV,NBAUM, 1965 ; MARKOVITZ and B~_KER, 1967). They have produced a linkage m a p of m u t a n t s which indicates t h a t the most resistant of the m u t a n t s (capR6) is most closely linked to proC while two more sensitive m u t a n t s capR66, capR9 are arranged in a linear order to the right of capR6. The capR6 m u t a t i o n m a y represent a third phenotypic class in t h a t it was found to be more resistant t h a n capR66 and capR9 (MARKOVITZ a n d BAKER, 1967) and more UV resistant t h a n either our Class A or Class B mutants, capR66 a n d capR9 would appear to belong to our Class A m u t a n t s except t h a t t h e y recombine with each other to produce nonmucoid recombinants. These m u t a n t s m a y belong to neither of our classes because of the unusual p r o p e r t y of exhibiting dominance for mucoidy when located on an episome a n d recessiveness when on the chromosome. This was not found true for capR6 which is always recessive. I t should be emphasized t h a t capR9 a n d capR66 were selected from among nonmucoid F'13 bacteria and m a y represent a class different from either of our classes of mucoids. Class B as well as Class A m u t a n t s are being studied with regard to the reversions to UV resistance following t r e a t m e n t with UV and 1-methyl-3-nitro-1-nitroso guanidine (MA~DELL a n d GR]~ENB]~BG, 1960). Theoretically four classes of m u t a n t s are possible: 1) reversion of lon locus to lon+; 2) mutations a t one or more loci responsible for mucoid synthesis (ADLER and HXRDIGREE, 1965; DONC~ and GREENBERG, in preparation); 3) m u t a t i o n s at a locus controlling radiation resistance allelic to the tad m u t a t i o n in B/r; 4) m u t a t i o n at as y e t unidentified loci controlling radiation sensitivity and mucoid production. A t least one such class of this t y p e of m u t a t i o n is predicted on the basis of the reversions obtained b y MARKOVITZ and BAKER (1967) which were found to be ochre suppressors.
Acknowledgements. We are grateful for the excellent technical assistance of Do~oTEY WILLIAMS and )/Irs. JANn~ DOVGDALE-BRADLEY, and for the volunteered assistance of Mrs. JAN NE~L.
Genetic Analysis of Ion Mutants of Strain K-12 of E. coli
115
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