Molec. gen. Genet. 131,159--171 (1974) © by Springer-Verlag 1974

Genetic Studies of Tolerance to Colicin E2 in Escherichia coli K-12 I I . Multiple Mutations as a Cause of the Various P h e n o t y p i c Properties of Cet- Mutants R. S. Buxton and I. B. Holland Department of Genetics, University of Leicester, Leicester, England Received January 15, 1974 Summary. The genetic nature of some of the phenotypic properties of Cet- mutants, which were selected as being tolerant to colicin E2, has been examined. In contrast to other reports, tolerance to colicin E2, which derives from a mutation at the cet locus, was found to segregate from the other phenotypie properties of Cet- mutants in genetic crosses. Thus, in two Cet- strains, sensitivity to ultraviolet (UV) light derived from a lou mutation; in another, UV-sensitivity mapped near the recA locus; in a fourth mutant, resistance to phage T4 was shown to map at a position distinct from cet and loosely linked to his and tyrA. An investigation of one of the properties of certain Cet- strains, viz., resistance to phage 2, revealed it to be due to poor adsorption of the phage.

Introduction We have recently reported (Buxton and Holland, 1973) the genetic location of the cet gene, mutation of which renders Escherichia coli tolerant to the protein antibiotic colicin E2, whereas treatment of wild-type cells with colicin E2 causes DNA degradation and the inhibition of cell division. The cet gene was found to lie between s e r B and thr. I t had previously been established that Cet- mutants could be grouped into two phenotypic classes, viz., CetB- which were E2 tolerant and CetC- which besides being E2 tolerant were also sensitive to ultra-violet (UV) light (Holland, 1967). I n addition, CetC- strains showed different combinations of several other phenotypic properties (Holland et al., 1970). These properties included reduced growth rates, detergent sensitivity, recombination deficiency, poor growth of phage ~ and filament formation. That all these properties were pleiotropic, i.e., the result of a single mutation, at the cet locus, was deduced from two pieces of evidence: (1) all the mutant properties were found to be 100% cotransducible with E2-tolerance, and (2) UVresistant revertants of two CetC- strains were found to have also reverted to E2-scnsitivity (Threlfall and Holland, 1970). I n consequence this appeared to be a profitable system for further more extensive genetic analysis, since the pleiotropic properties of CetC- mutants suggested that the cet + gene was concerned with both DNA metabolism and cell surface formation. I n the course of the current study, however, it soon became apparent that derivatives of CctC- strains could be isolated in which some of the mutant properties had reverted completely or had changed quantitatively, whilst other characters had not been altered. Thus when the strains were kept in stab cultures at room

160

R.S. Buxton and I. B. Holland

t e m p e r a t u r e for several years, faster growing derivatives of the m u t a n t s could be f o u n d which, although still E2-tolerant, were no longer d e t e r g e n t sensitive a n d only m a x i m a l l y expressed the UV-sensitive p h e n o t y p e at low t e m p e r a t u r e . I n addition, a s p o n t a n e o u s E2-scnsitive r e v e r t a n t of a CetC- s t r a i n (ASH 112) still r e t a i n e d its UV-sensitivity. Moreover, S a m s o n a n d H o l l a n d (1970) reported t h a t all U V - r e s i s t a n t r e v e r t a n t s of two CetC- strains (ASH 111 a n d A S H 114) rem a i n e d E2-tolerant, a n d a n E2-sensitive r e v e r t a n t of one s t r a i n (ASH 114) was still UV-sensitive. These findings were i n c o n s i s t e n t with the reversion analysis of Threlfall a n d H o l l a n d (1970) (see above). T h e present observations on a n E2sensitive r e v e r t a n t , a n d the observations of S a m s o n a n d H o l l a n d could of course be explained b y m u t a t i o n s at other loci, only suppressing the UV-sensitive phenot y p e for example. This could involve interactions b e t w e e n different proteins i n the cell envelope, as proposed b y S a m s o n (1970). A n a l t e r n a t i v e a n d simpler hypothesis is t h a t CetC- strains carry m u l t i p l e m u t a t i o n s . Since this was n o t compatible with the observations of Threlfall a n d Holland, a n e x p e r i m e n t was performed to d e t e r m i n e if the UV-sensitive a n d E 2 - t o l e r a n t p h e n o t y p e s of A S H 114 segregated out a m o n g s t r e c o m b i n a n t s from a n H f r X F - cross. A l t h o u g h only a small n u m b e r of r e c o m b i n a n t s (30) were tested, the results clearly d e m o n s t r a t e d t h a t these characters did i n fact segregate, a n d a similar segregation was also o b t a i n e d with A S H 111 (data n o t shown). These results indicated the subject to be w o r t h y of closer scrutiny.

