OF THE T-EVEN BACTERIOPHAGES
RICHARD L. RUSSELL
Division of Biology, California Institute of Technology, Pasadena, California 91109
Manuscript received April 25, 1974 Revised copy received July 5, 1974
A system of amber mutants has been developed for each of the T-even bacteriophages T2 and T6, to complement those already available in T4. I n
T2 these mutants identify 52 genes, of which 49 are homologous with T4
genes; in T6 they identify 45 genes, of which 42 have T4 homologs, and an additional one which is homologous to a T2 gene not yet identified i n T4.
In both T2 and T6, recombination between mutants is characterized by con- siderable negative interference, which is correctable by a mapping function designed for T4. Recombinational maps of T2 and T6 constructed with these mutants have the same gene order and nearly the same gene spacings as in T4,
with the exception of the tail fiber region; T2 and T6 appear to lack a localized recombinational expansion of this region found in T4. Homologous gene prod- ucts from all three phages are in general interchangeable, with the exception of those from two apparently "co-adapted" tail fiber genes, 37 and 38. The general genetic similarity of all three phages suggests that they are anal- ogous to races of higher organisms, retaining the capacity for genetic exchange despite some clear genetic differences and some incipient isolating mecha- nisms.
extensive set of conditional lethal mutations isolated i n bacteriophage
has been used to provide a description of its genome which is unsur-
passed in completeness
The related bacteriophages
ever, have received much less genetic attention
even though conditional lethal mutants can be isolated with
equal ease in all three phages.
report below the isolation and partial character-
ization of conditional lethal mutants of bacteriophages T2 and
have proved useful for a detailed genetic comparison of the three T-even bac-
teriophages (see below) and for a study of their interactions in mixed infections
1970; PEES 1970; RUSSELL
also been used to construct phage hybrids of defined composition for a variety
'Research supported by Public Health Service Training Grant GM 00086 and by grants to R. S. EDGAR from the National Foundation for Infantile Paralysis (CRBS-120) and the National Science Foundation (GB 3930). It was com- pleted in 1967 and submitted as part of a Ph.D. thesis at the California Institute of Technology. Although unpublished, it came to the attention of several interested workers who have subsequently investigated some of its aspects more fully.
968R . L. RUSSELL
MATERIALS A N D M E T H O D S
Phages; T4D wild type, T2L wild type, T6 wild type, and all mutants of T4D were obtained from the collection of DR. ROBERT S. EDGAR. Amber mutants of T 2 with the prefix FS were obtained from DR.
FRANKLINW. STAHL. All other mutants of T 2 and all mutants of T6 were isolated in this study and are described below.
Bacteria: All bacterial strains were obtained from the collections of DRS. ROBERT S. EDGAR and JEAN J. WEIGLE. The E . coli strain BB from these collections proved to be permissive for most T 2 and T4 amber mutants, in contrast to the BB strain maintained at the Virus Labora- tories of the University of California at Berkeley. The origin of this difference is not clear.
Media: Hershey broth contained 8 g DIFCO Nutrient Broth (dehydrated), 5 g DIFCO Bacto Peptone, 5 g NaC1, 1 g dextrose (glucose), and 1000 ml distilled H,O, and was adjusted to pH 7.4 with 4% NaOH before distribution and autoclaving. Enriched Hershey bottom agar
(used for most plates, 30-35 ml per plate) contained 10 g DIFCO Bacto-Agar, 13 g DIFCO Bacto-Tryptone, 8 g NaC1, 2 g sodium citrate dihydrate (Na3C,H,*2H,O), 1.3 g dextrose (glu- cose) and 1000 ml H,O. Enriched Hershey top agar (used for most plates, 2 ml per plate) con- tained 6.5 g DIFCO Bacto-Agar, 13 g DIFCO Bacto-Tryptone, 8 g NaC1, 2 g sodium citrate dihydrate, 3 g dextrose (glucose), and 1000 ml H,O. Slant Medium No. 1 contained 18 g DIFCO Bacto-Agar, 8 g DIFCO-Tryptone, 1 g DIFCO Yeast Extract, 5 g NaC1, and 1000 ml H,O, and was adjusted to p H 7.4 with 4% NaOH before distribution and autoclaving.
Chemicals: 5-bromodeoxyuridine (5-BUdR) was obtained from the California Corporation for Biochemical Research (A grade). Propylene glycol monolaurate, often used as an anti-foam- ing agent, was obtained from K & K Laboratories, Inc. All other chemicals were standard reagent grades.
Antisera: The antisera used in this study were gifts from various people, as follows: anti-T2, J. J. WEIGLE; anti-T3, R. S. EDGAR and J. E. FLATGAARD; anti-T6, R. S. EDGAR.
Mnintenance of bacterial strains: Bacteria were maintained on slants of Slant Medium No. 1 ;
transfers to fresh slants were carried out every six to eight weeks, followed by one day of growth at 37" and subsequent storage in the cold (4").
Stock cullures of bacteria were made by inoculating about 30 ml of Hershey broth with a loopful of cells from a slant and aerating for a period of about 16 hr at 30'. The final concen- tration of bacteria in such cultures varies from 2-8
xIO!)/ml, depending on the strain. Stock cultures were stored in the cold (4") and used for 7-10 days.
Plating bacteria were made by diluting a stock culture 100-fold into fresh Hershey broth, aerating for either 2.5 hr at 30" or 1.75 hr at 37", centrifuging the turbid suspension for 10 min a t 1500 X g in the cold ( 4 O ) , and resuspending the bacterial pellet in one-tenth the original volume of chilled Hershey broth. Bacteria prepared in this manner have a higher efficiency of plating than do saturated cultures, and maintain their higher efficiency for about two days if stored at 4". Two drops of this suspension were used for each plate.
