VIROLOGY 63, 147-159 (1975)

Isolation,

Characterization Mutations

MARLENE Department

and Deletion

of Amber

in the cll Gene of Phage X

BELFORT, DINA NOFF,

of Microbiological

Mapping

Chemistry,

AND

AMOS B. OPPENHEIM

The Hebrew University-Hadassah ha el

Accepted

Medical

School, Jerusalem,

August 27, 1974

(SUS) mutations in the The isolation and characterization of amber suppressor-sensitive Xc11 gene is described. A series of Xspi-uir-nin deletion strains, extending for various lengths into the Xc11 gene, was generated and characterized in order to facilitate the mapping of Xc11 point mutants. The eight CR amber mutants fall into two clusters with respect to map position and response to bacterial suppression. A unique feature of the cI1 amber mutants is their narrow range of sensitivity to amber and ochre suppressors. Moreover, relief of the cII- phenotype is highly dependent on the efficiency of chain propagation by an effective suppressor. The implications of the existence of cI1 amber mutants and of their highly specific patterns of suppressibility are discussed in terms of the structure and function of the Xc11 gene product.

INTRODUCTION

Establishment of lysogeny by bacteriophage X is dependent upon the timely synthesis of repressor protein, the product of the Xc1 gene (Ptashne, 1971; Eisen and Ptashne, 1971). Repressor synthesis is initiated in the y region and is under the positive control of the Xc11and Xc111gene products (Echols and Green, 1971; Reichardt and Kaiser, 1971). The lytic pathway is followed almost exclusively when there are mutations in any of these genetic elements, resulting in the clear plaques characteristic of XcI, XcII, Xc111and Xcy strains (Kaiser, 1957). The structure and function of the Xc1 repressor has been under intensive and fruitful investigation (Ptashne, 1971; Maniatis and Ptashne, 1973). Our knowledge of the action of the Xc11and Xc111products is much poorer, and while nonsense mutations’ exist in the Xc111gene (see in Signer et al., 1969) none has ever revealed ’ The terms “nonsense,” “amber” and/or “ochre,” and “suppressor sensitive (sus)” are used interchangeably. Copyright 0 1975 by Academic All rights of reproduction

Press, Inc. in any form reserved.

147

itself in ~11, despite extensive searches.2 There was therefore some uncertainty as to whether the functional cI1 product is indeed a protein. This prompted us to reinvestigate the existence of amber mutations within the cI1 gene by devising a rigorous selection for cI1 mutants and by screening these for sensitivity to suppression on a variety of permissive3 hosts. We were thus able to isolate 400 cI1 mutants, 8 of which are partially suppressed by the appropriate bacterial amber suppressor strains. The cI1 character of our mutants was established by their complementation behavior and confirmed by their map position. In order to verify and more precisely determine their position inferred from three-factor crosses, we developed a deletion map of the Xc11 region. Deletions extending from the Xatt site through the ‘The mutant Xc1128 has been referred to as amber (Echols and Green, 1971). It has not, however, been found to be suppressible by any of the many supressors used in this study. ‘The terms “permissive,” “amber and/or ochre suppressing” and “suX” are used interchangeably.

148

BELFORT.

NOFF AND OPPENHEIM

immunity region and for various lengths into the cI1 gene facilitated the mapping of the cI1 amber mutants and confirmed the positions of previously mapped cI1 mutants. This method together with two-factor crosses revealed two clusters of amber mutants within the cI1 gene. Investigations of plaque morphology, lysogenic response and repressor levels on a range of bacterial suppressor strains showed that the cI1 mutants of each cluster have a characteristic and highly specific pattern of suppressibility. This allows for interesting speculation on the structure and function of the Xc11protein. MATERIALS

AND

METHODS

Media and Buffers

Tryptone broth (TB) (1% Bacto tryptone; 0.5% NaCl), TB bottom agar (TB + 1% Bacto agar) and TB top agar (TB + 0.6% Bacto agar) were routinely used for growth of bacteria and phages. TB-ma1 contains 0.2% maltose, while LB is TB enriched with 0.5% yeast extract. EMBO plates contain EMB-agar base and 0.1% yeast extract. McConkey-lactose base supplemented with 7 g of Bacto agar/liter was used for identification of Lac+ revertants of M721ac-su-. Suspension of bacteria, dilution of phages and adsorption were routinely performed in 10m2M MgSO,. Tris-magnesium buffer (TM) is 10e2 M Tris (pH 7.4) and 1O-2 M MgSO,. Bacterial