Materials and Methods Bacterial Strains. Some of the E. coli K-12 strains used are shown in Table 1. Other strains were listed previously (Buxton and Holland, 1973). ASH 1 is an Hfr strain which apparently has two origins of transfer, one similar to Hfr C and one similar to Itfr B l l (Buxton, 1973). A comparable situation has been reported for another strain by Low (1967), who suggested that it was due to the presence of two sex factor affinity sites on one chromosome. Bacteriophage Strains. Phages ~, 2gv and T4 were from laboratory stocks. Phage 2ci857 was provided by Dr. B. M. Wilkins. Media were mostly as described previously (Buxton and Holland, 1973). ,~ tryptone broth was Oxoid tryptone broth plus 10-2 M MgSOt and 0.5% maltose; 2 tryptone agar was ,~ tryptone broth solidified with 1% Oxoid No. 1 agar, and ~ soft agar was solidified with 0.7% Oxoid No. 1 agar. Mating Conditions and P1 Traneduction Techniques were as described previously (Buxton and Holland, 1973). Spot Test Response ol Bacterial Strains to Ultraviolet (UV) Irradiation. A loopful of an overnight culture, or a cell suspension in buffer, was streaked onto a nutrient broth plate; different portions of the streak were then irradiated for 0, 5, 10, 15, 20, 30, 40 and 60 seconds with UV at a dose rate of 8.5 ergs/mm2/sec from a low pressure mercury lamp without filter (Hanovia Limited, Slough, Bucks.) at room temperature. Incident dose rates were measured by using a dosimeter constructed by R. Latarjet. Plates were incubated at the desired temperature in the dark, to prevent photo-reactivation. This procedure was used to test recombinants. Quantitative Response oI Bacteria to U V. Overnight cultures of cells grown in nutrient broth at 37° C without aeration were washed and resuspended in phosphate buffer at an A~so of 0.1. This suspension, as a thin layer in a petri dish, was irradiated with UV at room temperature, plated out on tryptone broth plates, and incubated overnight at 37° C. Microscopic Examination oI Bacteria was carried out on cell suspensions using a Wild phase contrast microscope (Wild Heerbrugg Limited, Heerbrugg, Switzerland). To measure

Colicin E2 Tolerance

161

Table 1. Genotype and origin of bacterial strains Strain

Mating type Genotype

Origin/Reference

RB29

HfrH

serB thi (2-) sup- cetl T41~ 2R

M u t a n t (2-aminopurine) of 14H

RB288

HfrH

serB + thi (2-) s u p - thyA drm cet3

RB28 by P1. RB268

RB290

HfrH

serB+ thi (2-) s u p - thyA dra real 2 R cet111

RB286 b y P1. RB243

RB292

Hh'H

serB + thi (4-) s u p - thyA dra + cet114

RB284 by P1. A S H 114

RB239

Hfr AB257

metB (2-) s u p - tax

T6 R m u t a n t (spontaneous) of AB257 (2-)

ASH 1

Hfr ASH 1

thyA metB str (2-) s u p -

See B u x t o n and Holland (1973)

ASH 111

HfrASH 1

As A S H 1, cet111 lon111

M u t a n t (2-aminopurine) of A S H 1/Holland (1967)

A S H 114

Hfr A S H 1

As A S H 1, cet114 U V s

M u t a n t (2-aminopurine) of ASH 1/Holland (1967)

RB49

F-

thr leu thi trp his urg ( E C B H ) lacY gal m a l t xyl mtl non + str supE + tsx

T6 R m u t a n t (spontaneous) of PA309/see B a c h m a n n (1972)

RB201

F-

thr leu trp his + urg lacY gal m a l T str met + non

A S H 111 x RB49

RB211

F-

thr leu arg str non 1on111

ASH 111 × RB201

A S H 112

F-

leu metB thyA lacZ str (2+) supE+ cet112 Ion 112 trp

Cet- m u t a n t (2.aminopurine) of A S H 10/Holland et al. (1970)

RB224

F-

As ASH 112, cet+

Cot + r e v e r t a n t (Spontaneous) of ASH 112

ASH l l 6

F-

As ASH 112, cet116 lon116 trp +

Cet- m u t a n t (2-aminopurine) of A S H 10/Holland et al. (1970)

ABl157

F-

PB314

F-

thr leu thi proA his argE str supE + (2-) lac thi purE tsx str non

B. M. Wilkins/sce B a c h m a n n (1972) P . A. Meacock

RB233

F-

As PB314, purE + lon111

A S H 111 × PB314

RB243

F-

As PB314, p u r e + cet111

A S H l l l × PB314

SA130

F-

his argF tyrA str thi

S. I. A h m a d

MC102

F-

leu purE trp laeZ gal str lon9

A. Markovitz/Markovitz a n d Baker (1967)

AT2092

F-

his thi purC pheA str

P. A. Meacock

RB272

F-

his thi purC+ pheA UVs

ASH

i14 × AT2092

cell filament lengths, bacteria were embedded in minimal agar (0.7 % ) a n d photographed using t h e Wild microscope with a Nikon microflex model AFM a t t a c h m e n t a n d a Nikon 35 m m camera.

Phage Adsorption Measurements were carried out using chloroform to inactivate the infected bacteria, essentially as described in Adams (1959).