Bacteria for infections, including crosses and liquid culture complementation tests, were made by diluting a stock culture 500-fold into fresh Hershey broth, aerating for either 2.5 h r at 30" or 1.75 hr a t 37", centrifuging the somewhat turbid suspension for 10 min at 1500
xg in the cold (4"), and suspending the bacterial pellet in one-fiftieth the original volume of chilled Hershey broth. By this procedure, early logarithmic phase bacteria were obtained at a final concentration of 1-2
xIO"m1, and could be diluted as desired.
T-EVEN P H A G E GENETICS
lated culture at 25". Incubation at higher temperatures resulted in lower stock titers and often higher reversion indices, probably because of the limited restriction exerted by CR63 and other
E . coli
Kstrains against T 2 (REVEL 1967). The resulting stocks were sometimes centrifuged for
10 min at 5800
xg to rid them of large bacterial debris, and were then filtered through a Mandler candle filter to complete the cleaning. I n the other method of stock-making, about
IO5phages were plated on each of several plates, and after 12-15 hr of incubation a t 25", 0.5 ml of chloroform was added to the surface of the confluent plate and allowed to evaporate. Five ml of Hershey broth were then added to the plate, and 2-3 hr at room temperature were allowed for the phage to diffuse from the top agar layer into the broth. The broth was then collected ( 3 4 m! could be recovered from a normal plate), and the resulting "plate stock" was centrifuged and filtered in the same way as the normal liquid culture stock described above. The Sagik effect
(SAGIK 1954) did not hamper use of T2 stocks, probably because a matter of weeks usually elapsed between the making of a stock and its use for experiments i n which its titer had to be accurately known.
Standard phage techniques (ADAMS 1959) were used for the dilution, plating and counting of phage samples, with the €allowing restrictions: (1) no more than 0.5 ml and no less than 0.05 ml of a phage sample was ever plated; ( 2 ) no plates were used later than 5 days after they had been poured (the use of older plates leads to smaller plaques and a n apparent drop i n efficiency of plating); (3) no plating bacteria were used later than 2 days after they had been prepared; and (4) no counts of less than 100 plaques were accepted.
Mutagenesis of phages was accomplished as follows. The phage to be treated was grown i n a restrictive host, to eliminate any large clones of amber mutants already present by chance. This stock was then used to infect permissive bacteria (CR63, 1-4
x108,"l) at a multiplicity of 5, and 5BUdR was added to bring the final concentration to 10 pg/ml. (This concentration had previously been found to give a reasonably high frequency of amber mutants in T4.) The in- fected cells were then aerated vigorously until they lysed, and the progeny were plated as de- scribed below
Selection of amber mutants was carried out by a method based on a suggestion by DR. RICH- ARD
HEPSTEIN. The phage sample plus 2 drops of normal CR63 plating bacteria were added to 2 ml of melted top agar, mixed, and poured over 2-3-day-old plates. After this initial layer had hardened, a second layer, consisting of 2 ml of melted top agar plus 2 drops of a IOO-fold dilu- tion of restrictive (B/5 or S/6/5) plating bacteria was poured over the first layer and allowed to harden. After incubation the bacteria in the upper layer gave rise to clearly separated micro- colonies which were easily distinguished from the bacterial lawn below. Over a wild-type plaque these microcolonies were missing from an area somewhat larger than the plaque. Over a plaque of an amber mutant, however, the microcolonies were present without reduction in number or size, giving the plaque a characteristic stippled appearance. With T 2 and T6, about 10% of the plaques selected for their stippled appearance contained amber mutants.
Stab testing of suspected mutants was carried out by stabbing into a stippled plaque a sterile pin which had been coated with a sterile mixture of vegetable dye and glycerine (to identify the plaque stabbed). The pin was then stabbed successively into defined positions on each of two plates, the first of which had been seeded with restrictive, and the second with permissive, bac- teria. Tests indicating the presence of an amber mutant were checked by picking the area of lysis from the permissive plate with a sterile glass capillary, transferring it to a few ml of chloroform-saturated Hershey broth, allowing a few hours for the phage to diffuse out of the agar plug, and then respotting the resulting suspension on two plates seeded. respectively, with restrictive and permissive bacteria.
970R . L. RUSSELL
a considerably greater number of plaques; controls and negative tests usually contained only a few scattered plaques, whereas in positive tests there was nearly confluent lysis of the bacteria. Rare intermediate tests were repeated or abandoned in favor of liquid culture complementation tests. (The numerous plaques in positive spot tests were due to wild-type recombinants formed in mixedly infected bacteria; since the bacteria were restrictive, the formation of these recom- binants depended on the ability of the two mutants to complement one another.) Spot tests be- tween mutants of the same phage worked well, whether the phage was T2, T4 or T6. Spot tests between mutants of different phages worked for T2-T4 tests and for T2-T6 tests, but considerably less well f o r T4-T6 tests. The latter could be markedly improved by a 100-fold reduction in the bacterial concentration, and this was adopted as the standard method for T4-T6 tests.
Liquid culture complementation tests were used to check the results of spot tests and to clarify situations in which these results were ambiguous. Restrictive bacteria were prepared for infection, at a final concentration osf 4
x1018/ml. Just before use, KCN was added to give a final concentration of 0.004 M. A test was performed by mixing together equal volumes of two amber mutant phage suspensions, each at 6
xIO~'m1, and then adding to one volume of the mixture an equal volume of bacteria. After 8 min osf adsorption at 30", anti-phage serum was added to give a final neutralization constant (k) of 1 min-1. The antiserum was allowed 5 min to inactivate unadsorbed phages, and the adsorption mixture was then diluted 40,000-fold into Hershey broth at 25O. Samples were quickly taken for measuring infective centers and residual unadsorbed phages. After 120 min of incubation, chloroform was added to ensure complete lysis. The progeny phage were then assayed, and the burst size was computed and compared to that of simultaneous controls. These controls were infections with wild-type phages a t the same total multiplicity (ca.15) used in the tests, and they were usually inserted at the beginning and at the end of a series of tests. When the tests were between mutants of different phages, the controls were mixed infections between the wild types of the same two phages. The liquid culture tests have advantage over spot tests in that the results do not depend upon the production of recombinants.