and Bacteriophage

Strains

The names and relevant characteristics of bacterial and bacteriophage strains used in this study are listed in Tables 1 and 2 respectively. Isolation

of Xspi-vir Deletion

Strains

A mid-log phase lvsogenic culture of was given an inducing dose of uv light and incubated at 37” for 2 hr. The resulting lysate was plated with a light suspension of W3350(hind -) indicator (ca. lOa cells/plate) onto fresh tryptone plates, and after an overnight incubation at 38” the small clear plaques which appeared were purified and tested for their ability to M72(Xnin5)

grow on W3350, W3350(Xind-) and M72(P2) and their inability to grow on W335OlX and 152recA. Growth on hindlysogens selects for deletions that extend from the attX site through the immunity region and toward and into the cII-O-P operon (Fig. 1). The deletion of the recombination region from the X chromosome is reflected in the ability of these virulent strains to grow on P2 lysogens (i.e., their spi- character; see Lindahl et al., 1970) and in their inability to grow on a recA strain (Zissler et al., 1971). The spi-uir character of 80 independent isolates was thus confirmed. Hydroxylamine

Mutagenesis

Wild-type phage stocks were mutagenized essentially according to the procedure of Scott (1968). Crude lysates were diluted lo-fold into LB containing 0.5 M hydroxylamine (pH 7.8) and incubated at 30”. Samples were withdrawn at intervals over 48 hr and diluted lOO-fold into TM buffer containing 10% (v:v) acetone, and stored at 4”. Each sample was plated on W3350 to determine percent survival and on ER437 to ascertain the number of clear-plaque forming phage. Those samples which yielded a 30- to loo-fold enrichment of clear-plaque formers per viable phage were used in mutant selection experiments. This enrichment was achieved at a level of approximately 99% killing. Mutant

Selection

and Screening

Mutagenized Xc+ and Ximm434c+ lysates were plated on a lawn of ER437, upon which c+ and ~111~phage do not form plaques (Rolfe et al., 1973; Oppenheim et al., 1974). ER437 therefore provides a useful selection for certain clear-plaque forming phage. Several hundred clear-plaque formers thus obtained were purified by streaking onto a lawn of W3350 and each purified plaque was inoculated into 0.7 ml of chloroform-saturated low2 M MgSO, in a well contained within a Teflon tray. The phages were transferred by a grid of 20 nails corresponding to the grid of 20 wells within each teflon tray. Twenty phages at a time could thus be easily “printed” onto a

149

THE Xc11 GENE TABLE BACTERIAL

Bacterial

1 STRAINS

Source and/or reference

Genetic markers and/or special properties

strain

used for

and Balbinder

Standard su-(pm-) phage plating

W335OlX W3350(Xind-)

Derivatives

of W3350

C606

Standard

su2+ indicator

strain”

Appleyard

(1954)

Ymel, QD5003

Standard

.su3+ indicator

strain

Bachmann

(1972)

ER437

Does not allow shutoff of h repressor synthesis or plaque formation by Xc+ or XcIII-. No suppressor activity was detected with a variety of Xsus mutants which are well suppressed by many amber and ochre suppressors.

Rolfe et al. (1973) Oppenheim et al. (1974)

UC4185(Xrim)

When used as indicator to many Ximm434cII

Belfort

NlOO. 152

recAsu-strR

M72

lcf-am

M721ac+ M72su2+ M72su3 +

trp-am su(lac-am trpam)+su2+ (lac-am trp-am)+su3+

M72(P2),

indicator

Campbell

w3350

This laboratory

imparts turbidity mutants

From J. Beckwith

su-

Ca13.B H13R7A Ca13.5 Ca13.12 (I Nomenclature

via P. Brachet

Derived as spontaneous Lac+ revertants on McKonkey agar. Assignment of su+ genotype was based on their ability to suppress a variety of characteristic hsus mutants. More information on request. This laboratory

M72(nin5)

Cal3suCa13.1 Ca13.2 Ca13.3 Ca13.6 Ca13.7

and Wulff (1973)

From M. Meselson

strains

trp-am

(1958)

F-try- am lac-am susul+, inserts serine for UAG su2+, inserts glutamine for UAG su3+, inserts tyrosine for UAG su6+, inserts leucine for UAG su7+, inserts glutamine for UAG suppressor carried on F’ element SUB+, inserts glutamine for UAA suC = su4+, inserts tyrosine for UAA su5+, inserts lysine for UAA sul2+, inserts glutamine for UAA of suppressors is that suggested by Gorini

variety of different hosts. In the initial screening the phages were printed onto lawns of W335O(su-), Cal3(sul+), C600 (su2+) and Ymel(su3+) to test suppressibility of their clear-plaque character

Ca13.1, Ca13.2, Ca13.3, Ca13.6 and Ca13.B are Pl transductants of Cal3su-, while the other members of the Cal3 series were obtained by Hfr crosses. The Cal3 series was kindly supplied by R. Thomas.

