- Email: [email protected]
Transposition mutagenesis of bacteriophage lambda
J. iiol.
Biol.
(1979) 132, 307-322
Transposition
Mutagenesis
of Bacteriophage
Lambda
A New Gene Affecting Cell Lysis RY YOUNG?,
JEFFREY
WAY,
AND M&HAEL
SUSAN WAY,
JERRY
YIN$
SYVANEN
Department of Microbiology and Molecular Genetics Harvard Medical Xchool, Boston, Mass. 02115, U.X.A. (Received 27 November 1978, alzd in revised form
8 March 1979)
Insertions of Tn903, a transposable kanamycin-resistance element, in bacteriophage lambda at 0.95 on the lambda physical map adversely affect growth of the phage. These insertion mutants are able to assemble particles, but are unable to lyse the infected cell properly. The mutants define a new genetic complementation group that we have designated as gene Rz. Cells infected with the XRz : :Tn903 isolates will, at the normal time of lysis, change their shape from a rod to a sphere. These spheres are stable in dilute buffers with Mgz’ but are lysed with EDTA. In addition, these results demonstrate the utility of transposition mutagenesis in refining the genetic map of even so intensely studied a genome as lambda.
1. Introduction Bacterial mutagenesis using the transposable drug-resistance elements is proving to be a very powerful tool in the genetic analysis of bacteria and their associated plasmids; this follows from the ability of these elements to insert into a very large number of bacterial and plasmid regions, including structural genes. Many different insertion mutations that inactivate bacterial genes have been obtained in this manner. The second property that makes these elements so useful in genetic analysis is that the insertion confers an antibiotic resistance to the bacteria. Hence, one can monitor the insertion directly by the antibiotic resistance; this is particularly useful in mapping genes whose complete inactivation does not affect cell growth and also in analyzing recessive-lethal insertion mutations. The advantages of using TnlO, a transposable tetracycline-resistance element, in bacterialgenetic analysis have recently been described by Kleckner et al. (1977). In addition to TnlO, other drug-resistant elements have been utilized, such as Tn5 (a kanamycin-resistance determinant; Berg, 1977), Tn3 and Tn9. In this paper we describe the use of Tn903, a kanamcyin-resistance determinant, for the genetic study of bacteriophage lambda. With bacteriophage like lambda, the size of the phage chromosome is limited by the packaging capacity of the phage ->*Ah-m< issspii..~s~d~ SEzaai.aL- -. . L r-as-. t Present address: Department Station, Texas 77840, U.S.A. $ Present address: Department 53706, U.S.A.
of Biochemistry,
Texas
A and M Medical
of Biochemistry,
University
of Wisconsin,
School,
College
Madison,
Wise.
307 0022-2836/79/230307-16
$02.00/O
0 1979 Academic
Press Inc. (London)
Ltd.
K. ‘YOUNG
308
ST
ii i
head. There-Lbre, in transposition mu.tagenesiu it is helpfu.1 to use a transposaible element that i8 relatively small in relation .to the phage genome. For example, TnlU, Tn3 and Tn5 are all larger than 5000 base-pairs. a considerable fraet,ion of the 46,500base-pair lambda chromosome. Hence ono advantage of Tn.903 is -thab it is small Tn903 has the characteristic flanking-inverted(3160 base-pairs). Furthermore, repeat, structure that gives rise to the striking iistem-and-LoopP’ structures in heteroduplexes. Tn9, the only drug-resistance transposon as small as X903, does not form stem-and-loops and is thus less convenienk for betSerodupiox analysis. Many mutants of bacteriophage lambda have been isolated that cause conditional or absolute defects in lambda growth (Campbell, 1961; Parkinson, 1968; Harris et al., 1967). We have isolated a large number of Tn903 insertions into la,mbda. In this isolation, we have required that the insertion mutants be able to assembIe phage particles and that they be able to establish themselves as lysogens. Among these, we found two classes that affected lambda growth a,dversely; one of these classes defines a new gene.
2. Materials and Methods (a,) Bacteria
and @iage
The Eschericlaia coli K12 strain W3110 (sup”) and Ymel (N~F) are used to follow phage growth. Bacteria are grown in TYM, a broth with tryptone (0.50/), yeast, extract (0.10/b), XaC1 (O.Sq/,) and malt,ose (0~2%). Plates conta,ined 1.6% agar of the above composition but without maltose. The phage strains Xb519cIts857Sam’i and Xb519b515cIts857nin5Sam7 were used as the recipients for Tn903 insertion. (b) Isolation
of Tra903 insertions
in, h
The self-transmissible drug-resistance plasmid R-6 (Cohen, 1977) was introduced into W3110 lysogenic for one of the lambda strains. After several days of subculturing, the lysogen was thermally induced and phage stocks containing 1011 to 1Ol2 p.f.u.t/ml were prepared. The pha,ge were adsorbed t,o W3110 (normally kanamycin-sensitive) at a ratio of 5 p.f.u./bacterium and lo8 infected cells plated on solid agar medium containing 40 pg kanamycin sulfate/ml. After 24 h at 3O”C, single kanamycin-resistant clones would a,ppear. All tested were lambda lysogens and t,hey gave rise to high-frequency transducing lysates for kanamycin resistance. Table 1 lists those we have used in this study.
TARLE I Xtrain
Fist
Phago designation
Complete genotype
x hpk3 hdk6 Xdk23 hpk26 hpk24 hpk22 XpkPl Xpk25 The number in parentheses following Tn903 give,,q the physical location of the insertion as measured from heteroduplex molecules or from the molecular weights of DNA fragments produced by SmaI and XfboI endonuclease digestion of purified DNA from each phage (see Results). t Abbreviation
used: p.f.u., plaque-forming
units.
LAMBDA
TRANSPOSITION
MUTAGENESIS
309
(c) Quantitative measures of XTn903 transduction to the measurement of plaque-forming units, the number of kanamycinresistant (kanR) transducing particles was measured by the ability to confer kanamycin resistance to bacteria. To lo8 cells of Ymel(X) is added lo5 to lo7 of XTn903 particles; after 5 min, 5 ml of TYM is added to the culture, which is then grown at 37°C overnight. The following day 0.05 ml of this culture is plated on to media containing 4Opg kanamycin sulfate/ml. Approximately 1 kanamycin-resistant clone appears for every lo3 XTn903 particles added to the original cells. The number of clones is linear over a lO,OOO-fold range with the number of p.f.u. added. In the determinations of the number of defective hTn903 particles we always include for calibration a non-defective hTn903, whose p.f.u. titer is known.
In addition
(d) Preparation
of lambda DNA
Lambda particles were purified from lysates by adding CsCl to a density of 1.5 g/cm3, centrifuging overnight at 30,000 revs/min in a Beckman SW50.1 rotor and extracting the turbid band of phage from the centrifuge tube with a syringe. The phage were dialyzed into 20 m&r-MgCl,, 20 mM-Tris (pH 8.0). DNA was extracted with redistilled phenol saturated with water. The DNA was dialyzed into O-1 M-KC& 20 mrvr-Tris (pH &O), 1 mM-EDTA. The endonuclease digestions of the DNA with SmaI, Hind111 and XhoI were performed as described in the instructions provided by BioLabs, from which these enzymes were purchased. Agarose gel electrophoresis will be described elsewhere. (e) Electron The hTn903 DNA was analyzed using the 40% formamide spreading
by forming technique
microscopy heteroduplexes with Ximm434 DNA described by Davis et al. (1971).
and
3. Results Tn903 is a transposable kanamycin/neomycin-resistant determinant found on the plasmid R6 (Sharp et al., 1973). It consists of a pair of flanking inverted repeat sequences 1130 base-pairs long, called 70 (Ohtsubo & Ohtsubo, 1977) and a unique central region 900 base-pairs long (Armstrong et al., 1977) coding for the drug resistance.
The physical
map
of Tn903
(a) Isolation
is shown
in Figure
of Tn903 insertions
1.
in lambda
We prepared 80 independent lysates by inducing W3110 parental-phage lysogens harboring the R6 plasmid. From each of these, a kanamycin-resistant lambda lysogen of Ymel was obtained that would yield high-frequency transducing lysates for
1600
II30
FIG. 1. Physical map of Tn903. The SlnaI and Hind111 cutting sites were determined by Armstrong et al. (1977). M. Casadaban (personal communication) informed us of the XhoI cutting site. The dimension of 18 was measured from the stem formed by self-annealing of the intrastrand complementary sequences from heteroduplex molecules such as the one shown in Fig. 4. The size standard for this measurement is the distance between b519 and the left edge of the imm434/immX junction and the distance between the right edge of the junction with the &n5 deletion (Fiandt et al., 1971).
.i 10
Ii.
YUUIUG
i!.!cP A1 e,
kanamycin resistance. Each of these lysogens \zas in.duced a;rld the lysat,es t,e&ed for plaque-forming ability on Ymel (supF). The V~/~~P-containing strain is used to test for plaque formation because the parental phage contains the RanL’i a;llele. Two !ysates contained almost no plaque-formers (hdk6 and hdk23’), a,nd five others gave very small plaques (Xpk21, Apk22, Xpk24, hpk25 and hpk26). The other stocks conof particle tained normal plaque-formers at high t’iter. The direct examinatjon production for each of t,hese seven XTn903 insertions and for Xpk3 (a normal plaqueforming derivat,ive) was done by inducing W3110 (SLY") iysogens of each phage. Phage growth for three hours after induction was permitted. (The 8nm’i mutation in these phages prevents cell lysis and permits particle assembly to continue well beyond the normal lysis time of 45 minutes.) Aft,er these cells were concent!rated and lysed by freezing and thawing, the lysat,es were adjusted to a density of 1.5 g!cm3 with CsCl. Sfter overnight centrifugation to establish CsCI gradients, dense phage bands were observed for all seven induced lysogens. The equal size for each band demonstrates that the defect in Xdk6 and Adk23 is not due to a gross inability is unto make particles, a.t least in growth conditions where the Earn7 mutation suppressed. This property is also evident ia Figure 2: which shows the kinetics of
Time after induction (min) P'IG. 2. Phage particle accumulation kinetics. W31 IO cells lysogenic for hpk5 or XdkB exponentially growing at 30°C wore diluted loo-fold into a prewarmed broth at 42°C after reaching A,,, = O”15. After 15 min the 25-1111 cultures were shifted to a 37°C aeration. Beginning at 30 min after induction, 2-ml samples were removed, vortexed with a few drops of CHCI,, and chilled. Samples were assayed for phage particle titer by the transduction assay, as described in Materials and Methods. The slopes of the accumulation curves varied signiScantJly from day to day, but the temporal shift in the curves was always 5 to 7 min., -o-o--, Xpk5; -e-e-, hdkli.
phage production for induced sup” lysogens of a normal plaque-forming XTn903 and hdk6. The kinetics of particle accumulation for hdk6 are identical to the plaqueformer, except for a five to seven-minute delay. (b) Heteroduplex
mapping
and restriction
enzyme
analysis
of
the Man isolates
The positions of the Tn903 insertion in four of t.he phages were determined by electron microscopic examination of heteroduplex molecules formed between the XTn903 and himm434 DNA. We examined Xdk6, hdk23; Xpk21 and Xpk24. Tn903 forms the stem-and-loop structure, indicating an invertled duplication in the DTw’A
LAMBDA
FIG. 3. A and hin~m434 deletion loop, stem-and-loop
TRANSPOSITION
MUTAGENESIS
311
heteroduplex DNA molecule formed between Xdk6 = b519cI857?tin5 Tn903 (0.951) DNAs. If one follows the molecule from the lower right to the upper left, the b519 the i,mmX/ivw~434 substitution “bubbles”, the nin5 deletion loop a,nd the Tn903 structure are encountered in order.
