Molec. gen. Genet. 150, 161 - 170 (1977) © by Springer-Verlag 1977

Role of dnaB43 in F'-plasmid Incompatibility Alan F. Jamieson and Peter L. Bergquist Department of Cell Biology, University of Auckland, Auckland, New Zealand

Summary. In order to perform complementation tests with mutations of D N A replication of F'-plasmids incompatibility must be overcome. We report our inability to duplicate the results presented by Palchoudhury and Iyer (1971) and Bezanson and Iyer (1975) who have claimed to demonstrate the autonomous replication of two incompatible F'-plasmids in a strain carrying the temperature sensitive dnaB43 allele. In addition, we describe experiments designed to measure complementation using transient heterozygotes and compatible plasmids. Assessment of our data and those of others in the light of a recent report by Uhlin and Nordstr6m (1975) suggests that new approaches will have to be developed for the successful employment of complementation analysis in Fplasmid genetics.

Introduction Strains of Escherichia coli that carry an F-plasmid resist infection by the same or by a related plasmid. Two separate barriers exist, surface exclusion and incompatibility (Novick, 1969). Although surface exclusion is still incompletely understood, techniques are available to diminish its effects. However, once a superinfecting plasmid successfully enters a cell already carrying a plasmid, the two plasmids may be incompatible and one or the other may not be able to replicate normally. Incompatibility has been used as the basis for the classification of plasmids into a large number of groups (see for example, Datta and Hedges, 1971). The basis of incompatibility is obscure. It was proposed that incompatible plasmids competed for specific maintenance and replication sites on the cell membrane (Jacob et al., 1963). More recently, it has been suggested that plasmid replication is under the

control of repressor molecules, which are specific for an operator that is common to incompatible plasmids (Pritchard et al., 1969; Uhlin and Nordstr6m, 1975). Incompatibility is probably closely linked not only to the prevention of replication of plasmids of the same class but also to the regulation of the number of copies of a plasmid per chromosome present in a cell and to all other aspects of the control and mechanics of the replication of the plasmid DNA. The rudimentary state of the genetics of F-plasmid replication largely results from the inability to overcome incompatibility, thus making complementation analysis impossible. Several approaches have been suggested to overcome incompatibility. One solution, to fuse two F-primes into one autonomous element, has been successfully achieved (Press et al., 1971 ; Willetts a n d Bastarrachea, 1972; Palchaudhuri etal., 1972). However, without extensive heteroduplex analysis, it is not clear whether such fused F-primes carry all the cistrons of both F factors. Palchaudhuri et al. (1972) have proposed that one of the sets of genetic determinants for incompatibility is lost on fusion and thus stable maintenance of the hybrid plasmid in the cell is permitted. Another approach is to look for mutants with altered incompatibility relationships. All inc plasmid mutations have been unable to express the Inc- phenotype unless the plasmid was integrated (Maas and Goldschmidt, 1969; DeVries and Maas, 1973; Wyman and Novick, 1974). Such results suggest that either reversion of the inc mutation was required for autonomous replication or that the inc locus may be repressed under certain circumstances when the plasmid carrying it is integrated. San Blas et al. (1974) described a chromosomallylocated mutation isolated in a recA strain that allows the replication of two autonomous F-primes. Unfortunately, the mutation was specific for F'-gal ÷ (F8) and F'-his + (F30). Palchoudhury and Iyer (1971)

162 reported that two F-plasmids were able to replicate autonomously in a strain carrying the dnaB43 mutation, which brings about an abrupt temperature-dep e n d e n t c e s s a t i o n o f D N A s y n t h e s i s a t 4 2 °. A t t h e p e r m i s s i v e t e m p e r a t u r e (31 ° C), cells c a r r y i n g d n a B 4 3 a n d F ' - l a c + ( F 4 2 ) s h o w e d a n i n c r e a s e d a b i l i t y t o all o w t h e e n t r y a n d r e p l i c a t i o n o f F ' - t h r + leu + ( F 1 0 1 ) as c o m p a r e d w i t h a n i s o g e n i c d n a B + s t r a i n . E v i d e n c e was presented to show that two physically separable classes of DNA molecules were present in the dnaB43 strain, which could also transfer either F'-lac ÷ or F ' - t h r + leu + t o a r e c A r e c i p i e n t . A l t h o u g h t h e s e e x p e r iments were performed with Rec + dnaB43 strains, a subsequent report claimed that two separate F-plasraids were present in recA1 derivatives of the dnaB43 s t r a i n s ( B e z a n s o n a n d I y e r , 1975).

