Genetic map of coliphage 186 from a novel use of marker rescue frequencies

NIG'G

Mol Gen Genet (1982) 187:87-95

© Springer-Verlag i982

Genetic Map of Coliphage 186 from a Novel Use of Marker Rescue Frequencies Stephanie M. Hocking and J. Barry Egan Department of Biochemistry, University of Adelaide, Adelaide, South Australia, 5000, Australia

Summary. A genetic map of phage 186 has been constructed, using the frequency of marker rescue from 186 mutant prophages for genes to the left of att, and int promoted recombination for genes to its right. At the left end of the genome lie 7 genes involved in the formation of the phage head, followed to the right by the lysis gene P, a gene (O) of unknown function, and a group of 11 genes involved in the formation of the phage tail. Gene B, the late control gene, lies to the right of this group but to the left of the phage attachment site. To the right of the a n site lie the non-essential genes (cI and cII) involved in lysogen formation and the gene (A) required for 186 D N A synthesis.

Introduction

Coliphages 186 and P2 are closely related (Bertani and Bertani 1971) and this relationship is mirrored in the problems encountered in the construction of their genetic maps, for both phages display very low recombination frequencies, some hundred-fold lower than that of coliphage 2. Initial attempts to generate a map of 186 involving two-factor crosses led to variable and inconsistent map orders, and in some cases the unlikely mapping of head genes among tail genes. Three-factor crosses, although satisfactory for mapping purposes despite very high negative interference (as with P2, Lindahl 1969b), necessitated the construction of a number of double mutants, and this proved impractical due to the low recombination frequency of 186 and the absence of a selective technique. We therefore investigated the possibility of deletion mapping, using as a set of overlapping deletions a series of P2.186 hybrid phages with deOffprint requests to: J.B. Egan

creasing 186 content (Hocking and Egan 1982c). We used a marker rescue approach, determining the ability of an amber mutant to form plaques on a nonpermissive lysogen of each hybrid - the amber mutant carried the vir mutation enabling it to bypass lysogenic immunity. From the results we realized that, while the presence/absence of marker rescue enabled the ordering of limited groups of genes, the frequencies of marker rescue could be used to give a consistent allele order, the frequency approaching zero as the allele locus approached to P2.186 junction (novel joint) of the hybrid. This fact implied that marker rescue occurred by recombination rather than complementation, which one would expect to show no relationship to the distance of the allele from the novel joint. We therefore predicted that the frequency of marker rescue from a prophage that is itself mutant could be exploited to generate a genetic map, and the rationale and results of this activity are presented in this paper. Materials and Methods

The media used and all general methods involving the preparation of phage stocks, phage assays and construction of lysogens have been described in Hocking and Egan (1982 a). Bacteria. The bacterial strains used are listed in Table 1. Bacteriophages. The 186 amber mutants used are described in Hocking and Egan (1982a), and cItsp is a temperature sensitive mutant of the repressor gene, while cII is a gene necessary in the establishment of lysogeny. A spontaneous clear plaque mutant appeared in a stock of 186cItspPam16 (W.H. Woods 1972, Ph.D. Thesis, University of Adelaide). This mutant is able to grow on a 186 lysogen and is therefore virulent. The new mutant is known as 186cItspPam-

Table l. Bacterial strains Collection number

Relevant phenotype

Genotype

Origin or reference

Su Su + Su + Su + Su- recA

F- galK galT str594 F- thr leu thi lacy tonA supE HfrC rel tonA T2rphoA supD HfrC rel tonA T2rphoA supF

Campbell (1965) Appleyard (1954) Garen et al. (1965) Garen et al. (1965) Gottesman and Yarmolinsky (1968)

E. coli K12

594 C600 S26RIe H12R8A 152

F - galK str recA56

0026-8925/82/0187/0087/$01.80

88 16vir2 and the vir2 mutation from this strain has been recombined into all the other 186cItsp am mutants.