Crosses. Bacteria were prepared for infection at a final concentration of 4
x108/ml, and just before use, KCN was added to give a final concentration of 0.004 M. A cross was performed by mixing equal volumes of two amber mutant phage suspensions, each at 6 X 108/ml, and then adding one volume of the mixture an equal volume of bacteria. Adsorption was allowed to proceed for 8 min if unadsorbed phage were to be eliminated with antiserum (occasionally) or 10 min if not (usually). Antiserum was used at a final
kof 1 min-1 fo'r 5 min. After adsorp- tion the mixture was diluted 40,000-fold into Hershey broth at 25", incubated f o r 120-150 min, and then treated with chloroform to ensure complete lysis. A sufficiently small number of crosses (usually 20) was done with any one batch of bacteria to ensure that no bacteria had been ex- posed to KCN for more than 22 min (such exposure tends to reduce the burst sizes obtained).
Progeny phage were plated at various dilutions on permissive and restrictive bacteria. The frequency of recombination was calculated by multiplying the titer on the restrictive strain by 2 (to compensate for the unscored double mutant recombinants), dividing by the titer on the permissive strain, and multiplying by a correction factor f o r the inherent differences, if any, between the two strains of efCiciency of plating. This correction factor was determined sep-
arately for each batch of plating bacteria by plating on them 0.5 ml of a large volume stock
of wild-type phage at a titer of 1.2
x1 0 3 / ~ l ; it was often quite significant, usually being, for example, about 0.7 with T 2 on CR63 and S/6/5.
Often a given cross was repeated a number of times. In general the agreement between the various values for the frequency of recombination was good, and an average value was taken. Occasionally, however, one of or even sometimes two of a series of crosses seemed out of line with the rest of the series. In these cases, a n average of the remainder of the values was taken, and the exclusion of some values from the average was noted.
From stocks of
and T6 grown in the presence of
T-EVEN P H A G E GENETICS
by a mixed-lawn screening technique described above. The isolated mutants
occurred after mutagenesis at frequencies of approximately 0.5
they were not backcrossed to wild type before subsequent testing and 12% of
them turned out to harbor more than one amber mutation. Thirty-five additional
were obtained from the collection of
these were given the prefix
them from mutants isolated in this study.
On the assumption that T2 and T 6 genes might
have homologs in T4, the T2 and T6 mutants were first tested in complementa-
tion spot tests with a series of T4 amber mutants. These tests, originally designed
for complementation testing
T4 mutants, proved satisfactory for testing T2
mutants as well; for T6 mutants, however, the initial testing procedure had to be
modified by a reduction in the bacterial concentration to achieve satisfactory
results. With the appropriate testing procedure, each new T 2 and T6 mutant
(with a few exceptions
be described below) could be unambigously assigned,
either to a gene with a T 4 homolog. o r to a T2
T6 gene for which a T4 homolog
was not yet known. The testing procedure was sequential; each new mutant was
first tested against a set of seven multiple amber mutants of T4 which, between
them, contained mutations in 24 T4 genes.
it failed to complement one of
these, it was tested next against the T 4 single mutant constituents of the un-
it complemented all the multiple mutants, it was
subsequently tested against a set of single and double mutants which, between
them, contained mutations in the remaining 46 identified genes of T4.
By this procedure 144 of the 152 T2 mutants, and 129 of the 146 T6 mutants
were assigned to identified genes. The remaining mutants were tested against
each other in all possible combinations; they proved to identify a total of 5 new
genes, of which one was found in both
and T 6 , 2 were found in
2 were found in T6 alone. All new mutants assigned to a given gene were tested
against each other in all possible combinations, and with the exception (2)
noted below, all such tests were negative, as expected.
summary of these gene
assignments is presented in Table 1.
During the spot testing, occasional exceptional results were observed; these
were of the following types.
M u l t i p l e mutants.