(1970).

by the given amber suppressor. Their complementation characteristics were also tested by printing onto plates seeded with W3350 and the particular clear-plaque phage against which the mutagenized can-

150

BELFORT,

NOFF AND OPPENHEIM

TABLE 2 BACTERIOPHAGESTRAINS Strain Xc+, XcIIIco2, XcIII67, XcIIIs~s611, Xcy42, Xcy2001 Ximm434c+, Ximm434 derivatives of the preceding clear mutants, XcI60, XcI90, XcIsus34, Ximm434cI XcIsus494, XcIsu.s5OO XcIIsus302, XcIIsus3O3, XcIIs~s307 Ximm434cIIsus41, Ximm434cIIsus60, Ximm434cIIsus75, Ximm434cIIsus133, Ximm434cIIsus211 XcII119, Xc1168 XcII2002, XcII2038, XcII3010, Xc113031 XcII28 XcIIfllsar sus0 and/or susPderivatives of the above cI1 strains Xspi7lnin5, Xspill3nin5, Xspil56nin5 Xspi-uir20nin5. Xsphir380nin5, Xspi-uir420nin5, Xspi-uiROOnin5, Xspi-uirGOnin5, Xspi-oir80nin5

Source and/or reference Standard

h strains in our collection

Derived from Xc+ by hydroxylamine (HA) mutagenesis Derived by HA mutagenesis of Xc+ as described in this manuscript Derived by HA mutagenesis of Ximm434c+ as described in this manuscript Brachet and Thomas (1969). Supplied by R. Thomas Supplied by H. Echols Isolated by N. Katzir, this laboratory Prepared by crossing a particular clear mutant with the appropriate sus phage of opposite immunity Supplied by G. Smith Derived by induction of M72 (Xnin5) lysogens as described in text

didates were to be tested (see below). Candidates bearing 434 immunity were also printed onto lawns of UC4185(Xriml), upon which a variety of cI1 mutants give turbid plaques (Belfort and Wulff, 1973). The putative cI1 amber mutants were further checked for complementation characteristics by other methods and their identity confirmed by map position (see below).

M MgSO, across an agar plate seeded with

Complementation

Mapping-Genetic

Tests for Lysogeny.

1. Spot test. Each phage was tested for ability to complement XcI, XcII, Xc111and Xcy for lysogeny. TB-agar plates were seeded with 10’ bacteria (W3350) and lo5 to 10’ phage particles of a known clearplaque genotype. Phages to be tested were printed in batches of 20 from teflon trays onto each of the 4 kinds of plate. After overnight incubation at 38” the seeding phage had caused confluent lysis of the W3350 lawn except for dense spots of bacterial growth in areas of positive complementation for lysogeny. This method was used for initial screening of large batches of phages. 2. Streak test. Phages at 5 x lo5 - 1 x 107/ml were tested for complementation by running a drop of phage suspension in 10m2

10’ W3350 in 2.5 ml of TB top agar. After the streak was allowed to dry, complementing phage drops at similar titer were run at right angles over the original streak. Positive complementation for lysogeny is indicated by a zone of bacterial growth at the intersection of two phage streaks after overnight incubation at 38”. Recombination

Phage crosses were routinely performed by infecting logarithmic cells which had been grown in TB-ma1 and resuspended in lo-’ M MgSO,, at a multiplicity of 3 of both input phages. After 20 min adsorption at room temperature the suspension was diluted lOO-fold into lo-’ M MgSOI, irradiated with uv light at a dose equal to half the maximal inducing dose of CGOO(X+), and further diluted lo-fold into TB. The cells were aerated for 90 min at 37”, then chloroform was added and the lysate containing recombinants plated under the appropriate selective conditions. In the large-scale deletion crosses, cells were irradiated prior to adsorption. After infection with a particular cIIsusP phage and a specific spi- deletion strain the cells

//

SPllU///

/ //

red

/ /1

clll

, ‘I

I

\

_.I

ori cl , x y, cll ,)O , P _ Q . S . R . A

.-.