Materials and Methods Bacterial Strains. The strains used are described in Table 1. A reeA1 dnaB43 strain was constructed by mating PB758 (Thy + recA1 str-s) with PB28 (dnaB43 thy Rec + str-r), followed by selection for Thy + str-r recombinants at 32 °. A Thy + recA1 dnaB43 clone was purified and numbered PB1526. Bacteriophage Strains. The donor-specific phage R17 was prepared by growth in liquid medium on an Hfr E. eoli K12 strain, while the female-specific phage qbII was prepared by confluent lysis on E. eoli B. Media. These were described by Bergquist and Adelberg (1972). Mating Conditions. Mating was carried out between growing cultures at a donor to recipient ratio of 1 : 1 at 32 °. Acridine Orange Curing. About 5 x 102 cells were inoculated into 5 ml of L broth, pH 7.85 with various concentrations of acridine orange and were incubated in the dark with shaking at 32 °. Suitable dilutions were spread to L plates and replicated to various selective media to determine the loss of F'-plasmid genetic markers. Isolation of Strains Carrying Genetic Markers from Two Donor U-strains. Broth matings were caried out at 32° between the strains carrying dnaB43, PB28 and PB1526, and strains carrying the F'plasmids, F42 (PB197) and F101 (PB665). The resulting purified F-ductants which carried either F101 or F42 in the PB28 or PB1526 chromosomal background were then grown in phenocopy at 30 ° C and were used as recipients in a second mating with PB197 when the phenocopied strain from the step above carried F101, or with PB665 when the phenocopied recipient caried F42. A donor to recipient ratio of 1 : 1 was usually used. Transient Heterozygotes. In preliminary experiments, the optimal conditions for transient heterozygote formation were determined using growing donors carrying wild-type F'-gal + (F8) and F~sll 4lac + derived from F42 in the phenocopied recipient. The cells were mated at a donor to recipient ratio of 10:I at 34°C in stationary cultures for 60 rain. The mating mixture was then diluted 100-fold into L broth plus streptomycin to kill the donor cells and to prevent further mating, and incubated with shaking for a further 2 h at 34 ° C. During this time, samples were removed

A.F. Jamieson and P.L.Bergquist: F'-plasmid Incompatibility to determine : (i) The time taken for the phenocopied recipient to resume exponential growth as measured by viable count; (ii) The fraction of the recipients to which the donor plasmids have been transferred (measured by the frequency of Gal + cells on minimal galactose medium); (iii) The stability and proportion of transient heterozygotes in the population as measured by the frequency of sectored colonies on MacConkey indicator plates (all of the sectored colonies were shown to contain both Gal ÷ and Lac+cells). Isolation of Plasmid DNA. Thirty or 50 ml cultures of cells in minimal medium plus appropriate supplements and containing Casamino Acids (1 mg/ml) were grown at 32° or 34 ° to 1 x 108 cells per ml. Deoxyadenosine to a final concentration of 250 gg/ml and 100 to 200 gc of (3H-methyl)-thymidine, were added to the cultures, which were harvested at 8 x 108 cells per ml and washed twice inTES buffer (5x 10 - 2 M Tris-(hydroxymethyl)-aminomethane, pH 8.0; 5x 10 -a M EDTA and 5x 10 .2 M NaC1). Cleared lysates were prepared by the method of Clewell and Helinski (1970) or supercoiled DNA was isolated directly (Freifelder et al., 1971). The cleared lysates were subjected to either ethidium bromide-cesium chloride buoyant density centrifugation or centrifugation in alkaline sucrose. The gradients were fractionated and portions were spotted onto filter paper discs, dried, immersed in 10% (w/v) trichloroacetic acid, washed in 80% (v/v) ethanol and counted in a Packard 3375 scintillation counter.

