X activity duringtransduction-associated recombination - Europe PMC

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We are grateful for the encouragement ofour colleagues, especially. Mary Stahl, Jean Crasemann, Lisa Young, Ichizo Kobayashi, and Ira. Herskowitz.
Proc. Natl Acad. Sci. USA Vol. 78, No. 11, pp. 7033-7037, November 1981

Genetics

X activity during transduction-associated recombination (PI phage/A phage/Escherichia coli/conjugation)

NANCY A. DOWER AND FRANKLIN W. STAHL* Department of Biology and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Contributed by. Franklin W. Stahl, July 13, 1981

ABSTRACT X is a genetic element that stimulates phage A recombination by the Escherichia coi recBC pathway during lytic infection [Stahl, F. W. (1979) Annu Rea Genet. 13, 7-241. Herein we show that X in A prophage influences exchange distribution in P1 phage-mediated transduction and in conjugation. This demonstration encourages the view that X may influence genetic exchange in E. coi in the total absence of A.

Generalized recombination promoted by the recBC pathway of Escherichia coli occurs more or less uniformly along bacteriophage A chromosomes (1). Mutations of A that create recombination hotspots have been isolated and called x (refs. 2-4; for review see ref. 5). These mutations, arising at four or more different sites in A (6), confer a growth advantage to red- gamphage, which must use the host recombination system to make concatamers from which virion chromosomes can be packaged (7, 8). Experiments on the effects of X in A lytic crosses have revealed the following: (i) x enhances recombination at and near its locale (2-4, 6). (ii) X can promote recombination when present in only one of the two participating chromosomes (3). (iii) X can stimulate recombination even when embedded in a stretch of DNA that has no homology with the other chromosome. In this case, x-enhanced crossing-over occurs in homologous DNA adjacent to the heterologous region (9, 10). (iv) X activity is dependent on products of the recA, recB, and recC genes, which define the recBC recombination pathway of E. coli. Neither A's red nor E. coli's recE or recF pathway utilizes X (11, 12). (v) X promotes recombination more to one side of itself than to the other (13). (vi) Complementary products of tpromoted exchange are recovered unequally (3, 13). In crosses between X0 and X+ phages, A°-containing recombinants are preferentially recovered from x-stimulated crossing-over. Circumstantial evidence suggests that X may play a role in E. coli recombination: (i) xcan utilize only E. coli's predominant (recBC) recombination pathway, and (ii) X exists in the DNA of E. coli at a frequency of 1 per 5 X 103 base pairs (8, 14). The work presented here shows that X can act in transduction- and conjugation-associated recombination in the absence of A lytic functions. The X sites examined are in A prophage, and they are seen to alter the distribution of exchanges in their neighborhood.

XA I J(h)

A

A

0

A

20

A

40

AB

nin5 - XD att red N dIO QS int gam rex A

60

A

80

A

100

FIG. 1. Abbreviated map of A showingpertinent genes, mutations, and landmarks. Solid bars represent deletions used. Positions of EcoRI restriction sites are identified by numbers enclosed in arrows. The fragments resulting from EcoRl cuts are identified by letters in circles. The scale is in percent physical distance from left end.

MATERIALS AND METHODS Phage and Bacterial Strains. See Tables 1 and 2 and Fig. 1. All lysogens were verified to be monolysogens; lysogens used as recipients in transduction or conjugation were made unable to adsorb A by the selection of mutants resistant to A vir. Experimental Conditions and Media. Most growth media have been described (26, 31). The minimal (OMB) plates were supplemented with biotin at 0.1 gg/ml, glucose or galactose