(Fig. 1) similar to that observed for Tn5 (Berg et al., 1975) and TnlO (Kleckner et aE.: 1975). For the normal plaque-formers, Tn903 insertions were mapped in many places in the lambda genome. The four isolates with altered plating properties all mapped between lambda co-ordinates 0.95 and 1.00; at the extreme right end of the lambda genet,ic map. Figure 3 shows an electron micrograph of a heteroduplex of Xdk6 with himm434; the deletion loops corresponding to b519 and nin5 and the characteristic double substitution-deletion loops corresponding to the imm434 region are clearly visible; as is the stem-and-loop structure of Tn903 at about 0.95 on the h molecule. (h co-ordinates are 0.000 at the left end and 1.000 at the right.) We measured the size of the stem at 1130 base-pairs and the loop at 900 base-pairs. In addition, the positions of each of the seven XTn903 insertions that alter lambda growth were determined with the use of two restriction endonucleases (SmaI and XhoI) and agarose gel electrophoresis. These two enzymes cut Tn903 DNA at sites shown in Figure 1; SmaI cuts in the duplicated r$ sequences and XhoI cuts in the unique region. The SmaI endonuclease cuts h at 0.825. Thus after digestion with this enzyme, the right end of X DNA will be found on a fragment that is 0.117 h lengths (since the phage carries the nin5 deletion of 0*058 units). When DNA from each of the seven Tn903 insertions is digested with SmaI and the size of the resulting fragments measured by means of agarose gel electrophoresis, the 0.117 h length piece is missing. Four new fragments appear, two very small pieces consisting entirely of Tn903 sequences, and two pieces, consisting of 78 and X sequences, totaling 0.137 X
$12
R. YOUSG
1;;T dL.
lengths in size. Each of these latter two fragments will haSva 450 base-pairs, or I)*61 A units of 78 sequences. The size for each of these two fragments (which is given in Table 2) shows that the insertion is at one of two possible locations, either near the right end or close to t’he #r/ha1 cutting site at 0~825. To decide between the two sites, each sample was cut with the XhoI nuclease and the size of the fragments determined. The XhoI nuclease cuts lambda once at 0~703 (our da,ta,) and ma,kes a single asymmetrical cut 0.025 X units from one edge of Tn903 (see Fig. 3). Based on the size of the smallest XhoI digestion fragment when the hTn903 D?JVAs are cut (Table 2), we conclude that Tn903 for all seven insertions must map at t)he site closest to the right end. These sites for the seven insertions are given in Table 2. Because the Xhof cutting site in Tn903 is asymmetric, the size of the smallest XhoI fragment uniquely determines the orientation of each Tn903 insertion (Table 2). Orientation I means that the direction from the Hind111 site toward the XhoI site (see Fig. 1) is from left to right on the h map. TABLE 2 Location
of T&O3
in the seven ATn903 altered
Phage
____-__ hdk6 hdk23 Xpk26 hpk22 hpk24 Xpk21 hpk26
Two smallest observable SmnI fragments 0.082 0.059 Not done 0.109 0.033 0.11 0.028 Not done 0.117
0.016
0.121
Smallest XhoI fragment
~-___ 0.073 0.074 0.046 0.045 0.046 0.032 0.027
de&atives
with
growth
~~
Position of insertion
Orientation insertion
of
~~ ..-~~--.. 0.952
$- o.oost
0.951 * o.oost 0.979 0.98 0.980 0.993 0.998
I I I I
= 0.003t & 0.002?
I I I
The position and orientation of Tn903 in each DNA was determined from the molecular weight of the 2 smallest observable fragments produced by SnaaI digestion and the smallest fragment produced by XhoI digestion. Molecular weights are expressed in fractions of h and the map positions are linear with A DKA beginning at 0 at the left end and going to I.0 at the right end. Molecular weights were determined by electrophoresis of the DNA in agarose gels. 7 Positions of Tn903 insertion were initially determined by heteroduplex analysis.
The important result from this Table is that the two defective Tn903 insertions both map at 0.95 on A, while the five small-plaque-forming variants are between 0.98 and 0.997. In Figure 4 we give the genetic map of the right end of the phage X chromosome and show the locations and orientations of the seven Tn903 insertions given in Table 2, as well as the location and orientation of Tn903 in Apk3 (mapping data not shown). This region of the X chromosome contains the genes S and R, which are required for cell lysis, and the Q gene, whose protein product is necessary t,o activate late gene transcription from the late promot!er pR’. Transcription from pR’ proceeds rightward in Figure 4 through the lysis genes, through the cos site, and transcribes all of the genes coding for head and tail structural proteins, beginning with A, W, B and extending through J (not shown in Fig. 4). Any insertion mutation that interferes with the transcription from pR’ could therefore be defective for phage growth by reducing expression of these late genes. However, this explana,tion is not sufficient to explain why hdk6 and Xdk23 arc defective, Given that all seven
LAMBDA
TRANSPOSITION
MUTAGENESIS
313
insertions are in the same orientation, it is hard to imagine why those at 0.95 interfere with transcription, whereas those at 0.98 and 0.99 do not. A more reasonable explanation is that the insertions at 0.95 have inactivated an essential h gene.
FIG. 4. Insertion sites of Tn903 in the h late transcriptional unit. Vertical arrows indicate positions of insertions as determined by physical analysis of the DNA molecules. Approximate positions of known genes are taken from Echols & Murialdo (1978). Numerical values are given in Table 1. Also shown is Xpk3, which maps at 0.923 and is a normal plaque-former. Horizontal arrows indicate orientation of insertion site, from the XhoI to the Hind111 site as shown in Fig. 1.
(c) Plaque-forming
revertants of h&6 have lost Tn903
Since hdk6 does not make plaques, normally particles were assayed by transduction of a bacterial strain already lysogenic for h+ (see Materials and Methods). The different XTn903 particles behave identically in such infections, presumably because the primary event leading to a kanamycin-resistant colony should be the recombination of the repressed, circular hkan molecule into the h+ prophage. However, at frequencies between 10e4 and 10m5 per particle, two types of plaque arise: small plaques, which grow equally well on supF and sup’ bacteria; and large plaques, which grow only on supF lawns. The parental h phage grows only on supF-containing bacteria because of the Sam7 mutation. For plaque-forming hTn903 phage, kanamycinresistant lysogens grow in the plaque centers at 30°C. However, neither type of Xdk6 revertant plaque yields such resistant cells. Hence, the plaque-forming revertants of Xdk6 have gained the ability to grow and have lost their ability to transduce kanamycin resistance. To further characterize hdk6 and its revertants, we determined the distribution of these particles in a CsCl sedimentation equilibrium gradient, as is shown in Figure 5. Phage particles of lambda have a characteristic buoyant density that reflects their DNA and protein content. Since the chromosome of X is 6.8% shorter than XTn903, the phage that have lost Tn903, i.e. the revertants, should band at a lower density. The results in Figure 5 show the peak of the non-plaque-forming hdk6 particles flanked by two plaque-forming revertant peaks. The large-plaque-forming revertant gives a peak separated from the hdk6 peak by six fractions to the lower density; this corresponds to a change of approximately -6% of the X genome when deletion standards a,re subjected to the same centrifugation (data not shown). Thus the observed particle density is as expected if the large-plaque-forming revertant is formed by simple excision of Tn903 from Xdk6. The small-plaque-forming revertants, which define the higher density peak, are most likely the product of a “QSR” deletion-substitution event. In this event, the region between co-ordinates 0.838 and 0.95 on h is deleted and substituted by a functionally equivalent but larger and non-homologous region derived from a cryptic lambdoid prophage resident in the E. coli chromosome (Herskowitz & Signer, 1974). The
sre
R ).
YOUNG
&it‘
,it
density increase of the small-plaque revertauta, with respect to the A~tk6, corresponds l;o an 80/b increase in the genome. This is the wize increase expected if Adk6 lost, t;he, X sequences between 0,838 and 0.95 (including the Tn903 insert,ion) and replaced this DXA with the “QXR” or p4 insertion (Fiandt, et ad.: 1971). This substitution would also explain why these revertants become am+, since tshe Sam7 lesion resides in the sub&itut,ed regi.on. ‘wary In addition, DNA from iive independent, Ilarge-play us-forming rev&ants prepared and examined in the electron microscope by forming hitjeroduplex mwlecules with himm434. The IieterodupIexes were featureless, except for t-he parental and Ximm434 delet’ion and substitution loops (dat,a not shown). We conclude that the excision of Tn903 is precise, at least, to the level of 100 nucleotjdes, which is t,he lower limit of resolution of the heteroduplex technique.
Fraction
number
FIG. 5. Equilibrium density centrifugation of ;\dk6 phage particles. A volume (2.55 ml) of 3 x 109 hdk6 particles was mixed with an equal volume of a saturated CsCl solution and centrifuged for 28 hat 20~000 revsjmin in a SWSO.1 rot&. A total of 95 sa.mples were collected after piercing\he bottom of the nitrocellulose tube. The number of plaque-forming phage in each fraction was det,ermined by titering aga,inst IV3110 (sup”) (-E-n-), (8~~8’) (--e-e--). The was determined as out,linetl number of kanamycin-tral2sducirlg particles (- I’i,-- c)-) in aterials and Methods.