Results C o - i n h e r i t a n c e o f F ' - l a c + a n d U - t h r + leu + in P B 2 8 , a Rec + dnaB43 Strain and PB1526, a recA1 dnaB43 Strain Results similar to those reported by Palchoudhury and Iyer (1971) were obtained when the dnaB strain u s e d b y t h e m w a s e m p l o y e d as a r e c i p i e n t in m a t i n g s w i t h s t r a i n s c a r r y i n g e i t h e r F ' - l a c + o r F ' - t h r + leu +. T a b l e 2, l i n e s 1 a n d 5 s h o w t h a t t h e t w o F ' - p l a s m i d s are transferred at reasonable frequencies to the Rec ÷ dnaB43 recipient. There was a substantial reduct i o n in t h e t r a n s f e r o f F ' - l a c + t o a p h e n o c o p i e d d n a B 4 3 r e c i p i e n t t h a t a l r e a d y c a r r i e d F ' - t h r + leu + ( l i n e 3), a n d the frequency of Lac ÷ Thr ÷ Leu ÷ F-ductants was about one half of the value of the Lac ÷ selection (0.45% and 0.85% respectively). There was an even greater reduction in the frequency of transfer of F101 t o a r e c i p i e n t t h a t c a r r i e d F 4 2 ( T a b l e 2, line 7, colu m n s D a n d E). I n t h e m a t i n g s h o w n o n l i n e 3, w h e r e Lac ÷ was the selected marker, only 26% of the F-duct a n t s w e r e a l s o T h r ÷ L e u ÷ ( l i n e 3, c o l u m n H ) , a n d only 20% of the Thr ÷ Leu + F-ductants from the cross of F101 into the phenocopied dnaB43, F42 recipient w e r e L a c ÷ ( T a b l e 2, l i n e 7, c o l u m n H ) . Both F42 and F101 were transferred to PB1526 (dnaB43, recA1) at similar frequencies to that found w i t h t h e a l m o s t i s o g e n i c s t r a i n , P B 2 8 ( T a b l e 2, l i n e s 2 a n d 6). W h e n t h e r e c A 1, d n a B 4 3 s t r a i n s t h a t c a r r i e d

163

A.F. J a m i e s o n a n d P.L.Bergquist: F ' - p l a s m i d I n c o m p a t i b i l i t y Table 1. List of b a c t e r i a l strains Strain

Mating type

Chromosomal markers

Other properties

D e r i v a t i o n or source

PB15

Hfr

thi, galTI2

F~-gal + i n t e g r a t e d (F8-10)

B e r g q u i s t and A d e l b e r g (1972)

PB28

F-

thi, thr, leu, thyA, lac, dnaB43, str

PB68

F-

thi-1, thr-1, leu-6, proA2, lacY1, galK2, mtl-1, xyl-5, ara-14, strA20

2+

PB197

F'

thi, AlacX74

F'-lac+(F42)

J. L a n g r i d g e

PB349

F

thi-1, thr-1, leu-6, proA2, lacY1, galK2, mtl-1, xyL5, ara-14, strA20, nalA12, recA1

).+

B e r g q u i s t and A d e l b e r g (1972)

PB665

F'

argE3, proA2, galK2, sup-37,

F ' - t h r + leu + (F101)

B. B a c h m a n n

PB758

Hfr

thi-28, A(proB-lae) X I I I sup-56, recA1, nalA12

PK191. Transfers

O u r collection

F'ts 1 l,-lac + (from F42)

F. B o n h o e f f e r

his-4, leu-6, thr-1, mtl-1, xyl-5, ara-14, lac Y1, str-31, recA13, tsx-33

~his, recA, thyA...

PB 1130

F'

galT12, lacZ32, thr, str

PB1526

F-

thr, leu, lac, dnaB43, recA1, thi

PB1527

F'

thr, leu, lac, dnaB43, recA1, thi

F'-lac ÷ (F42)

PB197 x PB1526

PB1528

F'

thr, leu, lac, dnaB43, recA1, thi

F'-thr, leu (F101)

PB665 x PB1526

PB1534

F'

thr, leu, lac, dnaB43, thyA, thi

F ' - l a c + (F42)

PB197 x PB28

PB1535

F'

thr, leu, lac, dnaB43, thyA, thi

F'-thr + leu + (F101)

PB665 x PB28

PB1536

F'

thr, leu, dnaB43, thyA, thi

F'-thr + leu + (F101)

PB665 x PB1534

PB1550

F'

lac, dnaB43, thyA, thi

F'-lac + (F42)

PB197 x PB1535

PB1571

F'

galTI2, lacZ32, thr

F'-gal + (F8)

J a c o b et al. (1963) PB758 x PB28

Table 2. T r a n s f e r frequencies a n d c o - i n h e r i t a n c e of F - p l a s m i d s in n o r m a l a n d in p h e n o c o p y m a t i n g s between dnaB43 Rec ÷ and dnaB43 recA1 strains Line

1

Donor

PB 197

A Donor F-prime

F42

B Recipient

Recipient genotype

C D E T r a n s f e r frequency Lac +

Thr + Leu +

F Selected marker

G Unselected marker

H Unselected marker frequency

T h r ÷ Leu ÷

26

T h r + Leu +

1

Lac + T h r + Leu +

PB28

dnaB43

24

.

.

.

.

.

25

.

.

.

.

.