mutants and 18 T6 mutants showed
noncomplementation with more than a single T4 tester mutant. These appeared,
by subsequent complementation testing with single mutants of T2, T4 and T6, to
harbor multiple amber mutations in different genes. In T2 there were a total of
15 apparent double mutants and 2 apparent triples, In T2,
11 of the apparent
double mutants were tested by backcrossing to T2 wild type; in all cases the
expected single mutant constituents were isolated. The high proportion of mul-
tiple mutants is probably a consequence of the relatively heavy mutagenesis
hpplied, but it should also be pointed out that the multiple mutants appeared at a
frequency much higher than expected by assuming independent origins for the
9 72R. L. RUSSELI,
Gene locations of T2 and T6 amber mutations
Gene T 1 nlolanl5 TG mutants
1 153,99a,166a 57
2 163 64 FS109 50 FS48
4FS114 53 72
5[38,12~,1~,117b,132b,166b] [FSIS] 6 I06,160,FS63,30a,l54a
7 14,27,77 8 37,140 9 98c
12 60,FS13 13
14 69,93,167,FS62 16 FS15
17 16,98a 18 117a 20 108 21 111,124 22 99b
51FS82,148b 27 156,FS57,30b 29 7,65,81,FS29 48
54 95,FS66 30
63 43,54,FS51,152a 32 8,51,145
34 12,67,73,74,75,78,79,135,FS4.01,25a,l10a 35 FS6
36 21,24,120,FS23 3 7 123,129,FS4,25b 38 35,91,125,FSf% 52  [89,5a]
5613,61,64,87,1 O12,133,165,FS2,FS 18,154b 58 100
41 82,86,137,FS99,98b 42 3,6,68,136,FS85,76a,i 10b
[ 10,58,64,109,13 1,124b,142b] [43,127] [9,27,29,50,51 ,59,60i,82,24bl [I321 80
28 102,96a 14,79,117
18,39,13 7,144,24c,l11 a
63,84,73a 7,54,94,34a,99b 17,21,71,138 6,124a 87,130,42b,124c 93,112 119 57 73b 20,118,31b
[ 11 3,116,1251 [44a]
33,83,9O,107,11O1,146,15b,42a 3 7,62,26 b
23,72,115a [65,96b] [I223 25,66,89,95,99c,1408b
T-EVEN P H A G E GENETICS
Gene TL mutant? ‘I’G mutants
46 FS98 133
46 31,59,115,122,155,FS32,70b,132a,152b,lO5b 32,76,136,52b,135a
47 4,80,92,FS7,FS45 21,61,60,101
49 2,FS87 115b
Mutants are listed with the genes to which their defects were assigned by spot testing. T2 mutants with prefix FS are from the set isolated by DR. F. W. STAHL. Multiple mutants are listed once for each defect, with suffixes a, b, and (where necessary) c to distinguish their different defects. Within a gene, brackets are used to separate groups f o r which the spot tests suggested either unusually high intragenic recombination or some degree of intragenic complementation.
against one another in all possible combinations; the
T2 mutants fell into two
clear complementation groups, and both
T6 mutants fell into one of these two
groups. The two groups apparently correspond to genes
and the in-
ability of the T2 and T6 mutants in them to complement either gene
mutants of T9 apparently results from a specific gene product incompatibility,
previously reported by STAHL
(1966). This point is discussed in
more detail below
3 ) Weak positiue tests due to exclusion:
Some of the apparently positive spot
tests results were considerably weaker than usual; this was observed usually in
tests involving mutants
T4 with those of T2 or
(i.e., not usually
T2, T4-T4, T6-T6, or T2-T6 tests), and only when the T4 mutants came from
the “early regions” of the T4 genetic map. Such results are easily interpreted as
of partial exclusion; the spot tests depend for positive results on the
production of a sizeable number
wild-type recombinants in mixed infections
between complementing phages. I n T4-T2 or T4-T6 mixed infections, partial
exclusion hampers the production of these recombinants, particularly those which
contain wild-type alleles from the strongly excluded T2 or T6 early regions (see
the accompanying paper,
1974). Thus tests involving T4
early gene mutants with mutants of
T2 and T6 produce fewer than normal wild-
type recombinants (i.e., only a weakly positive result) even when full comple-
mentation has occurred. (Similar results observed by STAHL
can be explained in the same way). Besides the ones listed above, no other types
of exceptional results were observed.
In total, the new mutants served
identify 52 genes in T2 and 45 genes in T6.
these genes are homologous
T4 genes by complementation, and the
success of the spot testing procedure further argues that most of the genes are,
9 744R. L. RUSSELL
phages which must contain a particular combination
and T4 genes. Appar-
ently any such combination of genes (whether from T2, T4
T6) is compatible
with viability, except for the case of genes 37 and 38, discussed in more detail
The success of the spot testing procedure also argues that the
genomes of T2 and T6 are likely to be organized very much like that of T4. To
investigate this conclusion further, genetic maps of T2 and T6 were constructed,
using one representative mutant from each mapped gene. I n each case the
working hypothesis was that the desired map would strongly resemble that of T4
in its gene order; consequently each mutant was crossed first with its three
expected nearest neighbors on either side, and subsequently, if necessary, with
more distantly linked mutants. Data from these crosses are presented in Figures
and 2. In each case the gene order in the figure is that
T4, and in both T2 and
T6 the data are consistent with the same order. However the T2 and T6 data do
not establish this as the unique order
the map as a whole; rather the data
divide the mapping genes into
set of well mapped groups (in T2, for example,
the well-mapped groups are genes 57-30, genes 32-39, and genes 56-49). Within
each of these groups
unique order is established, but the ordering of the groups
with respect to one another is much less clear from these data, because the groups
are only distantly linked. Fortunately, the DNA-hybrid studies