(d) The S+ allele makes hd1%6’CLGny-$aTue-forme? All of the Tn903 isolates carried the Xana7 allele: in order to facilitate preparation of high titer st,ocks. When the S+ allele was recombined into hdk6 and ;\dk23, we found these phage now made tiny pinpoint plaques that, can be seen on a lawn of W3110 by viewing through a dissecting microscope. When the S+ allele was introduced into the phages with the more distal insertions, t’he plaque size increased to the extent that t)hey were barely distinguishable from Aclts$%‘S + . (e) hdk6 dejines
a new conzplementation
group
The two defect’ive phages; Xdk6 and Xdk23, have the Tn903 ins&ion at 0.96 on t’he X map. This is immediately to the right, of gene R: according to the generally accepted X map (Davidson & Szybalski. 1971). We therefore t,ested whether the insertion in hdk6 canses a mutation that defines a new complement~ation group. As can be seen in Table :‘rl Xdk6 will complerrlc~nl arr~bc~ nlt~l at.ionx in genes N, P, Q, R, da W and B. In order to test fbr corn~le~rlentation of the S gent, we corl.structe(l 1ysogen.s of Xdk6S + and, as control, hpk25S + in tjhe sup” bacteria W3110. The plat,ing
LAMBDA
TRANSPOSITION
MUTAGENESIS
315
efficiency of different heteroimmune phage with amber mutations in genes 8, R, A and K was determined on each. These het~eroimmune phage should be able to grow TABLE 3 Cowqdementation Phage Xdk6 hNaml1 XPam3 XRam5 XAam32 hSam7
XNaml7 ++ -++ ++ ++ ++
XQam73
XPam3 ++ ++ -++ ++ + -1
++ ++ +I+ + ++
tests for
h&c6
XRam5
ASam -++ ++ ++ ++ --
XAam32
+ ++ ++
++ ++ ++ ++ -++
++ ++
xwam403
t:: is ++ ++ ++
XBam6 ++ ++ ++ ++ ++ ++
Phage stocks of lo7 and lOa particles/ml were by dilution from fresh plate lysates, except for hdk6, which was diluted from a high-titer CsCl-purified stock. Complementation tests were performed using 5.~1 drops of each phage on a W3110 lawn in top agar. Complementation was scored as positive if confluent lysis under a spot resulted from overnight incubation at 37°C. + +, Confluence using lo7 particles/ml stocks; +, confluence at lo8 particles/ml. The reduced complementation of hdk6 and hRam5, and of XQam73 for XRam5 and XAam32 was reproducible. Note Xdk6 carries the Sam7 allele, and thus does not complement /\Sam’i.
on the sup’ lysogens, since the superinfecting phage can transactivate the late genes from the resident prophage (Thomas, 1971) unless the resident prophage is defective in the same gene. As is shown in Table 4, lysogens of XdkGS+ and Xpk25S + will equally support the growth of all four of the heteroimmune ambers. The data in Tables 3 and 4 show that hdk6 has a functional R gene, and that the insertion is not significantly polar on h genes A through K. Thus the mutation caused by the insertion at 0.95 defines a new complementation group. (f) Insertion
site and late gene expression
Insertions of Tn903 into the X late transcriptional unit between co-ordinates 0.95 and 1.00 could be polar on the head and tail genes, since in the vegetative state the
TABLE
Tmn.sactivation
Ymel Test phage Ximm434 Sam7 himm434 Ram7 Ximm2 1 Aam Ximm434 Kam21
1.0 1.0 1.0 1.0
4
of )tS+dkG and hS+dk23 genes Efficiency W(hS+)
10-l 1.0 5x10-a 5 x 10-l
of plating W(hS+dkB)
W(XS ‘pk25)
IO-1 1.0 5x10-z 10-l
10-l 1.0 5x10-2 10-l
Five-p1 drops of 109, 107, IO5 and lo3 phage/ml of the test phage stocks were spotted on lawns of the indicated lysogens of W3110. The efficiency of plating was calculated from the number of plaques seen on each of the lysogens relative to the number seen on the suppressor (Ymel) lawn after overnight incubation at 3O’C. Plaque sizes vary significantly in the spot zones, presumably due to a wide spread in absorption times for the spotted phage. Data presented are the averages from 2 experiments.
R.
316
YOUNG
ET
ri L.
cohesive ends of h are joined together (see Pig. 4). In the course of evalua,ting Lhc ,eomplementation t#est we saw some indicat,ion that the Trr903 insertions in Adk6 and hpk25 are somewhat polar on the distal genes of the late operon. We therefore eonsidered in more detail whether the defect in Xdk6 was perhaps due $0 polarity. If there are no necessary genes between 0.95 and 11*00, we would have n.o reason to suspect, a difference in the viability of hdk6 a,nd Xpk26. However, if the insertion at O-95 is more polar on the head and tail genes tha,n the insertions from 0*98 and 0,997, then the defective character of Xdk6 and the contrasting plaque-forming character of the more distal insertions could be explained as a cumulative polarity effect, not detectable in the complementation tests of single amber mutants. Quantitative complementation tests in liquid culture were carried out to assess fnrther the effects of t#he insertion site on late gene expression. The results of these complementation tests were unusually variable. In some experiments, the ability of the late operon insertion phages to complement the immediately dist’al late genes (A, W and B) did not differ from the (uninserted) parental phage; in other experiments, there was as much as a tenfold decrease observed, relative to the parental (data not shown). However, there was no detectable difference between the late gene complementation activity of hdk6 and hpk25 in any experiment. We conclude the Tn903 inserti0n.s at h co-ordinates 0.95 and 0.998 are equally polar on X late gene expression. Confirmat,ion of this result was obtained when phage yields were measured, upon a single-step infection (Table 5). Particle production is reduced twofold or less from dhe uninserted parent levels for both hdk6 and Apk25. Thus t’he severely reduced ability of hdk6 to make plaques does not st,em from increased polarity on the X capsid structural genes, relative to the more distal insertions.
TABLE
Single-step Phage /is+ Xdk6S + Xpk25S +
6
infection phnge yields Yield 45 28 30
Relative -.___-.--
yield? 0.47 0~29 0.32
One ml of W3110 cells growing logarithmically was chilled at Aej,, = 0.35 and infected with one of the phages at a multiplicity of 0.3. After being left for 20 min at room temperature for preadsorption, the cells were washed by centrifugation and resuspended in room temperature broth, and diluted 10s3 into prewarmed 37°C broth. The diluted, infected cultures were aerated at 30°C for 70 min and then vortexad with a few drops of CHCI,, prior to storage at 4°C. The lysates were titered by plating with W3110 as an indicator on very fresh broth plates. Idk6S+ plaques were counted using a steromicroscope. Data are averages from 2 experiments. p XI&I+ gives a relative yield of 1.0, hS + ; the parental phage, carries the b519 and n&5 deletions
(g) Adk6 is defective in cell lysis The fact that hdk6 and the plaque-forming phages bearing the more distal insertions produced the same number of infective particles in a single cycle of infection made it possible that the defectiveness of hdk6 could reside in its ability to lyse cells. To test this idea, the turbidity of cultures was followed after infection with hdk6
LAMBDA
Time after
TRANSPOSITION
infection (a)
(min)
MUTAGENESIS
Time after
induction
317
(min)
(b)
FIG. 6. (a) Lysis defect in Xdk6 infection. W3110 was grown at 37°C in liquid media until A,,, = 0.4. Aeration was stopped and the culture was made 10 mm in MgCl, before dividing into 3 portions. One portion was infected with hdk6S +. another with hpk25S +, and the third with hS +, each at a multiplicity of 7. After 5 min, aeration was resumed and the absorbance at 650 nm measured at appropriate intervals. --o-n--, Xdk6; --a-@- -, Xpk25S+ or x3+ (curves indistinguishable); -O---O--, Xdk6S+ with no MgCl, added. Identical results were obtained when the Sam7 version of each phage was infected into Ymel (.supP). (b) Lysis and kinetics of induced lysogens. W3110 lysogens of Xdk6S +, hpk25S +, and XS+ were grown in TBr until A,,, = 0.3 to 0.5. The cultures were shifted to 42°C with aeration for 15 min, followed by continued aeration at 37”C, and the absorbance at 650 nm monitored at appropriate intervals. --O--O--, W3110 (hpk25S+) or W3110 (AS+); -O-O-, W3110 (hdkSS+); -O--a---, W3110 (AdkBS+) with 10 mM-MgCl, added at t = 0. Identical results were obtained with Ymel lysogens and the X7 virions of each phage.
(or Xdk23) (Fig. 6(a)). The turbidity was found to increase for a few minutes after infection, before reaching a plateau, which remained unchanged for several hours. In contrast, cultures infected with hpk25 or the parental phage would lyse within one hour after infection. The lysis defect in infected cultures contradicted our observation that induced cultures lysogenic for hdk6 showed clearing at the expected time (data not shown). To resolve this conflict, the kinetics of cell lysis were followed more closely by monitoring the turbidity of a thermally induced culture of Ymel (Xdk6). Lysis was observed, beginning at approximately the same time as lysis of the lysogens with either the prophage or with Xpk26 prophage, as shown in Figure 6(b). However, the kinetics of thermally induced lysis is reproducibly complex; the turbidity falls to one-third of its original value, remains constant for approximately 30 minutes, then resumes its decline. Thus, Xdk6 has a lysis defect that is more apparent during infection than induction. The apparent difference between the severity of the lysis defect in infection and induction was eliminated when the effect of Mg2+ on the lysis curves was tested. In the infect’ion experiments, 10 mM-Mgcl, had been added in order to facilitate was added to the induced lysogenic cultures, lysis phage adsorption. When Mg2+ + but completely unaffected for Apk26S +. When was nearly eliminated for Xdk6S
318 zag=-I was removed
R. Y-0UNG it 2’ ,-Irt,
from t’he in&&ion prod.UCCJ~~htik8kj r ”j niv b.yi uuit lJ.l”es iA‘I”lY‘ observed to lyse, although lysis was significantly slower %han in. hpk26S’ cultures. Thus Xdk6 has a subtle lysis defect, ~which is absolute in I& present of 10 rn~~-Xg~ + I As expected, the magnesium ion also efiminat’es t,he nlnility of hdk6S+ to form tiny, pinpoint piques (d.ata not shown).
Although the complementation spot, tests and the transa,cti~z&~n experiments showed that Xdk6 is unaffected with respect t,o Ghe expression of t,he B gene, t’he lysis defect observed. and the position of the T&O3 ins&tion so n.ea,r the b0uadar.y of the R gene make it necessary to eliminate .E as a candidate for the insertion lesion.. Figure 7 shows the lysis kinetics of cells infect,ed with hdk6 or .XRam,!XRamEiO~ in
-30
0 30 60 90 Time after infection (min)
FIG. 7. Functional complementation between hRam54RamGO and hdk6S ?. WV3110 was grown in TYM at 37°C until _4 850 = 0.35. Aeration was stopped and the culture mado 10 m~inXgC1, before it was divided into 3 parts. One part was infected with XdkGS+ at a multiplicity of 10, another with XBnnx54Ra~nGO at a multiplicity of 10, and the third part with both these phages; at a multiplicity of 6 each. After 5 min, aeration was resumed and the absorbance at 650 nm monil-orod -z-s--, /w&s+; --g-2--, at appropriate intervals. --A--A-, hRam54Eam60; both phages.
the presence of 10 mM-Mg2 +. Although neither hdk6 nor t,he R double amber mut)ant infections result in lysis, a culture infected with both phages does lyse, albeit, slowly. Thus Xdk6 and the R mutant complement each other for lysis. We conclude that hdk6 must be functionally R +. (i) IVormal endolysin
coatent
of induced
A&G
iysogen
The possibility that the defect in hdk6 is due to an alteration of the R gene was also tested by direct assays in vitro for endolysin activit’y, t,he product, of gene R. Extracts of induced lysogens of hdk6 and hpk26 were tested for the amount of endolysin and for the kinetics of reaction w&h the substrat’e. Table 6 shows that the endolysin activity, a,s measured by the lysis of sensitive ceils (Court r,t al., 1975) was identical in bot’h hdk6 and Xpk26. We conclude t,hat the defect in Xdk6 does not lie in the R gene, the nearest known gene to the insertion site of Tn903 in the defective phage.