rex

SPiOO////////////////////////////////////C

N

I

__-.. _-_

/////////////////////////////////Y/L

/ ///

Y

imm 21 c hvn434 (

SPlCO///////////////////////////////////~

spill6

splllO//////////////////////////////

srr200//////////////////////////////d

~~//////////////////////////1

~ltl/////////////////////I

wl30////////////////////

xls

att inf

I -

J

152

BELFORT, NOFF AND OPPENHEIM

ability to rescue turbid recombinants indicates that the specific ~11 mutation lies outside of the particular deletion. Under our conditions a turbid plaque is detectable amongst 10’ clears. We assumed that for a value of less than lo-‘turbid recombinants a clear mutation is covered by a particular deletion. Percent Lysogeny

Bacteria grown in TB-ma1 containing 0.1% yeast extract to the appropriate stage of growth were resuspended in lo-’ M MgSO, and infected at a phage multiplicity of 3. After 20 min adsorption at room temperature the infection mixture was diluted into prewarmed (30”) TB containing 0.1% yeast extract and incubated for 30 min. The infected cells were plated onto TB plates and the colonies were tested for lysogeny by spotting each onto two EMBO plates, one seeded with XcIcIIcIII to score for immune lysogens and the other with Xuir (log phage/plate) to detect possible resistant cells. Alternatively in the case of low-level lysogeny infections (2 2%) cells were plated directly onto unseeded EMBO plates and the phage which were released on the plates caused lysis of sensitive cells. Pale pink intact colonies, *after an overnight incubation at 38”, were scored as lysogens. Uninfected controls were done in both cases. The two methods of scoring lysogens were in good agreement.

Table 3, which indicates that the frequency of ~11 mutants among other clear-plaque mutants is 30-fold higher when the lysate is plated on ER437 than when plated on W3350. A similar enrichment was observed when we used immX phage. This selective capacity of strain ER437 is dealt with more explicity in the Discussion. Approximately 400 putative ~11mutants were tested for turbidity on sul+, su2+ and su3+ amber-suppressor hosts and, subsequently, also on su6 +. Eight suppressible ~11 mutants were thus isolated, five from Ximm434c+ and three from Xc+. In both casestwo types appeared: members of class A form very turbid plaques only on su3+ and not on sul+, su2’ or su6+ suppressor hosts, while class B mutants appear slightly turbid on su2+ but not on sul+, su3+ or su6+ hosts. No strains suppressed by sulf or su6+ suppressors were found. Class A includes strains designated XcIIsus41, XcIIsus75, XcIIsr~.sl33, XcIIsus211, XcIIsus302, and XcIIsus307, while XcIIsus60 and XcIIsus303 comprise Class B. Those strains with numbers below 300 were isolated from Ximm434c+ and those numbered above 300 from Xc+. A more detailed analysis of complementation characteristics and patterns of suppressibility of the strains is given below. 2. Complementation Behavior cll Amber Mutants

of Putative

The complementation data given in Table 4 show that all the suppressible clear

Repressor Synthesis

TABLE 3

Repressor levels were measured in ex- DISTRIBUTIONOF CLEARPLAQUESISOLATEDON W3350 tracts prepared 30 min after infection at AND ER437 FROMHYDROXYLAMINE-TREATED 30” using the filter-binding assay as deXimm434c+ scribed by Echols and Green (1971). CII Host

RESULTS

1. Isolation

of cII Amber Mutants

Number

Complement&ion pattern”

of phage tested CII-

CI- or

like

cy-like

CI t cy other

Hydroxylamine-mutagenized phage ly0.3 50 10 34 6 sates of Xc+ and himm434c+ were plated w3350 9.3 358 290 31 37 ER437 onto lawns of W3350 or ER437 bacteria. The spot-complementation analysis per“Perfectly clear plaques isolated on W3350 and formed on perfectly clear plaques, which ERA37 were restreaked on W3350 and their comhad been repurified on W3350, revealed plementation characteristics for lysogeny with ~1,cII, ER437 to be a strain highly selective for ~111 and cy were determined using the spot-test hcI1 mutants. The analysis of Ximm434 technique described in Materials and Methods with clear mutants is represented by the data in W3350 as an indicator strain.