162

R.S. Buxton and I. B. Holland Results 1. Identi]ication o/ the Site o / a Mutation Con/erring UV-ser~sitivity on the Cet- Strain A S H 111

When the H f r CetC- strain A S H 111 was crossed with the multi-auxotrophic F - recipient RB49 and selection made for various prototrophic recombinants, it was noticed t h a t all classes of recombinants, except for the His + class, contained a large proportion of mucoid colonies, although neither of the parents themselves were mucoid. I n a similar cross of RB49 and A S H 1 (the Cet+ parent of A S H 111), no mucoid rccombinants were obtained. These results were compatible with the simple assumption t h a t the donor strain, A S H 111, carried a mutation which rendered colonies mucoid, but t h a t this mucoidy was suppressed b y another mutation linked to his. Thus mating of A S H 111 with an F - strain leads to the segregation of these two markers so that mucoidy is expressed. A mutation closely linked to his, termed non, capable of suppressing the mucoidy of Lon- strains (Howard-Flanders, Simson and Theriot, 1964; Adler and Hardigree, 1964), without affecting the accompanying UV-sensitivity of such strains, has already been described (Adler and Hardigree, quoted by Donch and Greenberg, 1970; Radke and Siegel, 1971). By analogy it appeared probable t h a t ASH 111 was genetically non-. Similarly since A S H 111 was m u t a n t at a locus governing mucoidy, and was also UV-sensitive, this study implied that this strain was lon-. Since working with mucoid colonies tends to be difficult, an F - h i s + recombinant (RB201) from a cross between A S H 111 and the multi-auxotrophic F - strain RB49 was used as recipient in subsequent experiments. This was apparently nonbut UV-resistant, and had retained the other auxotrophic markers of RB49. From a cross of A S H 111 with RB201, a Trp+ Met+ F - recombinant (RB211) which was UV-sensitive but E2-sensitive was retained. Using this as recipient, UV-resistant recombinants were selected in an interrupted mating experiment with an H f r H strain. The results of this experiment (data not shown) suggested t h a t the time of entry of the gene conferring UV-resistance takes place at about 15-20 minutes, i.e., between positions 11 and 16 on the standard map (Taylor and Trotter, 1972). A four-point cross was carried out to determine the relative order of the gene controlling UV-sensitivity and the lac, tsx and p u r e genes {Table 2). The data show t h a t the site of the mutation giving rise to UV-sensitivity, unlike the cet locus, is linked to the lac and tsx loci. From the least frequent recombinant classes, the order deduced is: l a e - - t s x - - ( U V ) - - p u r E . All the UV-sensitive recombinants were also Fil+ (i.e., formed filaments when grown on NB agar). The site of this mutation therefore corresponds with previous reports of the position of the lon gene (Itoward-Flanders, Simson and Theriot, 1964; Adler and Hardigree, 1964; Donch and Greenberg, 1968). I n addition to the genetic evidence described above, the phenotypie properties of ASH 111 were also consistent with the idea t h a t this strain was m u t a n t at the Ion locus. These were: (1) recombinants carrying the mutation giving rise to UV-sensitivity in A S H 111 formed mucoid colonies in the presence of the non + allele; (2) recombinants inheriting the (supposed) lon- allele showed approximately the same degree of sensitivity to UV as a known Lonstrain (MC102, see Fig. 1) ; (3) the recombinant strain t~B233 (cet+ lon 111), when

163

Colicin E2 Tolerance Table 2. Linkage between cet111, lac, tsx, p u r e and the mutation giving rise to UV-sensitivity in ASH 111 Phenotypes of recombinants

Lae+ TsxR Lae+ Tsx~ Lac + Tsxs Lac+ TsxS Lac- TsxR Lac- Tsx1~ Lae- Tsxs Lac- TsxS Total CetCet+ CetCet+ CetCet+ CetCet+ UVs UVR

0 0

0 1

7 0

23 3

1 2

5 64

0 0

11 0

Analysis Selected marker

Unselected markers

1.

(UV+)

(UV-)

lac+ 4 lac- 66

30 17

(UV+)

(UV-)

tsx- 67 tsx + 3

6 41

pure +

Indicated order lac+ (UV-) --t I --i

2.

purE +

t

I

tsx + (UV-) --I --I

pure +

I

purE +

I

--

+I

I -

-

I

Strains ASH 111 (Hfr ASH 1 cet111 thyA metB str non UVs Fil+) and PB314 (F-lac p u r e str tsx non ?) were mated for 1 hour at 37° C. Selection was made for Pur + [Met+ Thy+] reeombinants, which were purified by single colony isolation and tested for unselected markers. All UVs recombinants were Fil+ whilst all UV1~ recombinants were Fil-.

grown on N B agar, formed long filaments a b o u t 50 ~ m long as well as cells of n o r m a l length, a b o u t 2-3 t~m. Such filaments were n o t present a m o n g cells of the p a r e n t s t r a i n PB314 (cet + lon+). F u r t h e r m o r e , after liquid N B grown cultures of RB233 were exposed to 170ergs/mm ~ of U V i r r a d i a t i o n followed b y overnight i n c u b a t i o n a t 20 ° C, filaments were m e a s u r e d to be u p to 180 ~ m long. After a similar t r e a t m e n t all the ceils of the wildtype strain PB314 were 2-3 t~m long. This p r o p e r t y of f i l a m e n t f o r m a t i o n is a well established characteristic of L o n strains (see for e.g., H o w a r d - F l a n d e r s , Simson a n d Theriot, 1964); (4) A S H 111 showed a n increased survival after UV-irradiation if i n c u b a t e d i n the presence of D L p a n t o y l lactone (J. M. Boyle, personal communication), a n effect which is characteristic of L o n - strains (van de P u t t e , W e s t e n b r o e k a n d R6rsch, 1963). I n view of the above genetic a n d physiological evidence, it is concluded t h a t s t r a i n A S H 111 e x a m i n e d i n this s t u d y is UV-sensitive because of a m u t a t i o n in the lon gene. No evidence could be f o u n d t h a t the cet m u t a t i o n itself c o n t r i b u t e d to the U V - s e n s i t i v i t y of the m u t a n t , since r e c o m b i n a n t s carrying cet111 were no more UV-sensitive t h a n the p a r e n t carrying eet+ (Fig. 1). I n addition, Ser+ transd u c t a n t s (see B u x t o n a n d Holland, 1973) i n h e r i t i n g cet111 were no more UVsensitive t h a n those i n h e r i t i n g cet3 (from a C e t B - m u t a n t ) or cet114 (from a n o t h e r CetC- m u t a n t ) . I t cannot, of course, be ruled out t h a t A S H 111 contains more m u t a t i o n s besides cet a n d Ion, a n d i n fact some evidence for this has also b e e n o b t a i n e d