(F'-lac +) 2

PB197

F42

PB1526

dnaB43, recA1

3

PB197

F42

PB1535

FlO1/dnaB43 (F'-thr + leu ÷)

4

PB197

F42

PB1528

FlO1/dnaB43, recA1

5

PB665

F101

PB28

dnaB43

6

PB665

F101

PB1526

7

PB665

F101

PB1534

8

PB665

F101

PB1527

F42/dnaB43, recA1

0.85 0.20

-

0.45

--

-

39

dnaB43, recA1

--

33

F42/dnaB43

-

0.14

-

0.14

Lac ÷

< 1 x 10 - 4 .

Lac + .

.

.

-0.05 < 1 x 10 - 4

--

--

Thr + Leu +

Lac +

20

T h r + Leu +

Lac +

1

M a t i n g s were carried o u t at 3 2 ° C using a d o n o r : recipient ratio of 1 : 1. T r a n s f e r frequency is expressed as the n u m b e r of colonies c a r r y i n g the selected m a r k e r × 1 0 0 / n u m b e r of d o n o r cells. C o l u m n H shows the f r e q u e n c y w i t h w h i c h the unselected F ' - p l a s m i d m a r k e r s in the recipient was present a m o n g s t the t r a n s c o n j u g a n t s t h a t carried the selected d o n o r F ' - p l a s m i d m a r k e r . One h u n d r e d ' r e c o m b i n a n t s ' f r o m each m a t i n g were purified by repeated s t r e a k i n g to m i n i m a l selective media. Single colonies were isolated a n d replicated to suitable m i n i m a l a g a r plates to test for the presence of F'-lac + or F'-thr + leu +

164

either F101 or F42 were used as recipients in the appropriate crosses to strains carrying the donor plasmids, the transfer frequency, as measured in single selections, was greatly reduced (Table 2, lines 4 and 8) and no F-ductants at all could be observed when selection was for Lac + T h r ÷ L e u ÷ (less than 1 x 1 0 - 4 % of the number input donor cells in the cross). No single colonies appeared on the Lac + Thr ÷ Leu ÷ selective plates although limited background growth was evident at low dilutions. These data and observations suggest to us that it is not possible to isolate dnaB43 recA1 strains carrying two a u t o n o m o u s F'-plasmids and that segregation of F42 and F101 occurred soon after formation of the heterozygotes. The limited growth appearing on the plates probably resulted from a mixture of Lac ÷ and Thr ÷ Leu ÷ cells maintained by cross feeding and continued mating within the cell population. No single colonies could be isolated on streaking out from the background growth to the selective plates. Our results confirm that genetic markers from each of the two parental plasmids are present in the dnaB43 Rec + cells. This observation can be accounted for by the following possibilities: (i) Both F-primes are a u t o n o m o u s ; (ii) One F-prime is a u t o n o m o u s and the other is either integrated or the c h r o m o s o m a l genes it carries have become integrated and some or all of the F genes have been lost; (iii) both F-primes are integrated; (iv) the F primes have fused; or (v) a mixed population is present containing F'lac + cells, F'-thr + leu + cells and F'-lac+/F'-thr + leu + transient heterozygotes. The last possibility is virtually eliminated by repeated single colony isolation. The other four possibilities are examined below. Properties o f Strains P B 1 5 3 6 and PB1550

If both F primes are autonomous, it would be expected that either plasmid could be transferred independently from the heterozygote to a recA1 recipient. Twenty purified Lac + Thr + Leu + isolates from the two matings shown in Table 2 (lines 3 and 7) were tested. They were mated with PB349 to check their ability to transfer independently F42 and F101 to a recA1 recipient. N o n e was able to transfer both F'-plasmids to PB349, although all donors were Lac + Thr + Leu + immediately prior to mating. F r o m these transfer tests, we ascertained that 20 out of 20 of the Lac + Thr + Leu + isolates which were derived f r o m the cross F42 x FlO1/dnaB43 still have an autonomous F'-lac +, while 9/20 of the Lac + Thr + Leu + isolates f r o m the cross F101 x F42/dnaB43 carried