show unequivocally that these groups must have the same
57 I 2 50 64 53 5 5' 6 7 8 12 (101 14 16 17 20 21 23 25 26
'-6 0 - 1 1 7 - 1 2 ~ 9 ~ 1 0 ~ 7 ~ 1 0 7 ~ 7 8 - 7 4 - 1 2 7 ~ 1 5 ~ 1 0 7 ~ 1 3 7 + 1 2 &78-12 6 1 1&7&17&94-* -162-1-36120-12 l----rc142-179-169-143-12 1-1934
-142 = : 112 134 -23 7 153
-66- 130- 105 194 z -OM 236-
-156 ~ 148- 176 100- -24 0 275-
23 25 26 51 27 29 54 30 63 32 33 34 35 36 37 38 52 52' 39 39' 56 ~173-94-49~82-ao-126cr154rr~7-25~17-152-91-75-66-14 I u 1 6 ~ 2 4 - 1 3 ~ 4 ~ 3 4 r
C l 582-31-4 0-35 1-196- 171-164----rC170-26 1-
-24 -3 313 = 306
-24 -5 24 I 222-
-23-20-444 = : 191 177 2 394-
-142- 40 I -41 6 5-12 205- 471-
39 39' 56 58 41 42 43 44 45 46 47 55 49 57 I
' * 4 5 ~ 3 1 4 ~ 3 3 ~ 1 4 3 ~ 1 ~ 9 ~ l 1 ~ 1 2 ~ 7 5 ~' ~ 8 ~ 1 1 ~ ~ 1 0 ~ 1 6 ~ ~ ~ ~
-324-136- 84-1-33 147-234-491 --377- 175.---CI43---r--94-172-
-3 3 9
- -163 22 0 252-
-404- 187 z =
152--274 : = 258- 21 6
T-EVEN P H A G E GENETICS
I 2 5.3 5 6 6 ' 7 8 IO I2 I 3 I4 I6 17 le 20 26 27 29 30
C l 2 . 5 - 8 . 3 - 7 . 8 - - r C 9 . 9 -6.7-0.8-7.4- 31.4- 8.9-
-172-. 154.- 14.6 __C_ 10.3
c-12.5 10.8 9.3
-8.3 ___c_ 32.3
'-11 3-7.3-35- a6-4.7-8.4.~ 95- a ~ a ~ s . ~ 4 . 7 - 6 . ~ 3 . 7 - 4 . 3 - 9 . 1 - 2 2 ~ ~ 8 . 3 - 8 . 2 - so-
-10.9 __C_ 32.7
27 29 30 63 32 34 35 37 39 56 4 2 43 44 45 46 47 (103) I 2 53
* : : : : : : : : : : : : :
+a2*9.o-12.8o 1 9 8 - 1 8 ~ 6 . 5 - 1 4 . 6 - 2 5 . ~ 1 9 . 2 ~ 1 3 8- 7.2-6.7-a-8.2-11.0- 4 I - 2 9 5 ~ 1 I 3 ~ 7 . 3 -
C l8 8-31.8--rC13.2-22.5----u-20.9- 15.6- 11.2-27.7 -12.5-
-15.5- 1 8 3 - - C C 2 0 . 4 - 2 0 . 4 - - r C 8.4- 16.5- 16.0-37.6-
-27.5 20.3- 46.8 13.1 16.9 28.3
-26.8- 21 5
_ -13.9 :I 9.4 29.1-
C-27.3 16.3 21 0 14.7
FIGURE 2.-Recombination data from T6 crosses. Presented as in Figure 1. The average number of determinations per number in the figure is 2.0.
relative orientations in all three phages, and hence the gene orders of
appear identical to that of
construct genetic maps from the data of Figures
it was necessary
devise a relationship between map distance and measured
recombination frequency. Several such relationships, called mapping functions,
have been devised for
to deal with the
extensive negative interference seen in
crosses. Similar negative interference
was observed in
crosses, as shown in Figure
in the absence of nega-
tive interference the data should have scattered about the drawn lines. In a n
attempt to circumvent this negative interference, an average
the two best
mapping functions, the Four-Parameter Switch Function and the Four-Param-
100 I l l I I I I , I
4 6 8 IO 15 20 30 40 60 80 100
PERCENTAGE OF RECOMBINATION, SUMMED FOR SHORTEST INTERVALS
976R . L. RUSSELL
FIGURE 4.-Better additivity of T2 and T6 computed map distances. (A) T2, ( B ) T6. All distances are expressed as fractions of the total map length, as computed from recombination percentages using the T4 mapping function described in the text. Presentation is analogous with that of Figure 3, and in both cases the fit to the drawn line is better than in Figure 3.
eter Modified Bernstein Function
applied to recombination data from T2 and T6. This attempt was partially suc-
and more so for T6, as shown in Figure 4, and the resulting map
distances were used to construct the T2 and T6 maps shown in Figures 5 and 6.
The most striking feature of the
and T6 maps is their general similarity to
T4 (shown in Figure 7). Gene spacings are, in general, very similar in all
three phages, with a few exceptions. By far the most notable of these exceptions
the relative shrinking
genes 34 and
in T2. and, to a lesser extent, in T6.
somewhat smaller shrinkage is seen between genes 23 and 25 in T2. Some
newly identified genes of
T2 and T6, denoted by arrows in the figures, fall,
within the already well mapped regions, and the T2 and T6 maps, like that of T4,
contain extensive “silent regions” in which conditional lethal mutants have not
yet been found.
The tail fiber region; a site
That the tail fiber region
(genes 34-38) might be one of relative divergence for the three T-even phages
was suggested by two lines of evidence:
(1) the anomalous spot test behavior
gene 37 and 38 mutants, and
(2) the relative genetic shrinkage of genes 34 and
35 in T 2 and T6. To investigate this possibility further, several types of experi-
ments were performed.
T-EVEN PHAGE GENETICS
FIGURE 5.-Genetic map of T2. Distances separating genes have been computed using the
T4 mapping function described in the text, with a final correction to provide circular additivity. The symbol (10) denotes the new T2 gene identified by T 2 am 10, and the symbol (gtl) denotes the mapped position of an a-glucosyl transferase deficient mutant of T2 kindly provided by H. R. REVEL and mapped both by RUSSELL (unpublished results) and by
MOLHOLTand DE GROOT
(1969). Because slight fluctuations in recombination frequency strongly influence the sizes of the unmapped gaps in the map, their true sizes are necessarily somewhat uncertain.
(see the accompanying paper,
The results, pre-
sented in Table
2, can be interpreted as follows.
978R . L. RUSSELL
FIGURE 6.-Genetic map of T6. Presentation analogous to that of Figure 5. The new gene identified by T6 am 103 is apparently the gene for a-glucosyl transferase, as described in the DISCUSSION.
Secondly, from tests involving the wild type of the three phages (Table 2b), it
is clear that the burst sizes of
and T6 are somewhat lower than that
that the T4-T6 mixed infection is characterized by a marked “depressor effect”
(see the accompanying paper, RUSSELL
1974) which confounds
analysis of T4-T6 mutant tests.