117

164

R . S . Buxton and

I.B. Holland

100 1

•

~

f

4

i! ~ ~

10

RB2~3(cet111)

.c_

(33 O tea

(lon9)

MC102

01

(cefflllon111)

ASH111

u 13-

IO0

m

'.. ~ ~ R

B 28B(cet3)

29~((:et114)~~.

: RB

o

10 0

I

100

I

200

I

I

300 100 UV dose in ergs per mm2

200 300 I

I

Fig. 1. UV-survival curves of ASH 111 and recombinants derived from it. The experiments were carried out as described in Methods. Survival (colony-forming units) is expressed as a percentage of an unirradiated control. ASH 111 is a Cet- mutant of ASH 1. RB243 and RB233 are Put+ [Met + Thy +] recombinants from a cross of ASH 111 (Hfr ASH 1 cet111lon111thyA metB) and PB314 (F-purE). RB290, RB288 and RB292 are Ser+ transductants of strains RB286, RB28 and RB284 respectively (all derivatives of strain 14H), carrying the mutant cet- alleles as shown. MC102 is a known Lon- strain (Markovitz and Baker, 1967). All strains are cet+and lon+except where indicated

(Buxton, 1973), b u t t h e m a j o r p a r t of t h e U V - s e n s i t i v i t y seems t o derive f r o m the

lon m u t a t i o n . 2. Identi]ication o] the Site o/the Mutation Con/erring UV-sensitivity on Strain ASH 112

A S H 112 was a n o t h e r C e t - U V s strain originally described b y H o l l a n d et al. (1970). I n t h e current study, this strain was f o u n d t o h a v e r e v e r t e d to Cet+ b u t was nevertheless still U V s (strain RB224). I t is certain t h a t strain R B 2 2 4 was d e r i v e d f r o m A S H 112 since like A S H 112, b u t unlike t h e p a r e n t A S H 1, it re-

Colicin E2 Tolerance

165

Table 3. The location of the site of the mutation conferring UV-sensitivity on strain RB224 Phenotypes of recombinants

UV s UV K

Leu+ Tsx s

Leu+ Tsx R

Leu- TsxS

Leu- Tsx 1~

2 1

0 24

10 l

1 11

Analysis Selected marker

lac+

Unselected markers Indicated order

tsxtsx +

(uv-)

(uv÷)

1 12

35 2

Compatible with lac-tsx-(UV) or lac.( UV)-tsx

Strains RB239 (ttfrC tsx) and RB224 (F- leu lac str UV s) were mated for 1 hour at 37 ° C, and selection made for Lae + [Str R] recombinants. After purification by single colony isolation, the recombinants were tested for unselected markers. All recombinants were E2-sensitive. All UVs recombinants were Fil+ and mucoid when grown on NB agar at 20 ° C. RB224 is a spontaneous Cet+ revertant of ASH 112 which is still UV-sensitive.

quired t r y p t o p h a n , p r e s u m a b l y t h e result of a m u t a t i o n i n d u c e d a t t h e t i m e of mutagenesis. To locate t h e site of t h e m u t a t i o n conferring U V - s e n s i t i v i t y on RB224, t h e cross described in T a b l e 3 was performed. The H f r used was a s p o n t a n e o u s p h a g e T6 r e s i s t a n t m u t a n t of RB239 (HfrC). Since o n l y one gene controlling t h e response to p h a g e T6 has been r e p o r t e d in E. coli (Taylor a n d T r o t t e r , 1972), i t is f a i r l y c e r t a i n t h a t this s t r a i n is m u t a n t a t t h e tsx locus, n e a r lon. H o w e v e r , when i n i t i a l l y t e s t e d none of t h e r e c o m b i n a n t s was T6 r e s i s t a n t (tested b y crossstreaking) a l t h o u g h w h e n t h e t e s t p l a t e s were e x a m m e d a f t e r only 2 - 3 hours inc u b a t i o n a t 37 ° C, a slight difference in response to T6 could be seen a m o n g t h e r e c o m b i n a n t s . I t is t h o u g h t t h a t t h e tsx m u t a t i o n is p r o b a b l y a n amber m u t a t i o n , which is s u p p r e s s e d w h e n t r a n s f e r r e d to RB224 (supE+). Some mis-scoring m a y t h u s a c c o u n t for t h e fact t h a t t h e d a t a in this cross on t h e p o s i t i o n of t h e gene g o v e r n i n g U V - s e n s i t i v i t y do n o t give an u n a m b i g u o u s answer (see T a b l e 3). Nevertheless, in view of t h e linkage of this m u t a t i o n to /ac, a n d t h e F i l + a n d m u c o i d p h e n o t y p e s (only o b s e r v e d in this m u t a n t a t low t e m p e r a t u r e ) , it seems p r o b a b l e t h a t this strain, like A S H 111, is m u t a n t a t t h e lon locus. This h y p o t h e s i s is f u r t h e r s t r e n g t h e n e d b y t h e fact t h e U V - s e n s i t i v i t y of s t r a i n 1%B224 has also been f o u n d to r e s p o n d to p a n t o y l l a c t o n e (J. M. Boyle, personal commurgcation). 3. Attempts to Locate the Mutation Causing UV-sensitivity in Strain A S H 114 T h e m u t a t i o n giving rise t o U V - s e n s i t i v i t y in A S H 114 was f o u n d t o segregate from E2 t o l e r a n c e ( d a t a n o t shown), a n d a possible loose linkage to trp was established. T h e a b e r r a n t n a t u r e of t h e t r a n s f e r p r o p e r t i e s of H f r A S H 1 (see 12a