LAMBDA
TRANSPOSITION
319
MUTAGENESIS
TABLE 6 Endolysin Lysogen W3110(Xdk6) W3110(hpk25)
Endolysin
content of induced lysates
units/ml
Azso units/ml
1680 2650
Endolysin
47 70
units/&se 36 38
Cultures (100 ml) of the indicated lysogens were induced in late logarithmic phase, concentrated to 1 ml in TBr, quick-frozen in a methanol/solid CO2 bath, and thawed at 30°C in the presence of 5 pg DNase/ml. After removing debris by centrifugation at 3000 g for 5 min, the extracts were chilled and assayed within the hour. Endolysin assays were performed as described in Materials and Methods.
(j) Sphere-formation
of infected cells
A novel aspect of the lysis defect in hdk6-infected cells was revealed by phase contrast microscopy. Beginning at 30 to 35 minutes after infection, hdk6-infected cells abruptly change from the elongated rod shape characteristic of E. coli to oblate spheroids. This sudden change is followed by a slower metamorphosis into spheres, examples of which are shown in the micrograph in Figure 8. The spheres seem to be, at least initially, motile, and they are extremely fragile, since gentle pressure on the coverslip causes their destruction. Cells infected with XX- or hR- mutants also do not lyse, but neither do they change the rod-like morphology. In the absence of 10 mM-MgCl,, these spheres will spontaneously lyse on the microscope slide. The addition of EDTA to a culture of spheres leads to immediate lysis.
FIG. 8. Morphology of Xdk6-infected cells. Logarithmically growing W3110 was infected with Xdk6S + at a multiplicity of 3, in the presence of 10 mix-MgCl,. Samples were removed and observed under phase contrast microscopy; magnification 630 x Spherical cells begin to accumulate at about 30 min; the photomicrograph is taken 90 min after infection. Some of the spherical cells have become transparent by this time. The rod-shaped bacteria are presumably uninfected cells; in cultures infected with higher multiplicities, the frequency of rod-shaped cells drops.
We have described. a new essential gene in phage A. A lesion in this gene was8 obtained by transposition mutagenesis with Tn903, a kanamycin-resista,nce transposon. Two independent insertions of 1’11903 into the region of h co-ordinate 095Z & 0.002 result in defective phages; the defect is co.mplement,ed by all known genes; in&ding the nearest cistron, gene R. Five other independent insertions more distal on the right arm of A (between co-ordinates 0.979 and 0,998) result, in plaque-forming ‘rhat it must lie phages. Thus we can define a new gene, an,d state unequivocally between h co-ordinates 0.951 (the right t’ermirms of gene R) and O-979. (b) Lysis-defective
phe,aotype
The phenotype of phages with the insertion lesion in the new gene is abnormal cells infected with the mutant phage do not lyse at all. Since lysis; in 10 mM-MgCI,, the insertion maps so near the known locus of the R gene (Davidson & Szybalski, 1971) and the R gene product is required for Iysis (Jacob & Fuerst 1958), it was important to test these mutants for R activity. We find %he cont!ent. of endolysin (the R gene product) to be unchanged from the parental phage, when measured in induced lysogens. Furthermore, a double nonsense mutant of t#he R gene complemented the insert’ion mutant for lysis of mixedly infected cells in 10 m&r-MgCl,. Thus genetically and §unct,ionally, the defect,ive insertion mutants define a new gene. Because it is an essential gene, and because it is a lysis gene mapping adjacent to the R cistron, we ea.11the new gene the Rz gene. (c) Spherical
cell formatim
Phase contrast microscopy of cells infecected wit’h t#he defective insertion muta,nts revealed t,hat inst,ead of lysing, the cells were transformed from rods to spheres, In 10 mM-MgCl,, the spheres were completely st’able; although very fragile mechanically. The observation t’hat the mutant-infected cells become spherical instead of lysing suggests that t,he sphere form is an intermediate in normal lysis. There is precedent for such a spherical lysis intermediate in the observations of Groma,n & Suzuki (1962), who observed short-lived spherical forms appearing immediately before lysis of induced X lysogens. In addition, Birdsell & Cota-Robles (1967) showed that plasmolyzed, lysozyme-treated E. coli cells became spherical, without losing turbidity, and that further osmotic shock or treatment with EDTA resulted in lysis. They suggested that the outer membrane cont’ributes to the maintenance of cellular integrity, since treatment with lysozyme alone results in sphere format,ion rather than complete lysis. It is possible then t,hat the attack of endolysin, the R gene product, on the pept,idoglyean is necessary but not sufficient for normal lysis. A endolysin is an endopeptidase, which cleaves the peptide bond between diaminopimelate and nalanine in the cross-linking polypeptides of the peptidoglycan (Taylor? 1971). Cleavage of these bonds should still leave the polysaccharide chains intact, if the outer membrane is also intact. Then one might expect t’he “relaxed” peptidoglycan outer membrane wall structure to lose much of its shape and rigidity. MgZ+ is required to stabilize t,he spheres, which is consistent with outer membrane involvement. The function of the
LAMBDA
TRANSPOSITION
MUTAGENESIS
321
Rx gene product may thus be needed to weaken this “relaxed” complex and ensure complete lysis. Taylor found evidence for two peptidoglyoan-degrading activities in h lysates, only one of which corresponds to the known endopeptidase activity of gene R (Taylor et al., 1975). It is possible that the Rx gene codes for a lysozyme-type protein, which attacks the polyglycoside backbone of the peptidoglycan. (d) Detection of a phage gene by transposition
mutagenesis
h is possibly the most extensively characterized genetic entity, principally because powerful methods exist for finding conditional lethal mutants. Why have no nonsense or temperature-sensitive mutations in Rx been found in the many mutant searches previously conducted on A? The answer to this is that hRz- phage under normal plating conditions makes very tiny plaques. It was not until we had worked with Xdk6 for an extended period of time that we realized that Mg2 + stabilized the highly unstable spheres and consequently XRz- phage would not make any plaques when the indicator bacteria were grown in the presence of 10 m&r-Mg2 +. Thus it was only by virtue of the fact that the original hdk6 mutant carried Tn903 at the site of the Rz gene that allowed us to isolate this mutant (by conferring kanamycin resistance to its lysogens) and to map its physical location (by the striking appearance of Tn903 in heteroduplex molecules) with the electron microscope. The phenotype of Rz- is much too subtle to allow its isolation by more traditional means. (e) Implication
for lysis control and cell ultrastructure
It is now apparent that h elaborates at least two enzymes to attack the cell wall. Presumbly neither can function from within the cytoplasmic membrane and in AS-infected cells the peptidoglycan is unimpaired (Reader & Siminovitch, 1971). Thus S effects the transport of two different enzymes to the periplasm, the R and Rz gene products. S-protein may then be a generalized t,ransport function, of wider biological significance than heretofore imagined. This work was supported by a grant 5ROlCA18217). One author (R. Y.) had Institutes of Health (IF 32 AT 05622-01).
from the National Institutes of Health (no. a post-doctoral fellowship from the National
REFERENCES Armstrong, K. A., Hershfeld, V. & Helinski, D. R’. (1977). science, 196, 172-174. Berg, D. E. (1977). In DNA Insertion Elements, Plasmids and Episomes (Bukhari, A. I., Shapiro, J. A. & Adhya, S. L., eds), pp. 205212, Cold Spring Harbor Laboratory, New York. Berg, D. E., Davies, J., Allet, B. & Rochaix, J. D. (1975). Proc. Nat. Acad. Sci., U.S.A.
72, 3628-3632. Birdsell, D. C. & Cota-Robles, E. H. (1967). J. Bacterial. 93, 427-437. Campbell, A. (1961). Virology, 14, 22-32. Cohen, S. N. (1977). In DNA Insertion Elements, Plasmids and Episomes (Bukhari, A. I., Shapiro, J. A. & Adhya, S. L., eds), pp. 672-673, Cold Spring Harbor Laboratory, New York. Court, D., Green, L. & Echols, H. (1975). I’ViroZogy, 63, 484-491. Davidson, N. 85 Szybalski, W. (1971). In The Bacteriophage Lambda (Hershey, A. D., ed.), pp. 45-82, Cold Spring Harbor Laboratory, New York. Davis, R., Simon, M. & Davidson, N. (1971). Methods Enzymol. 21, 413-418.
322
H.
YOUXG
WI’
.iI i
Echols, H. c!z Murialdo, 8.. l:l978). hficrobioE. Rw. 48, 577-69 L. Piandt, M., Hradecna, Z.; Loeeron, H. A. 8x Szybalski, W. (197 1). In ‘Z%e lisr*:ter~&&q,~s Lambda (Hershey, A. D., ed.), pp. 329-354, Cold Spring Harbor Laboratory, New York. Groman, N. B. & Suzuki, G. (1962). J. BacCe&l. 84, 596-597. Rarris, A. W., Mount, D. W. A., Fuerst, C. R. & Siminovitch, L. (,lfN7). R’il*oCoggr, 82, 553569. Herskowitz, I. & Signer, E. R. (1974). Viroloyg, 61, 112-,119, Jacob, F. & Fuerst, C. R. (1958). J. Gen. Microbial, 18, 518-526. Kleckner, N., Ghan, R. K., Tye, B. EL. & Botstein, D. (1975), J. Mol. Biol. 91, ,561.~575. Kleckner, N., Rot)h, J. & Botstein, D. (1977). J. Mol. Ri,ol. 116, 125-159. Ohtsubo, H. & Ohtsubo, E. (1977). In DNA Insertion Elernenfs, Plum&ds mm? ,!Gpiso~ur~es (Bukhari, A. I., Shapiro, J. A. & sdhya, S. L., eds), pp. 49-63, Cold Spring Harbar Laboratory, New York. Parkinson, J. S. (1968). Genetics, 59, 311-325. Reader, R. W. & Siminovitch, L. (1971). Tiirology, 43, 6233637. Sharp, P. A., Cohen, S. N. & Davidson, N. (1973). J. Alol. Biol. 75, 235-255. Taylor, A. (1971). Nature New Riol. 234, 144-145. Taylor, A., Das, B. & Van Heijenoort, J. (1975). EM. J. Biochem. 53, 47-54. Thoma,s, R. (1971). In The Racterio@age Lambda (Hershey, A. D., cd.), pp. 211-219, Cold Spring Harbor Laboratory, New York.