Thirdly, tests between the wild type of one phage and mutants of another
(Table 2c) reveal evidence for the partial exclusion which is the subject
accompanying paper (Russell and Huskey 1974). In T2-T4 infections, for ex-
ample, mixed infections between T2 wild type and T4 mutants give much lower
burst sizes than those between
T4 wild type and T2 mutants. This asymmetry
T-EVEN P H A G E GENETICS
FIGURE 7.-Genetic map of T4, from WOOD (1974)
T6 excludes T2, and both suggestions are confirmed in the accompanying paper
Fourthly, tests between mutants of different phages (Table 2c) reveal a num-
ber of instances of lowered complementation. Some
these appear to be further
the polarity effects described above, but one particularly strong
effect, involving genes
T2 and T4, is novel.
effect, the gene
mutants of T2 cannot be distinguished from one another on the basis
of their complementation properties with
mutant complements either mutant from the other phage.* Such a result might
have been due to a strong polarity effect involving genes
and 38, but there is
evidence f o r such a n effect when either T2
T4 is considered separately. The
R. L. R U S S E L L
34 0.4 0 . 0 0 . 1 0 . 5 8 . 1 0.1 6 . 1 3.4 7.5 7 . 1
100 74 142 122 1 2 1 1 3 2
Complementation between tail fiber mutants
T2 T4 T6
+ 34 35 36 37 38
38 183 1 4 1 1 2 9 105 118 0 1
371121 1 0 5 106 61 O.O+
t C ) T2 T6 T6
+ 34 35 36 37 3a
37 1 4 1 4 7 . 7 1.6 0.0 p.ol 38 19 20 1 7 4 . 6 l o . y T4 36 1 4 1 2 11 0.0 12
Each value is the burst size (total phage yield per infected bacterium) of a mixed infection of strain B/5 with the two indicated phages, at a multiplicity of at least five each; the numbers 34-38 indicate amber mutants in the corresponding genes of the indicated phage. The mutants used were (in gene order) T2 ambers 135, FS6, 24, 123, and 35; T4 ambers B25, B252, El, N52, C290; and T6 ambers 15, 26, and 126. All experiments were done in one batch to avoid possible bacterial variation. For ease of comprehension the table is divided into three parts, and some values necessarily occur in more than one part. The boxes denote the results which demonstrate the unusual complementation behavior of T2 and T4 genes 37 and 38.
result might also have been due
a celective inactivation
T2 genes 37 and 38
in mixed infection with T4, but the tests between T4 gene 37 or gene 38 mutants
and wild type show no evidence of such inactivation. The only remaining inter-
pretation appears to be that genes 37 and 38 are a pair of “coadapted” genes whose
products must interact rather closely during tail fiber assembly; when both
products are drawn from the same phage (T2 or T4) the interaction is successful,
but when the two products are €rom different phages (one from T2, and the
other from T4), it is not. This interpretation has also been arrived at by
(1966) on the basis of very similar results. With respect to this in-
terpretation, it is encouraging to note that
studies of tail fiber assembly
1969) show that the products of genes 37 and 38 are both
the initial step in assembly
the distal half-fiber
Although the gene 37 and 38 products
T2 are apparently incompatible with
T-EVEN PHAGE GENETICS
compatibility is quite limited. Whether the
products of these genes ,in turn,
are compatible with those of T4 cannot be determined from Table 2, because of
depressor effect. However, the spot tests suggest that they are not, since
T6 gene 37 mutants (like
gene 37 and gene 38 mutants) were anomalous in
spot tests against gene 37 and gene 38 mutants
T4. In short, then, the T2 and
these genes have limited compatibility with one another but none
Recombinational variation in genes 34 and
Another set of experiments
was performed to investigate further the apparent discrepancies in the relative
genes 34 and 35.
determine whether these discrepancies might
seriously result from fortuitously different mutant distributions within these
genes, maximum and minimum estimates
f o r
genes 34 and 35 were
made f o r each phage. As Table
shows, the maximum estimates for both T2 and
are well below the minimum estimates for T4, and the genetic size discrep-
ancy is therefore real. Such a genetic discrepancy might result from a true
physical size discrepancy, o r it might result from the presence of local factors
genetic exchange per nucleotide pair.
physical size discrepancy between the genes would imply a similar size discrep-
ancy between their products, and would make the demonstrated interchange-
these products difficult to understand. It would also imply that genetic
T h e genetic lengths of genes 34 and 35
T 2 m a x T 6 m d x Gene Estimate Phage map length TL/T4 TA/T4 T4 nnn T 4 min
of total ~ __-
Minimum T4 6.5 0.46 0.12
Maximum T4 9.9 0.34 0.46
__- 0.52 0.71
Minimum T4 4.7
Maximum T4 9.2 0.21 0.24
_-___ 0.40 0.47
~ _ _ _ _ _
Minimum estimates were obtained by taking the two most separated markers within a gene, measuring the frequency of recombination bstween them, and converting this frequency to a percentage of the total map length with the T4 mapping function described in the text
Maximum estimates were obtained in the same way, using the two markers lying closest to the gene but outside it an either end.
982R. L. RUSSELL
exchange between phages within genes 34 and 35 might be severely limited. The
presence of localized recombination-enhancing factors, on the other hand, would
be consistent with similar-sized gene products and with considerable genetic
exchange between the two phages in these genes.
To test these points, T2 and T4, which showed the greatest apparent discrep-
ancy, were selected, and their gene 34 mutants were crossed in all possible com-
binations. The results are shown in Table 4. Clearly there is considerable genetic
exchange between the phages within gene 34, suggesting considerable genetic
homology there. T o test this homology further, an attempt was made to order the
T2 and T4 mutants on a common map of gene 34. Because of the complications of
T2-T4 crosses, such a map could not be constructed by two-factor crosses alone.