~olec. gen Genet. 131

R . S . B u x t o n a n d I. B: Holland

166

Table 4. Linkage between pheA, his, thyA and the mutation giving rise to UV-sensitivity in ASH 114 Phenotypes of recombinants Phe + His+ Phe + His+ Phe + His- Phe + His- Phe- His+ Phe- His+ Phe- His- Phe- His- Total Thy + ThyThy + ThyThy + ThyThy + ThyUVs UV ~

1 2

7 0

5 5

5 3

0 2

1 0

4 13

0 1

49

Analysis Selected marker

Unselected markers

1. purC +

2.

purC +

Indicated order

(UV +)

(UV-)

pheA + pheA-

10 16

18 5

thyA + thyA-

(UV+) 22 4

(UV-) 10 13

~

<

(UV-) I Jr thyAI -F

pheA + purC+ I I---(UV-) I ~-

purO + I---

Strains ASH 114 (Hfr AStt 1 thyA metB UV s) and AT 2092 (F-his purC pheA) were mated for 2 hours at 37 ° C, and selection made for Pur+ [Met+]. Recombinants were purified by single colony isolation and tested for unselected markers. Methods) m a k e t h e results of linkage analysis s o m e w h a t difficult to i n t e r p r e t . H o w e v e r , a n u m b e r of lines o f evidence m a d e i t u n l i k e l y t h a t t h e m a j o r p a r t of t h e U V - s e n s i t i v i t y d e r i v e d from a m u t a t i o n in t h e lon gene. These i n c l u d e d : (1) t h e absence of a n y linkage of U V - s e n s i t i v i t y w i t h tsx in a cross analogous to t h a t described for A S H 111 in T a b l e 2, selecting for PurE+ r e c o m b i n a n t s ( d a t a n o t shown); (2) few or no m u c o i d r e c o m b i n a n t s were o b t a i n e d in a cross w i t h 1~B49, s i m i l a r to t h a t described in section 2. T h e a b o v e results d i d n o t of course exclude t h e p o s s i b i l i t y t h a t A S H 114 carries a n amber lon m u t a t i o n which was s u p p r e s s e d b y s u p E + in t h e F - s t r a i n ; (3) t h e U V - s e n s i t i v i t y of ASI-I 114 was n o t relieved b y i n c u b a t i o n of cells in t h e presence of p a n t o y l lactone (J. M. Boyle, personal communication), unlike L o n - m u t a n t s . On t h e o t h e r hand, t h e results of t h e cross described in Table 4 suggest t h a t t h e site of t h e m u t a t i o n giving rise to U V - s e n s i t i v i t y lies b e t w e e n t h y A a n d pheA. This region includes t h e recA, recB, recC a n d recH loci (see T a y l o r a n d T r o t t e r , 1972), all of which r e n d e r t h e cell UV-sensitive a n d r e c o m b i n a t i o n deficient. I n consequence a t e s t was carried o u t to d e t e r m i n e t h e r e c o m b i n a t i o n deficiency of a s t r a i n c a r r y i n g t h e UV-sensitive m u t a t i o n of A S H 114. A n F - U V s r e c o m b i n a n t from t h e cross described in T a b l e 4 was t e s t e d for its r e c o m b i n a t i o n deficiency b y selecting for H i s + r e c o m b i n a n t s a t 37 ° C in a cross w i t h a n H f r transferring t h e w i l d - t y p e UV-resistanee allele as a v e r y l a t e m a r k e r . T h e results o b t a i n e d ind i c a t e d t h a t this s t r a i n does h a v e a slight r e c o m b i n a t i o n deficiency of approxim a t e l y 2, b u t this is p r o b a b l y n o t large enough t o be significant. I n c o n t r a s t t h e r e c o m b i n a t i o n deficiency i n d e x (His +) for f e c A l 3 is 1 × 10 a, a n d for recB21 i t is 30

167

Coliein E2 Tolerance

Table 5. Linkage analyses to determine the location of the site of the mutation conferring phage T4 resistance on strain 1~B29 1. Selections made:

Leu+

Pro+

Lac +

Gal+

I-Iis+

Arg+

% recombinants/input Hfr No. Cet- recombinants No. T4I~ recombinants No. Gal+ recombinants No. His+ recombinants