Biol.
(1979) 132, 307-322
Transposition
Mutagenesis
of Bacteriophage
Lambda
A New Gene Affecting Cell Lysis RY YOUNG?,
JEFFREY
WAY,
AND M&HAEL
SUSAN WAY,
JERRY
YIN$
SYVANEN
Department of Microbiology and Molecular Genetics Harvard Medical Xchool, Boston, Mass. 02115, U.X.A. (Received 27 November 1978, alzd in revised form
8 March 1979)
Insertions of Tn903, a transposable kanamycin-resistance element, in bacteriophage lambda at 0.95 on the lambda physical map adversely affect growth of the phage. These insertion mutants are able to assemble particles, but are unable to lyse the infected cell properly. The mutants define a new genetic complementation group that we have designated as gene Rz. Cells infected with the XRz : :Tn903 isolates will, at the normal time of lysis, change their shape from a rod to a sphere. These spheres are stable in dilute buffers with Mgz’ but are lysed with EDTA. In addition, these results demonstrate the utility of transposition mutagenesis in refining the genetic map of even so intensely studied a genome as lambda.
1. Introduction Bacterial mutagenesis using the transposable drug-resistance elements is proving to be a very powerful tool in the genetic analysis of bacteria and their associated plasmids; this follows from the ability of these elements to insert into a very large number of bacterial and plasmid regions, including structural genes. Many different insertion mutations that inactivate bacterial genes have been obtained in this manner. The second property that makes these elements so useful in genetic analysis is that the insertion confers an antibiotic resistance to the bacteria. Hence, one can monitor the insertion directly by the antibiotic resistance; this is particularly useful in mapping genes whose complete inactivation does not affect cell growth and also in analyzing recessive-lethal insertion mutations. The advantages of using TnlO, a transposable tetracycline-resistance element, in bacterialgenetic analysis have recently been described by Kleckner et al. (1977). In addition to TnlO, other drug-resistant elements have been utilized, such as Tn5 (a kanamycin-resistance determinant; Berg, 1977), Tn3 and Tn9. In this paper we describe the use of Tn903, a kanamcyin-resistance determinant, for the genetic study of bacteriophage lambda. With bacteriophage like lambda, the size of the phage chromosome is limited by the packaging capacity of the phage ->*Ah-m< issspii..~s~d~ SEzaai.aL- -. . L r-as-. t Present address: Department Station, Texas 77840, U.S.A. $ Present address: Department 53706, U.S.A.
of Biochemistry,
Texas
A and M Medical
of Biochemistry,
University
of Wisconsin,
School,
College
Madison,
Wise.
307 0022-2836/79/230307-16
$02.00/O
0 1979 Academic
Press Inc. (London)
Ltd.
K. ‘YOUNG
308
ST
ii i
head. There-Lbre, in transposition mu.tagenesiu it is helpfu.1 to use a transposaible element that i8 relatively small in relation .to the phage genome. For example, TnlU, Tn3 and Tn5 are all larger than 5000 base-pairs. a considerable fraet,ion of the 46,500base-pair lambda chromosome. Hence ono advantage of Tn.903 is -thab it is small Tn903 has the characteristic flanking-inverted(3160 base-pairs). Furthermore, repeat, structure that gives rise to the striking iistem-and-LoopP’ structures in heteroduplexes. Tn9, the only drug-resistance transposon as small as X903, does not form stem-and-loops and is thus less convenienk for betSerodupiox analysis. Many mutants of bacteriophage lambda have been isolated that cause conditional or absolute defects in lambda growth (Campbell, 1961; Parkinson, 1968; Harris et al., 1967). We have isolated a large number of Tn903 insertions into la,mbda. In this isolation, we have required that the insertion mutants be able to assembIe phage particles and that they be able to establish themselves as lysogens. Among these, we found two classes that affected lambda growth a,dversely; one of these classes defines a new gene.
2. Materials and Methods (a,) Bacteria
and @iage
The Eschericlaia coli K12 strain W3110 (sup”) and Ymel (N~F) are used to follow phage growth. Bacteria are grown in TYM, a broth with tryptone (0.50/), yeast, extract (0.10/b), XaC1 (O.Sq/,) and malt,ose (0~2%). Plates conta,ined 1.6% agar of the above composition but without maltose. The phage strains Xb519cIts857Sam’i and Xb519b515cIts857nin5Sam7 were used as the recipients for Tn903 insertion. (b) Isolation
of Tra903 insertions
in, h
The self-transmissible drug-resistance plasmid R-6 (Cohen, 1977) was introduced into W3110 lysogenic for one of the lambda strains. After several days of subculturing, the lysogen was thermally induced and phage stocks containing 1011 to 1Ol2 p.f.u.t/ml were prepared. The pha,ge were adsorbed t,o W3110 (normally kanamycin-sensitive) at a ratio of 5 p.f.u./bacterium and lo8 infected cells plated on solid agar medium containing 40 pg kanamycin sulfate/ml. After 24 h at 3O”C, single kanamycin-resistant clones would a,ppear. All tested were lambda lysogens and t,hey gave rise to high-frequency transducing lysates for kanamycin resistance. Table 1 lists those we have used in this study.
TARLE I Xtrain
Fist
Phago designation
Complete genotype
x hpk3 hdk6 Xdk23 hpk26 hpk24 hpk22 XpkPl Xpk25 The number in parentheses following Tn903 give,,q the physical location of the insertion as measured from heteroduplex molecules or from the molecular weights of DNA fragments produced by SmaI and XfboI endonuclease digestion of purified DNA from each phage (see Results). t Abbreviation
used: p.f.u., plaque-forming
units.
LAMBDA
TRANSPOSITION
MUTAGENESIS
309
(c) Quantitative measures of XTn903 transduction to the measurement of plaque-forming units, the number of kanamycinresistant (kanR) transducing particles was measured by the ability to confer kanamycin resistance to bacteria. To lo8 cells of Ymel(X) is added lo5 to lo7 of XTn903 particles; after 5 min, 5 ml of TYM is added to the culture, which is then grown at 37°C overnight. The following day 0.05 ml of this culture is plated on to media containing 4Opg kanamycin sulfate/ml. Approximately 1 kanamycin-resistant clone appears for every lo3 XTn903 particles added to the original cells. The number of clones is linear over a lO,OOO-fold range with the number of p.f.u. added. In the determinations of the number of defective hTn903 particles we always include for calibration a non-defective hTn903, whose p.f.u. titer is known.
In addition
(d) Preparation
of lambda DNA
Lambda particles were purified from lysates by adding CsCl to a density of 1.5 g/cm3, centrifuging overnight at 30,000 revs/min in a Beckman SW50.1 rotor and extracting the turbid band of phage from the centrifuge tube with a syringe. The phage were dialyzed into 20 m&r-MgCl,, 20 mM-Tris (pH 8.0). DNA was extracted with redistilled phenol saturated with water. The DNA was dialyzed into O-1 M-KC& 20 mrvr-Tris (pH &O), 1 mM-EDTA. The endonuclease digestions of the DNA with SmaI, Hind111 and XhoI were performed as described in the instructions provided by BioLabs, from which these enzymes were purchased. Agarose gel electrophoresis will be described elsewhere. (e) Electron The hTn903 DNA was analyzed using the 40% formamide spreading
by forming technique
microscopy heteroduplexes with Ximm434 DNA described by Davis et al. (1971).
and
3. Results Tn903 is a transposable kanamycin/neomycin-resistant determinant found on the plasmid R6 (Sharp et al., 1973). It consists of a pair of flanking inverted repeat sequences 1130 base-pairs long, called 70 (Ohtsubo & Ohtsubo, 1977) and a unique central region 900 base-pairs long (Armstrong et al., 1977) coding for the drug resistance.
The physical
map
of Tn903
(a) Isolation
is shown
in Figure
of Tn903 insertions
1.
in lambda
We prepared 80 independent lysates by inducing W3110 parental-phage lysogens harboring the R6 plasmid. From each of these, a kanamycin-resistant lambda lysogen of Ymel was obtained that would yield high-frequency transducing lysates for
1600
II30
FIG. 1. Physical map of Tn903. The SlnaI and Hind111 cutting sites were determined by Armstrong et al. (1977). M. Casadaban (personal communication) informed us of the XhoI cutting site. The dimension of 18 was measured from the stem formed by self-annealing of the intrastrand complementary sequences from heteroduplex molecules such as the one shown in Fig. 4. The size standard for this measurement is the distance between b519 and the left edge of the imm434/immX junction and the distance between the right edge of the junction with the &n5 deletion (Fiandt et al., 1971).
.i 10
Ii.
YUUIUG
i!.!cP A1 e,
kanamycin resistance. Each of these lysogens \zas in.duced a;rld the lysat,es t,e&ed for plaque-forming ability on Ymel (supF). The V~/~~P-containing strain is used to test for plaque formation because the parental phage contains the RanL’i a;llele. Two !ysates contained almost no plaque-formers (hdk6 and hdk23’), a,nd five others gave very small plaques (Xpk21, Apk22, Xpk24, hpk25 and hpk26). The other stocks conof particle tained normal plaque-formers at high t’iter. The direct examinatjon production for each of t,hese seven XTn903 insertions and for Xpk3 (a normal plaqueforming derivat,ive) was done by inducing W3110 (SLY") iysogens of each phage. Phage growth for three hours after induction was permitted. (The 8nm’i mutation in these phages prevents cell lysis and permits particle assembly to continue well beyond the normal lysis time of 45 minutes.) Aft,er these cells were concent!rated and lysed by freezing and thawing, the lysat,es were adjusted to a density of 1.5 g!cm3 with CsCl. Sfter overnight centrifugation to establish CsCI gradients, dense phage bands were observed for all seven induced lysogens. The equal size for each band demonstrates that the defect in Xdk6 and Adk23 is not due to a gross inability is unto make particles, a.t least in growth conditions where the Earn7 mutation suppressed. This property is also evident ia Figure 2: which shows the kinetics of
Time after induction (min) P'IG. 2. Phage particle accumulation kinetics. W31 IO cells lysogenic for hpk5 or XdkB exponentially growing at 30°C wore diluted loo-fold into a prewarmed broth at 42°C after reaching A,,, = O”15. After 15 min the 25-1111 cultures were shifted to a 37°C aeration. Beginning at 30 min after induction, 2-ml samples were removed, vortexed with a few drops of CHCI,, and chilled. Samples were assayed for phage particle titer by the transduction assay, as described in Materials and Methods. The slopes of the accumulation curves varied signiScantJly from day to day, but the temporal shift in the curves was always 5 to 7 min., -o-o--, Xpk5; -e-e-, hdkli.
phage production for induced sup” lysogens of a normal plaque-forming XTn903 and hdk6. The kinetics of particle accumulation for hdk6 are identical to the plaqueformer, except for a five to seven-minute delay. (b) Heteroduplex
mapping
and restriction
enzyme
analysis
of
the Man isolates
The positions of the Tn903 insertion in four of t.he phages were determined by electron microscopic examination of heteroduplex molecules formed between the XTn903 and himm434 DNA. We examined Xdk6, hdk23; Xpk21 and Xpk24. Tn903 forms the stem-and-loop structure, indicating an invertled duplication in the DTw’A
LAMBDA
FIG. 3. A and hin~m434 deletion loop, stem-and-loop
TRANSPOSITION
MUTAGENESIS
311
heteroduplex DNA molecule formed between Xdk6 = b519cI857?tin5 Tn903 (0.951) DNAs. If one follows the molecule from the lower right to the upper left, the b519 the i,mmX/ivw~434 substitution “bubbles”, the nin5 deletion loop a,nd the Tn903 structure are encountered in order.