at 0.2%, vitamin-free casamino acids at 0.04%, streptomycin at 250 ug/ml, or individual amino acids at final concentrations of 20 Ag/ml as appropriate. Avidin (0.2 ml at 0.1 mg/ml) was spread on plates to ensure that they were effectively lacking in biotin. P1 Phage Transductions. A fresh overnight culture of the recipient strain in LB-H broth was diluted 1:100 in LB-H and grown for about 2.5 hr at 340C until the cell density was about 1.65 X 108 per ml. The culture was then made 0.01 M in MgS04. Aliquots of culture (0.2 ml) were combined with 0.1 ml of P1 phage (at 3 x 108 plaque-forming units/ml) grown on the donor for two cycles in liquid culture. After 20 min for adsorption at 340C, 0.25 ml of 25% sodium citrate was added before the tube's contents were spread on minimal OMB plates supplemented appropriately. Plates were then incubated at 340C. Testing Transductants for Nonselected Markers. Individual transductants were picked, then streaked on selective plates and incubated. When selection was for biotin independence (Bio'), these purified transductants were inoculated in a grid on an eosin/methylene blue/galactose plate and incubated overnight. Prophage markers (A', O+, h+, vs. tsA, susO, and h, respectively) were determined by replicating grid plates onto plates spread with C600, C600/A (A h-sensitive), or 594. The first plate was incubated at 400C and the other two at 34TC. Before incubation, the plates were exposed to UV light (0. 8 J/ mI per sec for 60 sec) to induce the prophage. After overnight incubation, the plates were scored for phage growth. Conjugation. Conjugation experiments used exponentially growing cultures of donors and recipients obtained by 2-hr growth at 37C of a 1:100 dilution of an overnight culture in LBH. Donor and recipient were mixed at a ratio of 1:20 and incubated at 37°C for 45 min. To disrupt the mating pairs, the mixed culture was diluted 1:5 and stirred vigorously in a Vortex mixer. Appropriate dilutions of the exconjugants were plated on OMB minimal plates containing streptomycin and appropriate nutritional supplements. Recombinant colonies arising on these plates were purified by streaking on selective plates.

The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement'? in accordance with 18 U. S. C. §1734 solely to indicate this ftact.

Abbreviations: Su' and Su-, suppressor and nonsuppressor; Bio+, biotin-independent; Gal+, galactose-utilizing. * To whom reprint requests should be addressed.

7033

7034

Genetics: Dower and Stahl

Table 1. Phage mutations employed Mutation Properties Deletions (in Agt-C') AB Deletion of EcoRP fragment B nin5 From 83.8 to 89.2 Other mutations tsAU4 Blocks growth at 420C h In gene J; allows growth on C600/A sus gam210 Suppressible gam mutation rex5a Unconditional rex c126 Unconditional cI susO29 Blocks growth on Su- host Avir Virulent (triple mutant) X+A131 X located between I811 and J486

X+D123 Agt-C'

X located between Q and S

EcoRI fragment B and nin5 deleted; EcoRI fragment C

Proc., Nad Acad. Sci. USA 78 (1981) Ref. 15 16 17 18 19 20 21 22 23, 24

Ref. 6; personal communication, A. Taylor and G. Smith, Univ. Oregon 6, 25 15

inverted Su-, nonsuppressing.

The purified recombinants were then placed in a grid pattern on a master plate, and the prophage genetic markers were determined as in the transduction experiments. Statistical Test. Comparisons between the frequencies of crossovers in given intervals were made with the contingency

x2 test. RESULTS Demonstration of X Effects in Transduction. In our standard protocol, based on that of Rothman (32), P1 infects E. coli that are galactose utilizers (Gal') and Bio' and that contain a single Table 2. Strains of bacteria employed Designation Relevant properties CSH63 HfrH, gal+ C600 rec+, SuIl C600/A A-resistant but Ahsensitive, Sull' 594 m+ SuND1* rec+ Su- gal-1,2 bioND13t recB21 sbcA20 gal+ bio

ND15* ND24§ MS6033 W602(AdbioR24-2)

JM1 JC4693

rec+ gal+ bio- Sull+ isogenic with ND13 HfrH, galU bio+ Sugal+ V(chID-pgl) bio+ Sugal-1,2 bio- Su+ rec+ (Adbio) recB21 sbcA29 Sui,+ rec+ Su- trp,, precursor Of JM1

Ref. or source 26 27 M. Meselson, Harvard 28 This study This study

This study This study 29 30 13 A. J. Clark, Univ.