30.9% 7/10

1 6 . 6 % 5.8% 4/10 4/10

5.9% 15/40

0.9% 16/40

0.017% 13/40

0/10

0/10

0/10

0/40

7/40

0/40

NT NT

NT NT

NT NT

40/40 1/40

13/40 40/40

NT NT

2. Selections made:

His+

Tyr+

% recombinants/input tIfr No. T4R recombinants No. His+ recombinants No. Tyr+ recombinants

0.0079 % 4/50 50/50 1/50

0.002 % 7/50 21/50 50/50

Strain RB29 (HfrH cetl sup- T4tt; orientation of transfer o-leu-lac) and an F - recipient strain were mated at 37o C. Selection was made as described above. Contra-selection was for Str 1¢. Reeombinants were purified as indicated, and tested for E2 tolerance on NB at 20° C, T4 resistance on TB at 37° C, and on MM at 37° C for unselected auxotrophie markers. 1. F - recipient: ABl157 (thr leu proA lac supE+ gal his argE str). Mated for 90 minutes. l~ecombinants were partially purified (see Buxton and Holland, 1973). 2. F - recipient: SA 130 (his tyrA str). Mated for 1 hour. Recombinants were purified by single colony isolation. NT = not tested.

(Clark, 1967). I t should be noted, however, t h a t A S H 114 is more UV-sensitive at 20 ° C a n d it m a y therefore be more R e c - a t this t e m p e r a t u r e t h a n at 37" C.

4. Location o] the Site o/the Mutation Con]erring Resistance to Phage T4 in Strain RB29 S t r a i n RB29 was a Cet- m u t a n t isolated i n the p r e s e n t study. A l t h o u g h n o t UV-sensitive, RB29 was f o u n d to be resistant to phage 2 a n d also to phage T4 (Buxton, 1973). The E2 tolerance of this s t r a i n was shown to m a p a t a position similar to t h a t of other cet m u t a t i o n s ( B u x t o n a n d Holland, 1973) a n d tests on T h r + r e c o m b i n a n t s from a cross RB29 {ttfrtt cetl thr+) × RB53 ( F - c e t + thr--) showed t h a t the T4 resistance p h e n o t y p e clearly segregated from the Cet- phenotype. T h u s again there was no evidence t h a t the cet m u t a t i o n was h a v i n g pleiotropic effects. Table 5 describes the results of two crosses performed to t r y to locate t h e site of the m u t a t i o n giving rise to T4 resistance i n 1~B29. These results indicate loose linkage of T4 resistance to his a n d tyrA. Since almost half of the Tyr+ r e c o m b i n a n t s also i n h e r i t his +, whereas only 1/50 of the His + r e c o m b i n a n t s are Tyr+, this confirms t h a t tyrA is inherited later t h a n his from H f r H . No d r a m a t i c correlation exists for T4-resistance. I t seems most likely therefore t h a t the locus controlling t h e response to T4 is transferred before tyrA b y H f r I t . Only one locus governing the response to phage T4 appears at present on the s t a n d a r d chromosomal m a p of E. coli, viz., t]rA a t position 8 (Taylor a n d Trotter, 1972), a l t h o u g h Curtiss (1965) f o u n d other T4-resistant m u t a t i o n s m a p p i n g at 12b

Molec. gen. Genet. 131

168

R.S. Buxton andI. B. Holland

distinct loci. The results presented here appear to rule out the possibility of a mutation in the t/rA gene in RB29. 5. Investigations into the Nature o/the Resistance to Phage 4 in Strains A S H 114 and R B 2 9 ASH 114 has previously been shown to produce poor plaques and reduced plating efficieneies when infected with phage 4 (Holland et al., 1970). Similarly the Cet- mutant RB29 also showed this phenomenon. Previous work (Holland et al., 1970) suggested that this "resistance" to 4 was due to a failure of normal multiplication of the phage within its host. The following results indicate, however, that in strain ASH 114 studied by Holland et al., and also in RB29, poor growth of 4 is due to reduced adsorption or to reduced adsorption and injection of phage DNA. Thus RB29 only adsorbed 86% of 4gv in 10 minutes, whereas its wild-type parent 14H adsorbed 97 %, and ASH 114 adsorbed only 68 % and its parent ASH 1, 91%. Similarly RB29 adsorbed only 55 % of phage T4 whereas 14H adsorbed 99 %. In order to test directly the ability of these strains to support the growth of phage 4, lysogens were prepared by infection with 2ci857. The one-step growth curves obtained by heat induction of these lysogens indicated that the burst size of 4ci857 in RB29 was only slightly lower than in its parent strain 14H (200 as against 230) (data not shown). I t is therefore concluded that these strains are not defective in the intracellular growth of 4. Discussion