(Fig. 1) similar to that observed for Tn5 (Berg et al., 1975) and TnlO (Kleckner et aE.: 1975). For the normal plaque-formers, Tn903 insertions were mapped in many places in the lambda genome. The four isolates with altered plating properties all mapped between lambda co-ordinates 0.95 and 1.00; at the extreme right end of the lambda genet,ic map. Figure 3 shows an electron micrograph of a heteroduplex of Xdk6 with himm434; the deletion loops corresponding to b519 and nin5 and the characteristic double substitution-deletion loops corresponding to the imm434 region are clearly visible; as is the stem-and-loop structure of Tn903 at about 0.95 on the h molecule. (h co-ordinates are 0.000 at the left end and 1.000 at the right.) We measured the size of the stem at 1130 base-pairs and the loop at 900 base-pairs. In addition, the positions of each of the seven XTn903 insertions that alter lambda growth were determined with the use of two restriction endonucleases (SmaI and XhoI) and agarose gel electrophoresis. These two enzymes cut Tn903 DNA at sites shown in Figure 1; SmaI cuts in the duplicated r$ sequences and XhoI cuts in the unique region. The SmaI endonuclease cuts h at 0.825. Thus after digestion with this enzyme, the right end of X DNA will be found on a fragment that is 0.117 h lengths (since the phage carries the nin5 deletion of 0*058 units). When DNA from each of the seven Tn903 insertions is digested with SmaI and the size of the resulting fragments measured by means of agarose gel electrophoresis, the 0.117 h length piece is missing. Four new fragments appear, two very small pieces consisting entirely of Tn903 sequences, and two pieces, consisting of 78 and X sequences, totaling 0.137 X
$12
R. YOUSG
1;;T dL.
lengths in size. Each of these latter two fragments will haSva 450 base-pairs, or I)*61 A units of 78 sequences. The size for each of these two fragments (which is given in Table 2) shows that the insertion is at one of two possible locations, either near the right end or close to t’he #r/ha1 cutting site at 0~825. To decide between the two sites, each sample was cut with the XhoI nuclease and the size of the fragments determined. The XhoI nuclease cuts lambda once at 0~703 (our da,ta,) and ma,kes a single asymmetrical cut 0.025 X units from one edge of Tn903 (see Fig. 3). Based on the size of the smallest XhoI digestion fragment when the hTn903 D?JVAs are cut (Table 2), we conclude that Tn903 for all seven insertions must map at t)he site closest to the right end. These sites for the seven insertions are given in Table 2. Because the Xhof cutting site in Tn903 is asymmetric, the size of the smallest XhoI fragment uniquely determines the orientation of each Tn903 insertion (Table 2). Orientation I means that the direction from the Hind111 site toward the XhoI site (see Fig. 1) is from left to right on the h map. TABLE 2 Location
of T&O3
in the seven ATn903 altered
Phage
____-__ hdk6 hdk23 Xpk26 hpk22 hpk24 Xpk21 hpk26
Two smallest observable SmnI fragments 0.082 0.059 Not done 0.109 0.033 0.11 0.028 Not done 0.117
0.016
0.121
Smallest XhoI fragment
~-___ 0.073 0.074 0.046 0.045 0.046 0.032 0.027
de&atives
with
growth
~~
Position of insertion
Orientation insertion
of
~~ ..-~~--.. 0.952
$- o.oost
0.951 * o.oost 0.979 0.98 0.980 0.993 0.998
I I I I
= 0.003t & 0.002?
I I I
The position and orientation of Tn903 in each DNA was determined from the molecular weight of the 2 smallest observable fragments produced by SnaaI digestion and the smallest fragment produced by XhoI digestion. Molecular weights are expressed in fractions of h and the map positions are linear with A DKA beginning at 0 at the left end and going to I.0 at the right end. Molecular weights were determined by electrophoresis of the DNA in agarose gels. 7 Positions of Tn903 insertion were initially determined by heteroduplex analysis.
The important result from this Table is that the two defective Tn903 insertions both map at 0.95 on A, while the five small-plaque-forming variants are between 0.98 and 0.997. In Figure 4 we give the genetic map of the right end of the phage X chromosome and show the locations and orientations of the seven Tn903 insertions given in Table 2, as well as the location and orientation of Tn903 in Apk3 (mapping data not shown). This region of the X chromosome contains the genes S and R, which are required for cell lysis, and the Q gene, whose protein product is necessary t,o activate late gene transcription from the late promot!er pR’. Transcription from pR’ proceeds rightward in Figure 4 through the lysis genes, through the cos site, and transcribes all of the genes coding for head and tail structural proteins, beginning with A, W, B and extending through J (not shown in Fig. 4). Any insertion mutation that interferes with the transcription from pR’ could therefore be defective for phage growth by reducing expression of these late genes. However, this explana,tion is not sufficient to explain why hdk6 and Xdk23 arc defective, Given that all seven
LAMBDA
TRANSPOSITION
MUTAGENESIS
313
insertions are in the same orientation, it is hard to imagine why those at 0.95 interfere with transcription, whereas those at 0.98 and 0.99 do not. A more reasonable explanation is that the insertions at 0.95 have inactivated an essential h gene.
FIG. 4. Insertion sites of Tn903 in the h late transcriptional unit. Vertical arrows indicate positions of insertions as determined by physical analysis of the DNA molecules. Approximate positions of known genes are taken from Echols & Murialdo (1978). Numerical values are given in Table 1. Also shown is Xpk3, which maps at 0.923 and is a normal plaque-former. Horizontal arrows indicate orientation of insertion site, from the XhoI to the Hind111 site as shown in Fig. 1.
(c) Plaque-forming
revertants of h&6 have lost Tn903
Since hdk6 does not make plaques, normally particles were assayed by transduction of a bacterial strain already lysogenic for h+ (see Materials and Methods). The different XTn903 particles behave identically in such infections, presumably because the primary event leading to a kanamycin-resistant colony should be the recombination of the repressed, circular hkan molecule into the h+ prophage. However, at frequencies between 10e4 and 10m5 per particle, two types of plaque arise: small plaques, which grow equally well on supF and sup’ bacteria; and large plaques, which grow only on supF lawns. The parental h phage grows only on supF-containing bacteria because of the Sam7 mutation. For plaque-forming hTn903 phage, kanamycinresistant lysogens grow in the plaque centers at 30°C. However, neither type of Xdk6 revertant plaque yields such resistant cells. Hence, the plaque-forming revertants of Xdk6 have gained the ability to grow and have lost their ability to transduce kanamycin resistance. To further characterize hdk6 and its revertants, we determined the distribution of these particles in a CsCl sedimentation equilibrium gradient, as is shown in Figure 5. Phage particles of lambda have a characteristic buoyant density that reflects their DNA and protein content. Since the chromosome of X is 6.8% shorter than XTn903, the phage that have lost Tn903, i.e. the revertants, should band at a lower density. The results in Figure 5 show the peak of the non-plaque-forming hdk6 particles flanked by two plaque-forming revertant peaks. The large-plaque-forming revertant gives a peak separated from the hdk6 peak by six fractions to the lower density; this corresponds to a change of approximately -6% of the X genome when deletion standards a,re subjected to the same centrifugation (data not shown). Thus the observed particle density is as expected if the large-plaque-forming revertant is formed by simple excision of Tn903 from Xdk6. The small-plaque-forming revertants, which define the higher density peak, are most likely the product of a “QSR” deletion-substitution event. In this event, the region between co-ordinates 0.838 and 0.95 on h is deleted and substituted by a functionally equivalent but larger and non-homologous region derived from a cryptic lambdoid prophage resident in the E. coli chromosome (Herskowitz & Signer, 1974). The
sre
R ).
YOUNG
&it‘
,it
density increase of the small-plaque revertauta, with respect to the A~tk6, corresponds l;o an 80/b increase in the genome. This is the wize increase expected if Adk6 lost, t;he, X sequences between 0,838 and 0.95 (including the Tn903 insert,ion) and replaced this DXA with the “QXR” or p4 insertion (Fiandt, et ad.: 1971). This substitution would also explain why these revertants become am+, since tshe Sam7 lesion resides in the sub&itut,ed regi.on. ‘wary In addition, DNA from iive independent, Ilarge-play us-forming rev&ants prepared and examined in the electron microscope by forming hitjeroduplex mwlecules with himm434. The IieterodupIexes were featureless, except for t-he parental and Ximm434 delet’ion and substitution loops (dat,a not shown). We conclude that the excision of Tn903 is precise, at least, to the level of 100 nucleotjdes, which is t,he lower limit of resolution of the heteroduplex technique.
Fraction
number
FIG. 5. Equilibrium density centrifugation of ;\dk6 phage particles. A volume (2.55 ml) of 3 x 109 hdk6 particles was mixed with an equal volume of a saturated CsCl solution and centrifuged for 28 hat 20~000 revsjmin in a SWSO.1 rot&. A total of 95 sa.mples were collected after piercing\he bottom of the nitrocellulose tube. The number of plaque-forming phage in each fraction was det,ermined by titering aga,inst IV3110 (sup”) (-E-n-), (8~~8’) (--e-e--). The was determined as out,linetl number of kanamycin-tral2sducirlg particles (- I’i,-- c)-) in aterials and Methods.