However, three-factor crosses could be performed by using the known host range
difference between T2 and T4 as
convenient outside marker; Table
the evidence which localizes this host range difference to the 37-gene 38 end of
the tail fiber gene cluster. Table 6 shows how the host range difference has been
used to order T2 and T4 mutants with respect to one another on a common map
of gene 39. That such
map can be constructed attests to the homology between
T 2 and T4 in gene 34; the intermingling of T2 and T4 mutants in the map
provides further evidence that homologous regions of gene 34 have quite different
Genetic exchange within gene 34
AhlBER AIIIBER AhlBEK AMBER AMBER AMBER AMBER AMBER AMBER ARlRER AhIBER 12 73 135 7 0 74. 75 W25 R265 N58 B25S A455
AMBER 11.7 A455 B258 AMBER 9.7 AMBER 8.7 AMBER 8.3 AMBER 4.4 T4 N58
AMBER 15.1 T2 79
AMBER 135 20.7 AMBER 73 16.8 AMBER
AMBER 24.8 AMBER 21
5.3 2.2 0.4 5.8 5.3 5.4 5.5 1.4 0.8 4.5 3.9 01.2 5.2 5.0 6.6 6.2 1.3 3.2 1.3 0.9 4.0 3.9 2.1 2.9
5.7 1.3 301.9 29.5 28.4 17.4 1.1 2.9 32.3 26.3 23.9 3.1 8.4 18.5 13.4 6.2 12.7 10.4 6.4 13.0 1.3
T - E V E N P H A G E GENETICS
Localization of host range determinants
Percentage of wild-type progeny with host range genotype of
T2 T4 T6
T2 Wild type
xT4 Wild type
T2 Wild type
xT4 Amber in 34 T2 Wild type
xT4 Amber in 35
T2 Wild type
xT4 Amber in 36 T2 Wild type
xT4 Amber in 37 T2 Wild type
xT4 Amber in 38
T2 Amber in 36
xT4 Amber in 37 T2 Amber in 36
xT4 Amber in 38 T2 Amber in 37
xT4 Amber in 36 T2 Amber in 38
xT4 Amber in 36 T2 Amber in 37
xT4 Amber in 38 T2 Amber in 38
xT4 Amber in 37
10 56 84 92 97 1 0 95 99 2 0 la0 0
T2 Amber in 35
xT6 Amber in 37 T2 Amber in 36
xT6 Amber in 37
T2 Amber in 37
xT6 Amber in 35 T2 Amber in 38
xT6 Amber in 35
1 0 100
T4 Amber in 35
xT6 Amber in 37
T44 Amber in 36
xT6 Amber in 37 T4 Amber in 37
xT6 Amber in 35 T4 Amber in 38
xT6 Amber in 35
9.0 44 16 8 3 0 5 1 98 100 0 100 0 0 98 99
Progeny phages were plated on B/5 ta select wild types, and host range genotypes were deter- mined by stabs to plates seeded with the appropriate indicator strains (resistant strains used were B/2, S/4, and S/6). Occasional stabs growing on both indicators were assumed to be due to heterozygotes and were included in the table as having both host ranges. 100 plaques were stabbed for each determination. The wild-type progeny of the crosses between T2 and T4 gene 37 and gene 38 mutants were quite rare (0.1-0.3%), as might have been expected from the incompati- bilities described in the text; consequently it is not certain that they represent the products of single exchange events between T2 and T4. Wild-type progeny from crosses of gene 37 and gene
38 mutants of T2 and T6 or T4 and T6 grew so poorly that their host ranges could not be determined.
These results localize the host range d e t e h a n t s to the gene 37-gene 38 end of the tail fiber gene cluster, but because of the gene 37-gene 38 product incompatibility, they cannot localize them further.
probabilities of genetic exchange per nucleotide pair in the two phages. From the
anomalously large genetic sizes of genes 34 and 35 in T4, it seems likely that T4
harbors local factors which selectively enhance recombination in this part of its
The amber mutants of
T2 and T6 described above have provided a useful tool
with which to extend previous genetic work
9844R . L. RUSSELL
Each value in the table is the percentage of wild-type recombinants having the T2 host range genotype in a cross between the two indicated mutants. Since progeny of a T2-T4 cross selected to contain the gene 34 of T 2 are only 58% T2 in host range, this is the maximum figure expected in the table. Values close to this maximum suggest that the T 2 mutant lies to the gene 33 side of the T4 mutant, while values close to zero suggest the opposite ordering. Intermediate values are likely to occur when the T2 and T4 mutants are close to one another; in this case a definitive order cannot be deduced. The map indicates with respect to each other as best fits the T2 mapping data of Table 4.
are homologous with known T4 genes, and gene orders, as well as most
gene spacings, are apparently identical in all three phages. With a few excep-
tions, gene products
of one phage are interchangeable with the homologous
products of the others, and almost any combination of mixed genes derived by
recombination between different phages is compatible with viability. Such close
genetic similarity supports previous investigations on the comparative particle
1959), serological cross reactivity
base sequence homology
1971) of these three phages.
It also agrees fully with exhaustive
electron-microscopic studies of
hybrids subsequently carried out by
The limited number of genetic differences among these phages are of interest,
partly because they can serve as useful genetic markers, and partly because their
nature may suggest how the phages have come to differ. The most striking dif-
ferences occur within the region of the genome controlling tail fiber formation.