The results of the genetic mapping with strains originally designated CetCpresented in this paper have shown that: (1) the Cet- strain ASH 111 and the Cet + revertant of the Cet- strain ASH 112 are mutant at a locus, almost certainly identical with the lon locus, which renders them UV-sensitive, Fil + and mucoid, although this latter property is suppressed in ASH 111. Another CetC- strain, ASH 116, also has similar phenotypie properties to Lon- mutants, viz., UVsensitivity and mucoid colony morphology (Holland et al., 1970, and confirmed in the present study), and it seems reasonable to assume that this is also lon-, although no genetic mapping has been carried out in this strain; (2) the Cet- strain ASH 114 is mutant at a locus, probably near the recA locus, which renders it UV-sensitive; (3) in no UV-sensitive Cet- strain tested did the site of the mutation conferring UV-sensitivity map at or near the cet locus. This is in sharp contrast to the earlier reports (Holland and Threlfall, 1969; Threlfall and Holland, 1970), which in some cases ostensibly utilised the same CetC- mutants as the present study, that mutations at the cet locus could give rise to pleiotropic properties including UV-sensitivity. How may we reconcile these conflicting findings ? As already indicated, some of the phenotypic changes accompanying E2-tolerance in the original isolates have been lost, a feature which is reported to be frequently encountered amongst other colicin-tolerant mutants (Holland, 1974). In the case of the slow growth rate of the original isolate of ASH 111, transduction of cet111 to other strains did not lead to the reappearance of the original slow growth rate. This may be because secondary mutations had occurred at the cet locus, suppressing some of the pleio-

Colicin E2 Tolerance

169

tropic effects of the original cet mutations, and this cannot be ruled out on the data available. However, some evidence was obtained (Buxton, 1973) t h a t A S H 111 contained another mutation linked to leu but distinct from cet, which when transferred to a second strain, caused very slow growth rates. I n the case of the UVsensitivity of CetC- mutants, it must remain a possibility that the positioning o] the mutation giving rise to U V-sensitivity was initially in error (Holland and Threlfall, 1969). I t is possible t h a t more than one mutation is contributing to the UV-sensitivity of all these strains but in view of the consistency of the data from three-point crosses, this is probably unlikely. Of the other properties, the aberrant recombination frequencies of the CetCH f r strains A S H 111 and A S H 114 (Holland and Thrclfall, 1969) have been shown (Buxton, 1973) to be probably a characteristic of the particular Hfr (see Methods) and not associated with the cet mutation. However, this study has confirmed t h a t selection of Cet- m u t a n t s after the same method of mutagenesis (with 2-aminopurine) does result in the isolation of mutants having various defects conceivably located in the cell surface. At least in the case tested, viz., T4 resistance, this is clearly the result of a mutation unlinked to the cet gene. Of major interest therefore is the reason why so m a n y Cet- mutants are also m u t a n t at other loci, particularly at the Ion locus. Three basic explanations are proposed: (1) t h a t these other mutations are simply due to the effect of excessive mutagenesis causing random double or multiple mutations; (2) t h a t some selective pressure favours the survival of Cetm u t a n t s which are also m u t a n t at other loci; (3) t h a t Cet- strains undergo mutation more readily than Cet + strains, i.e., if cet is a m u t a t o r gene. The last possibility has not been subjected to rigorous tests, but no increase in the spontaneous rate of phage T6 resistant mutants in a CetB- strain (ASH 102) has been noticed compared with its Cet + parent (ASH 10). I n support of 1, one Cet- strain (ASH 112), upon initial isolation, was found to have also m u t a t e d to T r p - (Holland and Threlfall, 1969). Colicin E2 m a y in fact be causing some degradation of DNA even in a Cot- strain but this m a y normally be repaired. If this is so, the possibility exists t h a t E2 m a y itself be having a mutagenic effect on the selective plates. However, the fact t h a t so m a n y of the mutations appeared to affect the cell envelope does suggest the existence of some selective pressure, either for increased E2 tolerance, altered growth rates or increased survival of mutants under the prevailing conditions. I t is also well known t h a t selection for resistance to various phages (e.g., T6) results, fortuitously, in the selection of mucoid (often Lon-) colonies, i.e., phenotypically T6 resistant (Walker and Pardee, 1967). Presumably the mucoid capsule covers the phage receptors. Whilst it is possible t h a t some surface change in L o n strains allowed t h e m to better survive the E2 treatment, one Cot- L o n - m u t a n t (ASH 111) had the major p a r t of its mucoidy suppressed b y the non mutation, which suggests t h a t the mucoid property itself m a y not be the only selective factor in the occurrence of L o n - strains among Cet- mutants. A further complication is t h a t the phenotypes of Cet- UV-sensitive strains have been found to be somewhat unstable. I n particular, strains A S H 111 and A S H 114 have both become temperature-conditional in their response to UV since they were first isolated, i.e., they are more sensitive when the postirradiation

170

R.S. Buxton and I. B. Holland

t e m p e r a t u r e is a t 25 ° C (for A S H 111) or 20 ° C (for A S H 114), r a t h e r t h a n 37 ° C. A S H 111 has also been f o u n d (Holland et al., 1970) t o be m u c h m o r e UV-sensitive t h a n w h e n originally t e s t e d (Holland, 1967), a n d f o r m e r l y h a d a v e r y slow g r o w t h r a t e . I t is t h o u g h t l i k e l y t h a t t h e original m e t h o d of storing t h e strains in sealed a g a r s t a b t u b e s a t r o o m t e m p e r a t u r e m a y h a v e led t o t h e selection of faster growing derivatives, which m a y also h a v e a l t e r e d t h e response of t h e cells to UV. Therefore, p e r h a p s a note of c a u t i o n should be m a d e r e g a r d i n g t h e isolation a n d h a n d l i n g of m u t a n t s t h o u g h t to h a v e defective envelopes. T h u s m a i n t e n a n c e of such m u t a n t s u n d e r conditions where selective pressures m a y continue should be avoided, a n d freeze d r y i n g or freezing cell suspensions in glycerol is advisable. E x p o s u r e to t h e selective a g e n t should be as brief as possible, a ~ d t h e survivors grown u p a n d purified in t h e absence of this agent. F i n a l l y , w h e n dealing w i t h a p p a r e n t l y pleiotropic m u t a t i o n s , t h e use of m u t a g e n i c a g e n t s inducing closely linked m u l t i p l e m u t a t i o n s should be avoided, a n d t h e effect of t r a n s d u c t i o n of t h e m u t a t i o n s t o a n otherwise isogenic recipient, t h e r e b y a v o i d i n g p r o b l e m s of differential suppression, m u s t be v e r y carefully analysed. Acknowledgements. We thank Dr. Howard J. Rogers for his valuable comments on this manuscript, Miss Pamela Addams for kindly performing the experiments described in Fig. 1, and Dr. J. M. Boyle for the communication of unpublished results. R.S.B. gratefully acknowledges the award of a Scholarship for Training in Research Methods from the Medical Research Council.