(d) The S+ allele makes hd1%6’CLGny-$aTue-forme? All of the Tn903 isolates carried the Xana7 allele: in order to facilitate preparation of high titer st,ocks. When the S+ allele was recombined into hdk6 and ;\dk23, we found these phage now made tiny pinpoint plaques that, can be seen on a lawn of W3110 by viewing through a dissecting microscope. When the S+ allele was introduced into the phages with the more distal insertions, t’he plaque size increased to the extent that t)hey were barely distinguishable from Aclts$%‘S + . (e) hdk6 dejines
a new conzplementation
group
The two defect’ive phages; Xdk6 and Xdk23, have the Tn903 ins&ion at 0.96 on t’he X map. This is immediately to the right, of gene R: according to the generally accepted X map (Davidson & Szybalski. 1971). We therefore t,ested whether the insertion in hdk6 canses a mutation that defines a new complement~ation group. As can be seen in Table :‘rl Xdk6 will complerrlc~nl arr~bc~ nlt~l at.ionx in genes N, P, Q, R, da W and B. In order to test fbr corn~le~rlentation of the S gent, we corl.structe(l 1ysogen.s of Xdk6S + and, as control, hpk25S + in tjhe sup” bacteria W3110. The plat,ing
LAMBDA
TRANSPOSITION
MUTAGENESIS
315
efficiency of different heteroimmune phage with amber mutations in genes 8, R, A and K was determined on each. These het~eroimmune phage should be able to grow TABLE 3 Cowqdementation Phage Xdk6 hNaml1 XPam3 XRam5 XAam32 hSam7
XNaml7 ++ -++ ++ ++ ++
XQam73
XPam3 ++ ++ -++ ++ + -1
++ ++ +I+ + ++
tests for
h&c6
XRam5
ASam -++ ++ ++ ++ --
XAam32
+ ++ ++
++ ++ ++ ++ -++
++ ++
xwam403
t:: is ++ ++ ++
XBam6 ++ ++ ++ ++ ++ ++
Phage stocks of lo7 and lOa particles/ml were by dilution from fresh plate lysates, except for hdk6, which was diluted from a high-titer CsCl-purified stock. Complementation tests were performed using 5.~1 drops of each phage on a W3110 lawn in top agar. Complementation was scored as positive if confluent lysis under a spot resulted from overnight incubation at 37°C. + +, Confluence using lo7 particles/ml stocks; +, confluence at lo8 particles/ml. The reduced complementation of hdk6 and hRam5, and of XQam73 for XRam5 and XAam32 was reproducible. Note Xdk6 carries the Sam7 allele, and thus does not complement /\Sam’i.
on the sup’ lysogens, since the superinfecting phage can transactivate the late genes from the resident prophage (Thomas, 1971) unless the resident prophage is defective in the same gene. As is shown in Table 4, lysogens of XdkGS+ and Xpk25S + will equally support the growth of all four of the heteroimmune ambers. The data in Tables 3 and 4 show that hdk6 has a functional R gene, and that the insertion is not significantly polar on h genes A through K. Thus the mutation caused by the insertion at 0.95 defines a new complementation group. (f) Insertion
site and late gene expression
Insertions of Tn903 into the X late transcriptional unit between co-ordinates 0.95 and 1.00 could be polar on the head and tail genes, since in the vegetative state the
TABLE
Tmn.sactivation
Ymel Test phage Ximm434 Sam7 himm434 Ram7 Ximm2 1 Aam Ximm434 Kam21
1.0 1.0 1.0 1.0
4
of )tS+dkG and hS+dk23 genes Efficiency W(hS+)
10-l 1.0 5x10-a 5 x 10-l
of plating W(hS+dkB)
W(XS ‘pk25)
IO-1 1.0 5x10-z 10-l
10-l 1.0 5x10-2 10-l
Five-p1 drops of 109, 107, IO5 and lo3 phage/ml of the test phage stocks were spotted on lawns of the indicated lysogens of W3110. The efficiency of plating was calculated from the number of plaques seen on each of the lysogens relative to the number seen on the suppressor (Ymel) lawn after overnight incubation at 3O’C. Plaque sizes vary significantly in the spot zones, presumably due to a wide spread in absorption times for the spotted phage. Data presented are the averages from 2 experiments.
R.
316
YOUNG
ET
ri L.
cohesive ends of h are joined together (see Pig. 4). In the course of evalua,ting Lhc ,eomplementation t#est we saw some indicat,ion that the Trr903 insertions in Adk6 and hpk25 are somewhat polar on the distal genes of the late operon. We therefore eonsidered in more detail whether the defect in Xdk6 was perhaps due $0 polarity. If there are no necessary genes between 0.95 and 11*00, we would have n.o reason to suspect, a difference in the viability of hdk6 a,nd Xpk26. However, if the insertion at O-95 is more polar on the head and tail genes tha,n the insertions from 0*98 and 0,997, then the defective character of Xdk6 and the contrasting plaque-forming character of the more distal insertions could be explained as a cumulative polarity effect, not detectable in the complementation tests of single amber mutants. Quantitative complementation tests in liquid culture were carried out to assess fnrther the effects of t#he insertion site on late gene expression. The results of these complementation tests were unusually variable. In some experiments, the ability of the late operon insertion phages to complement the immediately dist’al late genes (A, W and B) did not differ from the (uninserted) parental phage; in other experiments, there was as much as a tenfold decrease observed, relative to the parental (data not shown). However, there was no detectable difference between the late gene complementation activity of hdk6 and hpk25 in any experiment. We conclude the Tn903 inserti0n.s at h co-ordinates 0.95 and 0.998 are equally polar on X late gene expression. Confirmat,ion of this result was obtained when phage yields were measured, upon a single-step infection (Table 5). Particle production is reduced twofold or less from dhe uninserted parent levels for both hdk6 and Apk25. Thus t’he severely reduced ability of hdk6 to make plaques does not st,em from increased polarity on the X capsid structural genes, relative to the more distal insertions.
TABLE
Single-step Phage /is+ Xdk6S + Xpk25S +
6
infection phnge yields Yield 45 28 30
Relative -.___-.--
yield? 0.47 0~29 0.32
One ml of W3110 cells growing logarithmically was chilled at Aej,, = 0.35 and infected with one of the phages at a multiplicity of 0.3. After being left for 20 min at room temperature for preadsorption, the cells were washed by centrifugation and resuspended in room temperature broth, and diluted 10s3 into prewarmed 37°C broth. The diluted, infected cultures were aerated at 30°C for 70 min and then vortexad with a few drops of CHCI,, prior to storage at 4°C. The lysates were titered by plating with W3110 as an indicator on very fresh broth plates. Idk6S+ plaques were counted using a steromicroscope. Data are averages from 2 experiments. p XI&I+ gives a relative yield of 1.0, hS + ; the parental phage, carries the b519 and n&5 deletions
(g) Adk6 is defective in cell lysis The fact that hdk6 and the plaque-forming phages bearing the more distal insertions produced the same number of infective particles in a single cycle of infection made it possible that the defectiveness of hdk6 could reside in its ability to lyse cells. To test this idea, the turbidity of cultures was followed after infection with hdk6
LAMBDA
Time after
TRANSPOSITION
infection (a)
(min)
MUTAGENESIS
Time after
induction
317
(min)
(b)
FIG. 6. (a) Lysis defect in Xdk6 infection. W3110 was grown at 37°C in liquid media until A,,, = 0.4. Aeration was stopped and the culture was made 10 mm in MgCl, before dividing into 3 portions. One portion was infected with hdk6S +. another with hpk25S +, and the third with hS +, each at a multiplicity of 7. After 5 min, aeration was resumed and the absorbance at 650 nm measured at appropriate intervals. --o-n--, Xdk6; --a-@- -, Xpk25S+ or x3+ (curves indistinguishable); -O---O--, Xdk6S+ with no MgCl, added. Identical results were obtained when the Sam7 version of each phage was infected into Ymel (.supP). (b) Lysis and kinetics of induced lysogens. W3110 lysogens of Xdk6S +, hpk25S +, and XS+ were grown in TBr until A,,, = 0.3 to 0.5. The cultures were shifted to 42°C with aeration for 15 min, followed by continued aeration at 37”C, and the absorbance at 650 nm monitored at appropriate intervals. --O--O--, W3110 (hpk25S+) or W3110 (AS+); -O-O-, W3110 (hdkSS+); -O--a---, W3110 (AdkBS+) with 10 mM-MgCl, added at t = 0. Identical results were obtained with Ymel lysogens and the X7 virions of each phage.
(or Xdk23) (Fig. 6(a)). The turbidity was found to increase for a few minutes after infection, before reaching a plateau, which remained unchanged for several hours. In contrast, cultures infected with hpk25 or the parental phage would lyse within one hour after infection. The lysis defect in infected cultures contradicted our observation that induced cultures lysogenic for hdk6 showed clearing at the expected time (data not shown). To resolve this conflict, the kinetics of cell lysis were followed more closely by monitoring the turbidity of a thermally induced culture of Ymel (Xdk6). Lysis was observed, beginning at approximately the same time as lysis of the lysogens with either the prophage or with Xpk26 prophage, as shown in Figure 6(b). However, the kinetics of thermally induced lysis is reproducibly complex; the turbidity falls to one-third of its original value, remains constant for approximately 30 minutes, then resumes its decline. Thus, Xdk6 has a lysis defect that is more apparent during infection than induction. The apparent difference between the severity of the lysis defect in infection and induction was eliminated when the effect of Mg2+ on the lysis curves was tested. In the infect’ion experiments, 10 mM-Mgcl, had been added in order to facilitate was added to the induced lysogenic cultures, lysis phage adsorption. When Mg2+ + but completely unaffected for Apk26S +. When was nearly eliminated for Xdk6S
318 zag=-I was removed
R. Y-0UNG it 2’ ,-Irt,
from t’he in&&ion prod.UCCJ~~htik8kj r ”j niv b.yi uuit lJ.l”es iA‘I”lY‘ observed to lyse, although lysis was significantly slower %han in. hpk26S’ cultures. Thus Xdk6 has a subtle lysis defect, ~which is absolute in I& present of 10 rn~~-Xg~ + I As expected, the magnesium ion also efiminat’es t,he nlnility of hdk6S+ to form tiny, pinpoint piques (d.ata not shown).
Although the complementation spot, tests and the transa,cti~z&~n experiments showed that Xdk6 is unaffected with respect t,o Ghe expression of t,he B gene, t’he lysis defect observed. and the position of the T&O3 ins&tion so n.ea,r the b0uadar.y of the R gene make it necessary to eliminate .E as a candidate for the insertion lesion.. Figure 7 shows the lysis kinetics of cells infect,ed with hdk6 or .XRam,!XRamEiO~ in
-30
0 30 60 90 Time after infection (min)
FIG. 7. Functional complementation between hRam54RamGO and hdk6S ?. WV3110 was grown in TYM at 37°C until _4 850 = 0.35. Aeration was stopped and the culture mado 10 m~inXgC1, before it was divided into 3 parts. One part was infected with XdkGS+ at a multiplicity of 10, another with XBnnx54Ra~nGO at a multiplicity of 10, and the third part with both these phages; at a multiplicity of 6 each. After 5 min, aeration was resumed and the absorbance at 650 nm monil-orod -z-s--, /w&s+; --g-2--, at appropriate intervals. --A--A-, hRam54Eam60; both phages.
the presence of 10 mM-Mg2 +. Although neither hdk6 nor t,he R double amber mut)ant infections result in lysis, a culture infected with both phages does lyse, albeit, slowly. Thus Xdk6 and the R mutant complement each other for lysis. We conclude that hdk6 must be functionally R +. (i) IVormal endolysin
coatent
of induced
A&G
iysogen
The possibility that the defect in hdk6 is due to an alteration of the R gene was also tested by direct assays in vitro for endolysin activit’y, t,he product, of gene R. Extracts of induced lysogens of hdk6 and hpk26 were tested for the amount of endolysin and for the kinetics of reaction w&h the substrat’e. Table 6 shows that the endolysin activity, a,s measured by the lysis of sensitive ceils (Court r,t al., 1975) was identical in bot’h hdk6 and Xpk26. We conclude t,hat the defect in Xdk6 does not lie in the R gene, the nearest known gene to the insertion site of Tn903 in the defective phage.