153

THE Xc11GENE TABLE

4

COMPLEMENTATION ANALYSTS OF PUTATNE cI1 AMBER MUTANTS-C•

Complementingphage

XcIIsu.s

Class A (~41, ~75, ~133, ~211,~302, ~307) Class B (~60, ~303)

MPLEMENTATIONFOR LYSOGENY”

xc1

AC111

ACY

~60

~61 co2

~42 c2001

c3010, c2002 c3031, cl19 ~28, ~68

c susclassA

c susclassB

c90

(c41, c75 c133, en1 ~302, ~307)

(~60, ~303)

+

(+)

+

-

-

+

(+)

+

-

-

xc11

a Complementation was performed on W3350 at 38” using the streak method as described in Materials and Methods. Strong-positive, weak-positive and negative complementation is denoted t, (+) and -, respectively.

mutants of both classes A and I3 complement for lysogeny, like classical ~11’s with XcI, MII and Xcy. Furthermore none of the sus clear mutants was able to complement any of the cI1 mutants tested, some of which, e.g., Xc1168and XcII3010, complement each other (Brachet and Thomas, 1969). No cI1 amber mutant complemented any other cI1 amber mutant. Analysis of repressor levels following infection showed that the mutants we have isolated are unable to stimulate repressor synthesis (Table 5). However, when the cI1 gene product is supplied by a heteroimmune co-infecting phage, X r.tpressor is synthesized (Table 5). These complementation studies strongly indicate that the suppressible clear mutants are in fact cI1 mutants, their identity as such being confirmed by our mapping study. 3. Mapping An examination of recombinants resulting from three-factor crosses between the clear amber mutants bearing 434 immunity and XsusP3 or XsusN7N53 (data not shown) indicated that the sus clear mutations lie outside of the 434 immunity region and to the right thereof (see Fig. 1). Further, in no case could the c mutation be transferred from the putative XcIIsus phage to Ximm2lc+ (by crossing with Ximm2lc+sus029susP3) under conditions in which 10e6 clears were detectable (unpublished observations). From this it was inferred that the clear amber mutants lie within the region covered by imm21, or

TABLE

5

COMPLEMENTATION ANALYSIS OF PUTATIVE ~11 AMBER MUTANTS-C• MPLEMENTATION FOR REPRESSOR SYNTHF~HS

Infecting phage

xc+s7 Xc&us41 (class A) XclIsus60 (class B) XcIIsus41 + himm434cII68 XcIIsus41 + Ximm434cIIIco2 XcIIsus60 + Ximm434cIIIco2

Repressor binding activitya units/mg protein 30.7 0 0.2 0 10.9 22.5

“Repressor binding activity was measured in extracts prepared 30 min after infection of M72su-, using the assay described by Echols and Green (19711, which measures the capacity of repressor to bind to 32P-labeled X DNA. Repressor-bound [s*P]DNA is retained on nitrocellulose filters. One unit of binding activity retains 0.5 rg of X DNA on the filter. The data was normalized to mg protein/extract, and a background activity of an uninfected control culture was subtracted.

very close to it. This places them between the right-hand ends of imm434 and imm21, namely in the y region or the cII gene (see Fig. 1). The mutations were finally localized within the cII gene by developing a deletion map of this region. The virulent deletion strains Xspi7lnin5 and Xspil56nin5, isolated and kindly supplied to us by Dr. G. Smith, extend all the way from the left arm into the cI1 gene. These strains prompted us to exploit the property of virulence in order to isolate additional spi deletions extending for various lengths into the ~11gene. Curiously, 75

154

BELFORT,

NOFF AND OPPENHEIM

out of 80 independent spi virulents, which were isolated from induced lysates of M72(Xnin5), were of the largest deletion type (like spi uir60), extending all the way to the extreme right-hand end of the ~11 gene. Fortunately the remaining five deletions extend randomly into the ~11 gene, thus allowing us to confirm the map position of previously isolated ~11 mutants (Brachet and Thomas, 1969) and to ascertain the position of the putative ~11amber mutants within the ~11gene. The 8 suppressible ~11mutants, as well as other ~11 mutants to be mapped, were crossed against each of the spi deletions shown in Fig. 1 and plated under recombinant-selective conditions. The recovery of turbid recombinants with a given deletion indicated that a particular clear mutant lies outside this deletion. A sample of representative data is given in Table 6, while the mapping data are summarized in Fig. 1. It should be noted that the ~11

amber mutants cluster into two groups, class A falling into segment IV and class B into segment II. Each member of a particular cluster is separable from non-amber ~11 mutants within the same segment on the basis of the recovery of turbid recombinants (0.050.2%) in two-factor crosses (data not shown). We have, however, not been able to separate amber mutations of the same cluster on this basis (