A.F. J a m i e s o n a n d P . L . B e r g q u i s t : F ' - p l a s m i d I n c o m p a t i b i l i t y

an a u t o n o m o u s F'-lac + and 11 carried an a u t o n o m o u s F'-thr÷ leu +. Hence superinfecting F42 tends t o exclude resident F101 more readily than vice versa. This result suggests that in each case the cells contain one a u t o n o m o u s F' and one integrated F' (or at least that its c h r o m o s o m a l gene complement has become integrated). One Lac ÷ Thr + Leu + isolate from the mating of PB197 and PB1535 that was able to transfer F42 but not F101 was retained and called PB1550, and one similar isolate from the mating of PB665 and PB1534 that was able to transfer F101 but not F42 was retained and called PB1536. These two strains were considered to be typical of the products of the crosses and were used to examine the status of the two F factors and their associated genetic markers. These two isolates and the dnaB43 strains carrying either F42 or F101 were examined for their ability T a b l e 3 . T r a n s f e r o f genetic m a r k e r s f r o m dnaB43 strains c a r r y i n g F' p l a s m i d s to isogenic Rec + a n d recA1 recipients Line D o n o r

Recipient

Selected marker

Transfer or recomb i n a n t frequency c

1

(F42/dnaB43)

PB68 Rec +

Lac + Pro +

29.0 2.9

98 38

PB1534

PB349

L a c +"

28.0

100

PB68

Lac + T h r + Leu +

22.0 1.6

94 68

2

PB1534

% recombinants which were donors d

(fecAl) 3

PB1550

(F42/dnaB43 Thr + Leu +) 4

PB1550

PB349

Lac +" 37.0 Thr+ Leu+ < 1 0 2

100 NT b

5

PB1535 (F101/dnaB43)

PB68

Thr + Leu + 30.0 Pro + 1.6 Xyl + < 10- 3

96 52 NT

PB1535

PB349

T h r + L e u +a 11.0

100

PB1536 (F 101/dnaB43, Lac +)

PB68

Thr + Leu + 16.5 Lac + 0.5 Pro + 1.1 Xyl + < 10 -3

96 46 66 NT

PB1536

PB349

T h r L e u +a 12.0 Lac ÷ < 10 2

100 NT

B r o t h m a t i n g s were carried out at 32 ° with a 1 : 1 d o n o r to recipient ratio a only F - d u c t a n t s for this m a r k e r a p p e a r e d on selective plates b N T = i n s u f f i c i e n t r e c o m b i n a n t colonies for testing c T r a n s f e r f r e q u e n c y refers to the p e r c e n t a g e o f recipient cells receiving the d o n o r F ' - p l a s m l d genetic m a r k e r . R e c o m b i n a n t f r e q u e n c y is the n u m b e r of colonies receiving the p a r t i c u l a r m a r k e r believed to be carried on the c h r o m o s o m e of the d o n o r as a result of F - m o b i l i z a t i o n and i n t e g r a t i o n via r e c o m b i n a t i o n . Both values are expressed as % of i n p u t d o n o r cells d Tested b y ability of purified r e c o m b i n a n t or F - d u c t a n t colonies to plate donor-specific b a c t e r i o p h a g e R I 7 at 3 2 ° C

A.F. Jamieson and P.L.Bergquist: F'-plasmid Incompatibility to transfer genetic markers to a Rec ÷ and a recA1 recipient. Table 3 shows that each of the four strains was able to transfer both the a u t o n o m o u s F plasmid and c h r o m o s o m a l genes to PB68 (Rec ÷) at relatively high frequencies, indicating frequent occurrence of exchanges between the plasmid and the c h r o m o some in the Rec + strain. Transfer of Thr ÷ Leu ÷ by PB1550 to the Rec ÷ but not to the recA1 strain (lines 3 and 4) suggests that these two markers are integrated. Similarly, the transfer o f Lac ÷ to PB68 but n o t to PB349 (lines 7 and 8) suggests that Lac + is n o t a u t o n o m o u s in strain PB1536. The presence of recombinants susceptible to the donor-specific bacteriophage R17 in the recombinant classes not f o r m e d by either F'-lac + or by F'-thr ÷ leu ÷ is unexpected but m a y be explained by the frequent occurrence o f F-genote enlargement (Bergquist and Adelberg, 1972; Jamieson and Bergquist, submitted for publication). A n analysis of the genetic markers carried by individual recombinants showed that F-genote enlargement had occurred (data not shown). Thus the low frequently o f Lac ÷ transfer by PB1536 to PB68 and PB349 (lines 7 and 8) suggests that some of the genetic material o f F42 has been lost on integration of the plasmid D N A to f o r m PB1536 (and similarly, F101 for PB1550).

165 Tab|e4.

Efficiencies of plating of bacteriophage R17 and ~II

Strain

Temperature (°C)

PB15

30 34 37

Role of dnaB43 in F'-plasmid incompatibility.

Molec. gen. Genet. 150, 161 - 170 (1977) © by Springer-Verlag 1977 Role of dnaB43 in F'-plasmid Incompatibility Alan F. Jamieson and Peter L. Bergqui...
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