LAMBDA
TRANSPOSITION
319
MUTAGENESIS
TABLE 6 Endolysin Lysogen W3110(Xdk6) W3110(hpk25)
Endolysin
content of induced lysates
units/ml
Azso units/ml
1680 2650
Endolysin
47 70
units/&se 36 38
Cultures (100 ml) of the indicated lysogens were induced in late logarithmic phase, concentrated to 1 ml in TBr, quick-frozen in a methanol/solid CO2 bath, and thawed at 30°C in the presence of 5 pg DNase/ml. After removing debris by centrifugation at 3000 g for 5 min, the extracts were chilled and assayed within the hour. Endolysin assays were performed as described in Materials and Methods.
(j) Sphere-formation
of infected cells
A novel aspect of the lysis defect in hdk6-infected cells was revealed by phase contrast microscopy. Beginning at 30 to 35 minutes after infection, hdk6-infected cells abruptly change from the elongated rod shape characteristic of E. coli to oblate spheroids. This sudden change is followed by a slower metamorphosis into spheres, examples of which are shown in the micrograph in Figure 8. The spheres seem to be, at least initially, motile, and they are extremely fragile, since gentle pressure on the coverslip causes their destruction. Cells infected with XX- or hR- mutants also do not lyse, but neither do they change the rod-like morphology. In the absence of 10 mM-MgCl,, these spheres will spontaneously lyse on the microscope slide. The addition of EDTA to a culture of spheres leads to immediate lysis.
FIG. 8. Morphology of Xdk6-infected cells. Logarithmically growing W3110 was infected with Xdk6S + at a multiplicity of 3, in the presence of 10 mix-MgCl,. Samples were removed and observed under phase contrast microscopy; magnification 630 x Spherical cells begin to accumulate at about 30 min; the photomicrograph is taken 90 min after infection. Some of the spherical cells have become transparent by this time. The rod-shaped bacteria are presumably uninfected cells; in cultures infected with higher multiplicities, the frequency of rod-shaped cells drops.
We have described. a new essential gene in phage A. A lesion in this gene was8 obtained by transposition mutagenesis with Tn903, a kanamycin-resista,nce transposon. Two independent insertions of 1’11903 into the region of h co-ordinate 095Z & 0.002 result in defective phages; the defect is co.mplement,ed by all known genes; in&ding the nearest cistron, gene R. Five other independent insertions more distal on the right arm of A (between co-ordinates 0.979 and 0,998) result, in plaque-forming ‘rhat it must lie phages. Thus we can define a new gene, an,d state unequivocally between h co-ordinates 0.951 (the right t’ermirms of gene R) and O-979. (b) Lysis-defective
phe,aotype
The phenotype of phages with the insertion lesion in the new gene is abnormal cells infected with the mutant phage do not lyse at all. Since lysis; in 10 mM-MgCI,, the insertion maps so near the known locus of the R gene (Davidson & Szybalski, 1971) and the R gene product is required for Iysis (Jacob & Fuerst 1958), it was important to test these mutants for R activity. We find %he cont!ent. of endolysin (the R gene product) to be unchanged from the parental phage, when measured in induced lysogens. Furthermore, a double nonsense mutant of t#he R gene complemented the insert’ion mutant for lysis of mixedly infected cells in 10 m&r-MgCl,. Thus genetically and §unct,ionally, the defect,ive insertion mutants define a new gene. Because it is an essential gene, and because it is a lysis gene mapping adjacent to the R cistron, we ea.11the new gene the Rz gene. (c) Spherical
cell formatim
Phase contrast microscopy of cells infecected wit’h t#he defective insertion muta,nts revealed t,hat inst,ead of lysing, the cells were transformed from rods to spheres, In 10 mM-MgCl,, the spheres were completely st’able; although very fragile mechanically. The observation t’hat the mutant-infected cells become spherical instead of lysing suggests that t,he sphere form is an intermediate in normal lysis. There is precedent for such a spherical lysis intermediate in the observations of Groma,n & Suzuki (1962), who observed short-lived spherical forms appearing immediately before lysis of induced X lysogens. In addition, Birdsell & Cota-Robles (1967) showed that plasmolyzed, lysozyme-treated E. coli cells became spherical, without losing turbidity, and that further osmotic shock or treatment with EDTA resulted in lysis. They suggested that the outer membrane cont’ributes to the maintenance of cellular integrity, since treatment with lysozyme alone results in sphere format,ion rather than complete lysis. It is possible then t,hat the attack of endolysin, the R gene product, on the pept,idoglyean is necessary but not sufficient for normal lysis. A endolysin is an endopeptidase, which cleaves the peptide bond between diaminopimelate and nalanine in the cross-linking polypeptides of the peptidoglycan (Taylor? 1971). Cleavage of these bonds should still leave the polysaccharide chains intact, if the outer membrane is also intact. Then one might expect t’he “relaxed” peptidoglycan outer membrane wall structure to lose much of its shape and rigidity. MgZ+ is required to stabilize t,he spheres, which is consistent with outer membrane involvement. The function of the
LAMBDA
TRANSPOSITION
MUTAGENESIS
321
Rx gene product may thus be needed to weaken this “relaxed” complex and ensure complete lysis. Taylor found evidence for two peptidoglyoan-degrading activities in h lysates, only one of which corresponds to the known endopeptidase activity of gene R (Taylor et al., 1975). It is possible that the Rx gene codes for a lysozyme-type protein, which attacks the polyglycoside backbone of the peptidoglycan. (d) Detection of a phage gene by transposition
mutagenesis
h is possibly the most extensively characterized genetic entity, principally because powerful methods exist for finding conditional lethal mutants. Why have no nonsense or temperature-sensitive mutations in Rx been found in the many mutant searches previously conducted on A? The answer to this is that hRz- phage under normal plating conditions makes very tiny plaques. It was not until we had worked with Xdk6 for an extended period of time that we realized that Mg2 + stabilized the highly unstable spheres and consequently XRz- phage would not make any plaques when the indicator bacteria were grown in the presence of 10 m&r-Mg2 +. Thus it was only by virtue of the fact that the original hdk6 mutant carried Tn903 at the site of the Rz gene that allowed us to isolate this mutant (by conferring kanamycin resistance to its lysogens) and to map its physical location (by the striking appearance of Tn903 in heteroduplex molecules) with the electron microscope. The phenotype of Rz- is much too subtle to allow its isolation by more traditional means. (e) Implication
for lysis control and cell ultrastructure
It is now apparent that h elaborates at least two enzymes to attack the cell wall. Presumbly neither can function from within the cytoplasmic membrane and in AS-infected cells the peptidoglycan is unimpaired (Reader & Siminovitch, 1971). Thus S effects the transport of two different enzymes to the periplasm, the R and Rz gene products. S-protein may then be a generalized t,ransport function, of wider biological significance than heretofore imagined. This work was supported by a grant 5ROlCA18217). One author (R. Y.) had Institutes of Health (IF 32 AT 05622-01).
from the National Institutes of Health (no. a post-doctoral fellowship from the National
REFERENCES Armstrong, K. A., Hershfeld, V. & Helinski, D. R’. (1977). science, 196, 172-174. Berg, D. E. (1977). In DNA Insertion Elements, Plasmids and Episomes (Bukhari, A. I., Shapiro, J. A. & Adhya, S. L., eds), pp. 205212, Cold Spring Harbor Laboratory, New York. Berg, D. E., Davies, J., Allet, B. & Rochaix, J. D. (1975). Proc. Nat. Acad. Sci., U.S.A.
72, 3628-3632. Birdsell, D. C. & Cota-Robles, E. H. (1967). J. Bacterial. 93, 427-437. Campbell, A. (1961). Virology, 14, 22-32. Cohen, S. N. (1977). In DNA Insertion Elements, Plasmids and Episomes (Bukhari, A. I., Shapiro, J. A. & Adhya, S. L., eds), pp. 672-673, Cold Spring Harbor Laboratory, New York. Court, D., Green, L. & Echols, H. (1975). I’ViroZogy, 63, 484-491. Davidson, N. 85 Szybalski, W. (1971). In The Bacteriophage Lambda (Hershey, A. D., ed.), pp. 45-82, Cold Spring Harbor Laboratory, New York. Davis, R., Simon, M. & Davidson, N. (1971). Methods Enzymol. 21, 413-418.
322
H.
YOUXG
WI’
.iI i
Echols, H. c!z Murialdo, 8.. l:l978). hficrobioE. Rw. 48, 577-69 L. Piandt, M., Hradecna, Z.; Loeeron, H. A. 8x Szybalski, W. (197 1). In ‘Z%e lisr*:ter~&&q,~s Lambda (Hershey, A. D., ed.), pp. 329-354, Cold Spring Harbor Laboratory, New York. Groman, N. B. & Suzuki, G. (1962). J. BacCe&l. 84, 596-597. Rarris, A. W., Mount, D. W. A., Fuerst, C. R. & Siminovitch, L. (,lfN7). R’il*oCoggr, 82, 553569. Herskowitz, I. & Signer, E. R. (1974). Viroloyg, 61, 112-,119, Jacob, F. & Fuerst, C. R. (1958). J. Gen. Microbial, 18, 518-526. Kleckner, N., Ghan, R. K., Tye, B. EL. & Botstein, D. (1975), J. Mol. Biol. 91, ,561.~575. Kleckner, N., Rot)h, J. & Botstein, D. (1977). J. Mol. Ri,ol. 116, 125-159. Ohtsubo, H. & Ohtsubo, E. (1977). In DNA Insertion Elernenfs, Plum&ds mm? ,!Gpiso~ur~es (Bukhari, A. I., Shapiro, J. A. & sdhya, S. L., eds), pp. 49-63, Cold Spring Harbar Laboratory, New York. Parkinson, J. S. (1968). Genetics, 59, 311-325. Reader, R. W. & Siminovitch, L. (1971). Tiirology, 43, 6233637. Sharp, P. A., Cohen, S. N. & Davidson, N. (1973). J. Alol. Biol. 75, 235-255. Taylor, A. (1971). Nature New Riol. 234, 144-145. Taylor, A., Das, B. & Van Heijenoort, J. (1975). EM. J. Biochem. 53, 47-54. Thoma,s, R. (1971). In The Racterio@age Lambda (Hershey, A. D., cd.), pp. 211-219, Cold Spring Harbor Laboratory, New York.