References Adams, M. : Bacteriophages. New York: Interscience Publishers Inc. 1959 Adler, H. I., Hardigree, A. A. : Analysis of a gene controlling cell division and sensitivity to radiation in Escherichia coll. J. Bact. 87, 720-726 (1964) Bachmann, B. J. : Pedigrees of some mutant strains of Escherichia coli K-12. Bact. Rev. 86, 525-557 (1972) Buxton, R. S. : A genetic analysis of colicin E2 sensitivity in Escherichia coli K-12. Ph. D. Thesis, University of Leicester (1973) Buxton, R. S., Holland, I. B. : Genetic studies of tolerance to colicin E2 in Escherichia coli K-12. I. Re-location and dominance relationships of cet mutations. Molec. gen. Genet. 127, 69-88 (1973) Clark, A. g. : The beginning of a genetic analysis of recombination proficiency. J. cell. Physiol. 70 (Suppl. 1), 165-180 (1967) Curtiss, R. : Chromosomal aberrations associated with mutations to bacteriophage resistance in Escherichia coli. J. Bact. 89, 28-40 (1965) Donch, J., Greenberg, J. : Protection against lethal effects of ultraviolet light on Escherichia coli by capsular polysaccharide. Mutation Res. 10, 153-155 (1970) Holland, I. B. : The properties of UV sensitive mutants of Escherichia coli K12 which are also refractory to colicin E2. Molec. gen. Genet. 100, 242-251 (1967) Holland, I. B. : Action of colicins. Advane. microbiol. Phys., in press (1974) Holland, I.B., Threlfall, E. J. : Identification of closely-linked loci controlling ultraviolet sensitivity and refractivity to colicin E2 in Escherichia coli. J. Bact. 97, 91-96 (1969) Holland, I.B., Threlfall, E. J., Holland, ]~. M., Darby, V., Samson, A. C. R.: Mutants of Escherichia coli with altered surface properties which are refractory to coliein E2, sensitive to ultraviolet light and which can also show recombination deficiency, abortive growth of bacteriophage 2 and filament formation. J. gen. Mierobiol. 62, 371-382 (1970) Howard-Flanders, P., Boyce, R . P . : DNA repair and genetic recombination: studies on mutants of Escherichia coli defective in these processes. Radiat. Res. (Suppl.) 6, 156-184 (1966) Howard-Flanders, P., Simson, E., Theriot, L. : A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics 49, 237-246 (1964)

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Low, B. : Inversion of transfer modes and sex-factor chromosome interactions in conjugation in Escherichia coli. J. Bact. 93, 98-106 (1967) Markovitz, A., Baker, B.: Suppression of radiation sensitivity and capsular polysaccharide synthesis in Escherichia coli K-12 by ochre suppressors. J. Bact. 94, 388-395 (1967) Radke, K. L., Siegel, E. C. : Mutation preventing capsular polysaccharide synthesis in Escherichia coli K-12 and its effect on bacteriophage resistance. J. Bact. 106, 432-437 (1971) Samson, A. C. R. : The mode of action of colicin E2 with regard to the structure of the Escherichia coli cell envelope. Ph.D. Thesis, University of Leicester (1970) Samson, A. C. R., Holland, I. B. : Envelope protein changes in mutants of Escherichia coli refractory to colicin E2. FEBS Letters 11, 33-36 (1970) Taylor, A. L., Trotter, C.D.: Linkage map of Escherichia coli strain K-12. Bact. Rev. 36, 504-524 (1972) Threlfall, E. J., Holland, L B. : Co-transduction with serB of a pleiotropic mutation affecting coliein E2 refractivity, ultraviolet sensitivity, recombination proficiency and surface properties of Escherichia coli K-12. J. gen. Microbiol. 62, 383-398 (1970) van de Putte, P., Westenbroek, C., R6rsch, A.: The relationship between gene-controlled radiation resistance and filament formation in Escherichia coll. Biochim. biophys. Acta (Amst.) 76, 247-256 (1963) Walker, J . R . , Pardee, A. B. : Conditional mutations involving septum formation in gscherichia coli. J. Bact. 98, 107-114 (1967) C o m m u n i c a t e d b y G. B e r t a n i Dr. R. S. Buxton Division of Microbiology :National Institute for Medical Research Mill Hill London NW7 1AA England

Dr. I. B. Holland Department of Genetics University of Leicester Leicester LE1 7RH England