Construction of 186 am vir Double Mutants

Two methods have been used to cross the vir2 mutation of the phage 186cItspPam16vir2 into the other 186cItsp am mutants, generally referred to here as 186cItsp Yam. Two-factor Crosses. 186cItspPaml 6vir2 and 186cItsp Yam were used as parental phages in the standard two-factor cross procedure. The progeny phages were plated on the supF suppressor strain H12RSA lysogenic for the mutant Paml6 and the plates were incubated overnight at 30 ° C. Since Pam16 is not suppressed by supF suppressors the major plaque-former will be the recombinant 186cItsp Yam vir2. (Int-promoted recombination predominates in 186 two-factor crosses, see results.) vir2 derivatives of the mutants Dam26, Fam20, Mam19, Oam6] and Waml5 were prepared by this method. As an alternative the mutant Gam27vir2 was used in place of Pam16vir2. In this case the progeny phages were assayed at 30 ° C on C600 lysogenic for the mutant Gam27 since the mutation Gam27 is not suppressed by supE suppressors. This was the procedure used to produce vir2 derivatives of the mutants Qaml, Ram51 and Uam37. Eam7vir2 was prepared from a cross between 186cIts pEam7 and 186cItspPam16vir2 as described above except that the selective indicator strain used was H12R8A lysogenic for the mutant Jam41. In addition to the Eam7vir2 recombinants, Jam41 vir2 and am +vir2 phages were also obtained, as a result of marker rescue from the prophage of the indicator strain. All vir2 derivatives constructed were purified by three successive single plaque isolation steps and tested for the presence of the particular am mutation by assays on a 594 lysogen of the same am mutant and a 594 lysogen of a different am mutant. Failure to obtain marker rescue from a particular am mutant prophage confirmed the presence of the same allele in the superinfecting phage. Marker Rescue. vir2 derivatives of the 186 am mutant phages can be prepared by assaying a stock of the phage 186cItspPam16vir2 on a H12R8A lysogen of the amber mutant, with incubation at 30 ° C. The superinfecting phage will not grow on this indicator since the supF suppressor of strain H12R8A is unable to suppress the Paml 6 mutation and any phage which appears on this indicator must be the result of rescue of the aml6 + allele from the prophage. These phages will also have thevir2 mutation since the presence of the prophage in the indicator bacteria will prevent the growth of any non-virulent phages. In recovering the aml6 + allele from the prophage, coincident recovery of the am allele of the prophage may also occur giving rise to the required am vir double mutant. Alternatively the am mutation of the prophage may not be recovered and the resultant phage will have the genotype 186cItspvir2. These two genotypes can be distinguished by testing the phages for ability to grow on an Su- strain. This procedure was used to construct a 186cItspGam 27vir2 phage. A similar procedure was used to construct 186cItspNam47vir2 but, due to the lack of an H12RSA

lysogen of Nam47, an S26Rle lysogen was used instead. The mutant Paml6 shows a very reduced plating efficiency on strain S26Rle and the low level of plaques formed are very tiny. Nam47vir2 phages were obtained by selecting larger plaques superimposed on a background of these tiny plaques. A variation of the above procedure involved assays of the phage 186cItspGam27vir2 (prepared as described above) on C600 lysogens of various 186 am mutant phages. This was the procedure used for the construction of vir2 derivatives of all 186 am mutants other than those discussed above. It was the preferred method as C600 lysogens had been prepared for most mutants. This procedure is simpler than the two-factor recombination method and since the frequency of am vir double mutants was high the presence of the am + vir phages was not a problem, am vir double mutants could usually be selected successfully on the basis of plaque size since the plaques produced by these phages were usually smaller than those produced by am + vir phages. The vir2 derivatives obtained were purified and tested as described above.

Construction of 186cItsp am Mutants from 186c! am or 186cH am Mutants

Several of the 186 am mutant phages also had a non-conditional mutation in either the cI or cII gene and it was necessary to cross this mutation out and replace it with a cIts pcII + combination so that lysogens of these phages could be made. The mutants Qaml and Ram51 have such additional clear plaque mutations and, while the mutant Uam37 gave turbid plaques on C600 at 30 ° C, attempts to lysogenize C600 and 594 strains failed. This might be due to an additional mutation in the int gene but this has not been examined. These mutants were first converted to am vir double mutants as described above. The double mutants were then recombined with the mutant 186cItspGam27 and the progeny phages plated on C600 at 30 ° C. Two plaque types were produced; clear plaques produced by the parental am vir phages and turbid plaques produced by the desired recombinant, 186cItspQaml, 186cItspRam51 or 186cItspUam37. Turbid plaque isolates were purified by three successive single plaque isolation steps and tested by plating on C600, H12R8A and 594. The non-conditional clear plaque mutation present in the phage Oam62 was removed directly by recombination with 186cItspPam16 and plating of progeny phages on strain HI2R8A. Two plaque types again resulted; clear plaques produced by parental Oam62 phages and turbid plaques produced by the 186cItspOam62 recombinants. A turbid plaque isolate was purified and tested as described above. The non-conditional clear plaque mutation has not been crossed out of the mutant Wam52cItspcII. The cII mutation of the phage 186Ham56cII was removed by recombining this mutant with 186cItspGam27 and plating the progeny phages on C600 at 30 ° C. 11 turbid plaques were obtained, although the frequency was very low (1 turbid plaque for every 55,000 clear plaques) due to the presence of the cI + allele in the Ham56cII phage (see under int-promoted recombination). These 11 plaques were all produced by 186cItspHam56 phages and one was selected, purified and tested as described above.