Calif., Berkeley

Su+, suppressing. * W602(AbioR24-2) cured of Adbio. tgal+ and bw- were cotransduced from agal+ bio- derivative of ND1 into agai- derivative of JM1 obtained by 2-deoxygalactose selection. tgal' and bw- were cotransduced from agal+ bio- derivative of ND1 into agal- derivative of JC4693 obtained by 2-deoxygalactose selection. Next Sul+ was transduced into this strain from JM1 and selected for by its ability to suppress the trp,, mutation. § A gal- derivative obtained by 2-deoxygalactose selection of CSH63 was first transduced to gal+ bio- from a gal+ derivative of ND1 and then to gal-1,2 bio+ from a bio+ derivative of ND1.

prophage marked with three mutations-tsA, h, and susO. The lysate produced by this infection is used to infect recipient lysogens that are gal- and bio- and that contain wild-type alleles of the A, J, and 0 genes of A. The injected DNA can recombine with the recipient chromosome to yield Bio+ transductants. Among these transductants, one crossover is in a unmonitored region to the right of bio (see Fig. 2), while the position of the other crossover(s) is determined by identifying other markers acquired from the donor. For example, if a transductant has the markers gal- O+ A' h, the monitored exchange has occurred in the interval A-h (Fig. 2). Some of the transductants enjoy quadruple exchanges. A transductant of genotype gal- O- A+ h+ would be scored as crossover in intervals bio-h, A-0, and 0-gal. xcan be introduced into the prophage, and the crossover distribution obtained can be compared to the control lacking x Some of the experiments involve modifications of this pro-

tocol as noted. The frequency of crossovers in a given interval is expressed as a percentage of all Bio+ (or Gal') transductants. Thus, an apparent increase in crossover rate in a given interval may actually be a result of an absolute decrease in other intervals. In this paper, apparent increases may be referred to as "increases" rather than "relative increases." Wild-Type Orientation of Prophage A. The circular A chromosome integrates into the E. coli chromosome by reciprocal exchange between the attachment site (att) of A and the A attachment site of E. coli. The att site is located near the middle of the virion chromosome, so the gene order of the prophage is a circular permutation of the virion gene order. When att is in its standard ("wild type") orientation in A, the order of our markers is gal-0-A-h-bio. P1 lysates were made on the Gal+ Bio+ C600(AtsA h susO), C600(A tsA V+A h susO), and C600(AtsA h susO V+D) strains and used to transduce the rec+ recipient Gal- Bio- ND1(A+)/ A. The distributions of exchanges that generated the Bio+ transductants are presented in Table 3. Our statistical tests show that the X+A and X+ D results are not significantly different from the

x ones. An explanation for the lack ofa X effect in these experiments invokes two features of X activity found in A lytic crosses, nonreciprocality and directionality (see Introduction). If X is a prophage acts as it does in a lytic cross, the X stimulation could go undetected according to whether X and the selected marker (bio+ in this case) are in coupling or repulsion and according to the orientation of X with respect to the selected marker. If we presume that the directionality ofV in transduction has the same sense as in A lytic crosses (toward gene A for A+A and toward 0 for V+D), then the experiments described in this section are

unlikely to have detected X activity (see Fig. 3). Agt'C' Prophage Orientation. One way to circumvent the problem of nonreciprocality/directionality is by inverting the X site. To do this, we used an in vitro derivative of A called Agt C' (15), which integrates into the E. coli chromosome with most of its genes in the opposite orientation to those of wildtype A. In these experiments, our markers are in the order gal-h-A-0-bio. ISUSO tsA h , bi,

,

_

_

I -9b , :gal-i + ~~~::} a _,i _

_ _.-

_

_

,

_

_

__

_

_

Donor

Recipient

FIG. 2. Consequences of recombination events during transduction for Bio+. One occurs to the right of bio in an unmonitored region. The other exchange(s) occurs in intervals defined by the gal gene of E. coli and the 0, A, and J (h) genes of prophage A. Squares indicate the prophage termini.

Genetics: Dower and Stahl

Proc. Natl Acad. Sci. USA 78 (1981)

Table 3. Distribution of exchanges that generated Bio+ transductants

Table 4. Distribution of exchanges that generated Bio+ transductants, with XD in Agt-C' orientation Total

Total

Exchanges, % %A in A orientation

gal

xO

6.9 X+ 4.4

0 39.5 39.7

0.00

X2 0.92 gal

X0 6.9 X+ 6.7 X2 0.01

9.2 13.2

19.6 16.2

1.72

gal

bio 24.8 26.5

0.75

0.14

X0 7.1 306 204

3.02

bio 306 313

1.40

Entries are percent of all exchanges occurring in the indicated interval. Contingency x2 values are given for each interval (1 degree of feedom). None of theP values is