89

186 Marker Rescue

(a)

186 am vir double mutants were assayed on C600, 594 and 594(186cItsp) by the procedure described in Hocking and Egan (1982a) except that the 20 min preincubation was at r o o m temperature and the plates were incubated overnight at 30 ° C. The indicator strains were grown to log phase at 30 ° C. Results

.... v-Jr

I

•. - . |

I I...

--

mrf

o¢ b ( c ÷ d - b )

mrf

04, a ( c + d - b )

]r

c --4~1d. b _ll... d ~

(b) x

Genes to the Right o f att - The Use o f Int Promoted Recombination A l t h o u g h low recombination frequencies (usually 0.01%) were obtained in two-factor crosses involving mutants from genes B to W, frequencies 10 to 50-fold higher (0.13 to 0.45%) were obtained in crosses in which one of the mutants was from gene A. This resulted in the appearance o f a " l o n g b l a n k r e g i o n " in the genetic map, with gene A m a p p i n g at one end (the right h a n d end) and genes B to W clustered at the other. By analogy with the " l o n g blank r e g i o n " found for the related phage P2 (Lindahl 1969a, b) this was assumed to reflect the existence of a relatively efficient int-promoted recombination system acting at a site (the att site) between gene A and genes B to W. The att-int region o f phage 186 has been located 70% from the left h a n d end o f the genome (Younghusband et al. 1975). This efficient recombination system a p p e a r e d to be under repressor control since relatively high recombination frequencies were only obtained if b o t h parental phages carried m u t a t i o n s in the cI (repressor) gene. I f one o f the parental phages carried a wild-type cI gene the recombination frequency was reduced 10-fold (0.016 to 0.048%). This reduction in frequency in the presence of the c + allele demonstrates that the " l o n g blank r e g i o n " is not a physical entity but is due to a b n o r m a l l y high recombination in this region. Besides gene A, the genes cI, eII and vir2 all m a p to the right o f the att-int region (i.e. the " l o n g blank region"), since in the crosses cItspAm5 x cI-cItspRam51, cItspAam 5 x cII-cItspWam52 and cItspAam5 x vir2cItspBam17 a large majority of the am + recombinants (97% of 684, 96% o f 238 and 96% of 670, respectively) had clear plaque morphologies at 30 ° C. In the last cross all 100 of the clear plaque recombinants tested were shown to be virulent since all were able to grow on a 186 lysogen.

Genes to the Left o f att - The Use o f the Relative Marker Rescue Frequency (relmrf) f o r Mapping In this w o r k the relmrf ~ is defined as the frequency of m a r k e r rescue for a particular 186 am vir double m u t a n t from an S u - indicator lysogenic for a 186 m u t a n t prophage, relative to the frequency of m a r k e r rescue for the same 186 am vir phage from an Su indicator lysogenic for a 186 wild-type prophage. In practice it is expressed as a percentage and, for a particular am vir mutant, is calculated from the pfu obtained on S u - ( 1 8 6 Xam) multiplied by 100 and divided by the pfu obtained on S u - (186). F r o m Fig. 1 it can be seen that the relmrf is directly p r o p o r t i o n a l to a/b, if the p r o p h a g e m u t a t i o n X lies to the right of the

Abbreviations used. relmrf relative marker rescue frequency; turf marker rescue frequency; pfu plaque forming units

__..~..,

L. _ . _

+

vlr

a(c*d-b) rel mrf~.b~c+d_b~

ka-I

(c)

a

x

,,-- --] -F--:~:'"J vlr

' L.-. "~P Fa.~

mrf

oCab

relmrfiK ab b(c * d - b ) a o(

c +d-b

Fig. I a-e. Marker rescue from 186 am mutant prophages. The ability of a 186Yamvir phage to form a plaque on Su-(186) or Su-(186Xam) was considered to be due to the formation of an am + vir recombinant from a double crossover. The figure represents a successful working model, and is not intended to inidicate insight as to the actual mechanism of recombinant formation. In the figure__I-I__ represents the cross-over,-----.~--- the cohesive ends, N---.)-----Nthe wild-type prophage and N-----) x N the mutant prophage, v~r o) Y is a linear representation of a circularised superinfecting Yam vir phage. X and Y represent amber mutations in essential genes, a is the distance between X and Y, b the distance from Y to the phage attachment site (att) and e the distance from vir to the cohesive ends. a Marker rescue from a wild-type prophage, b Marker rescue from a mutant prophage, where X lies to the right of I1. c Marker rescue from a mutant prophage, where X lies to the left of Y

superinfecting phage m u t a t i o n Y, or to a / ( c + d - b ) if X lies to the left of Y; where a represents the distance between X and Y, b the distance between Y and the right-hand phage-bacterial D N A junction, c the distance between vir and the cohesive ends and d the distance between the cohesive ends and the right-hand phage-bacterial D N A junction. F o r a constant superinfecting phage m u t a n t Y with variable p r o p h a g e m u t a n t s X, b, c and d are constant and a is variable, so that the relmrf is directly p r o p o r t i o n a l to a, the distance between X and Y. However, even though the relmrf is directly p r o p o r t i o n a l to a, relmrfs for crosses with X to the right o f Y cannot be related to the relmrfs for crosses with X to the left o f Y since the d e n o m i n a t o r for a given Y differs for the two situations. This p r o b l e m can be overcome by comparing the relmrfs only for a Y mutation m a p p i n g at one end o f a set of X mutations or by considering the relmrfs for X mutations on either side of particular Y separately. F o r a constant p r o p h a g e m u t a n t X, with variable superinfecting phage mutants I7, c a n d d are constant but both a and b are variable so that the relmrfis not directly p r o p o r tional to a, the distance between mutations. In theory it would a p p e a r that this type o f experiment could still be

9O Table 2. Marker rescue frequencies for 186 amber mutants from a 186cItsp prophage"

Table 3. Relative marker rescue frequencies for alleles of 186 genes B, D and E a

Mutant b % m r f c

Mutant

%mrf

Mutant

%mrf

Bam17 Barn57 Dam14 Dam23 Dam26 Dam48 Eam35 Eam46 Eam7 Fam20 Gam9 Gam27 Gain25 Gam29 Ham50 Ham56 Iam40a

Jam41 Kam42 Kam22 Kam58 Lain2 Lam21 Mam19 Mam31 Mam60 Nam47 Oam61 Oam62 Pam 36 Pare45 Pam65 Pam16 Pam66

0.12 0.19 0.19 0.17 0.18 0.19 0.35 0.24 0.20 0.33 0.50 0.52 0.61 0.61 0.51 0.82 0.29

Pam67 Qam49 Qaml Ram6 Ram51 Sam34 Sam4 Sam18 Tam8 Uam37 Uam64 Uam63 Vam38 Warn15 Warn39

0.66 0.58 0.48 0.37 0.23 0.42 0.55 0.88 0.11 0.16 0.36 0.37 0.38 0.21 0.25

SuperProphage ~ infecting phage b Eam7 Earn46 Dam26 Dam23 Dam14 Barn57 Baml7

23 16 0.09 0.12 0.18 0.25 0.22 0.35 0.26 0.12 0.21 0.17 0.12 0.10 0.32 0.29 0.18

a The figure given for each mutant was the average result of between 2 and 8 separate experiments, except for Eam46 (average of 10 experiments) and Barn57, Pam65 and Sam18 (one experiment each) b Each mutant also carried the vir2 mutation m.,~-=pfu,~on Su-(186cItsp)x 100 c Calculated a s ~ % pfu on Su + The Su- strain used was a 594 lysogen of 186eItsp. The Su + strain used was C600 for all mutants except Gam27 and Nam47 for which H12R8A and S26RIe were used respectively

used to provide a m a p order but in practice this was found not to be possible and in this p a p e r no comparisons have been made between relmrfs obtained for different superinfecting phage mutants from the same p r o p h a g e mutant. The relmrfrather than a simple m a r k e r rescue frequency (mrJ) was used because it eliminated any influence due to the plating efficiency o f an amber m u t a n t on an Su + indicator. Such an influence was seen in the mrfs, recorded in Table 2, for 186 Yam vir mutants plated on Su (186). The frequencies obtained (excluding the results for B I 7 2 and B57 which a p p e a r to represent a unique example of rescue by complementation) were generally between 0.1% and 0.7%, but considerable variation was seen even for mutants in the same gene. F o r example the m r f for P16 was 0.82% while that for P66 was 0.29%. Since the two mutants belong to the same gene, a similar m r f could be expected if the m r f w e r e solely dependent u p o n recombination. This large variation therefore p r o b a b l y reflects a difference in the d e n o m i n a t o r rather than in the numerator, that is in different plating efficiencies of P a m l 6 and Pare66 on the Su + indicator. Use of the relmrfavoids this complication since the plating efficiency on Su + indicator does not contribute to the expression. Initial confirmation that the relmrfs were p r o p o r t i o n a l (qualitatively) to the distance between markers was obtained by using 186 mutants which h a d already been mapped. M a r k e r rescue experiments from P2-186 hybrid prophages (Hocking and Egan 1982c) provided the order z In this paper the amber mutants will generally be referred to by their gene letter and allele number only, e.g. Barn] 7 will be referred to as B17

Eam7