The already known host range differences between
T2, T4 and T6 have been
shown to map to the tail fiber genes
this evidence clearly suggests that
the distal portion of the tail fiber controlled by these genes
responsible for the
bacterial adsorption specificity of the phage, a conclusion also arrived at by a
study of host range mutants in
unpublished results, quoted in
T-EVEN P H A G E GENETICS
The products of genes 37 and 38 apparently interact closely in forming the
distal half-fiber; when both products are drawn from the same phage the inter-
action is successful, and
functional half-fiber is produced in good yield. When
the products come from different phages, however, the interaction is much less
successful, ranging from
marginal success in the case of
and T6 to a n abso-
lute failure in the case of T2 and T4. It appears, then, that genes 37 and 38 are
a ‘Lco-adapted” pair, showing much greater diversity from phage
does the rest of the genome. This point is discussed at greater length by STAHL
The diversity among T2, T4 and T6 in these genes might have been an artifact
their selection, since they were initially distinguished from one another (and
thereby deemed worthy of beng separately kept and studied) by these very dif-
ferences in adsorption specificity.
however, one would expect that three T-
even phages drawn at random from wild populations would show
than do T2, T4 and T6.
preliminary test of this notion has been made by exam-
ining T-even phages drawn from nature (Long Island sewage treatment plant
among thirty-one such phages,
least ten distinct adsorption specificities
could be detected, suggesting that genetic diversity
host range is common
among natural populations of T-even phages as well (RUSSELL
Rationalizing such diversity is not difficult. Because of the T-even phages’
absolute dependence on their bacterial hosts and because of the apparent ease
with which the bacterial surface can be altered by mutation so as to resist these
phages, the adsorption phase
the phage life cycle is subject to considerable
selective pressures. The various bacterial derivatives resistant to one or another
the T-even phages can be viewed as different ecological niches which are poten-
tially exploitable by the T-even “species’’ as a whole. In order to exploit these
niches, however, the T-even species must exhibit sufficient diversity of adsorption
specificity so that bacterial mutants resistant to
of the T-even phages will be
very unlikely to arise. The differences in genes 37 and 38 of the tail fiber region
can be viewed as the result of this selection for adsorption diversity. The absence
comparable differences elsewhere in the genome may indicate less intense
selection for diversity in other aspects of the phage life cycle.
The detailed structure of genes 37 and 38 has subsequently been investigated
more fully by
KIM and LIELAUSIS
(1973), using both genetic
crosses and electron microscopy
hybrids. They have shown that these
genes, in contrast
the genome, are mostly heterologous between
T 2 and T4, and that the functional differences within these genes map to hetero-
logous regions. The extent of the heterologous regions suggests that they may
have arisen by insertion of heterologous
rather than by successive small
mutations. Interestingly, KIM and DAVIDSON
(1974) have found that T2 and T6,
whose gene 37 and gene 38 products are at least partially compatible, also show
heterology in these genes.
986R. L. RUSSELL
(1968, 1970) and a direct comparison of gene product sizes
1971) suggest that their actual physical
sizes are much smaller. Thus T 4 apparently possesses localized factors enhancing
recombination in this region. T2 apparently lacks these fectors, since the genetic
size of T2 gene 34 is consistent with the physical, as opposed to the genetic size
of gene 34 in
This interpretation has been explored further and confirmed by
(1972), who have shown that the enhanced genetic
size of gene 34 in T4 is due
a recombination gradient in which the probability
of recombination per nucleotide pair increases dramatically in the distal part of
the gene nearest gene 35. Additional experiments by
(1974) suggest that
this gradient results from a discontinuity near the junction of genes 34 and 35;
the gradient present in T4 can be eliminated by substituting this junctional
region (and only this region) with the homologous region from T2. The evo-
lutionary and functional significances
such a localized recombination enhance-
ment are unclear.
Other detected genetic differences among these T-even phages are much less
notable than those in the tail fiber region; slight differences in apparent gene
spacing have been found elsewhere in the genome, and reduced burst sizes sug-
gesting imperfect gene product compatibilities have sometimes been noted in
liquid culture complementation tests between mutants of different phages. In
both cases, however, the effects are relatively minor.
Some known differences among the phages have not shown up in this genetic
analysis. In particular the absence of a “collar” in T2
1965), and the known internal protein diversity of these phages
1972) have not produced detectable effects either on genetic
exchange between the phages or on the interchangeability of their gene products.
Perhaps even more striking, the multiple short regions
(1974) have not been detected genetically, and must there-
fore have, at most, only slight effects on the interchangeability of gene products
The new genes found in T2 and T6, but not in T4, may be related to some of
the known differences among the phages. In particular, the gene found in both
T2 and T6 is almost undoubtedly the gene for the enzyme a-glucosyl transferase,
needed for attachment of glucose in a-linkage to the hydroxymethylcytosine resi-
due of T-even phage
The evidence is (a) that T2 mutants wholly o r par-
tially defective in a-glucosyl transferase (T2gt mutants) map to the region
between genes 47 and 55
results) as do T2 and T6 mutants of the new gene, and (b) that T 2 and T6
mutants in this new gene, unlike those in any other gene, form perfectly normal
strains which lack amber suppressors but instead contain
mutations rendering them tolerant of T-even phages with non-glucosylated
An homologous gene for a a-glucosyl transferase exists in T4
T-EVEN P H A G E GENETICS
from being lethal. Consequently, amber mutants in the T4 gene have not been
recovered as conditional lethals.
The significance of the other new genes is much less clear, but one of them
(identified by T2 amlO) maps in a region suggesting that it may be involved in
the “collar” difference between T2 and the other two phages.
The results described above suggest that
T4, and T6 are related to one
another in the same way as are races of higher organisms; genetic exchange
between them is considerable, but there are clear genetic differences. Co-adapted
gene pairs have been found, and the genetic diversity they represent is easily
rationalized as providing efficient exploitation of diverse ecological niches. The
accompanying paper describes what may be a n incipient isolating mechanism
by which genetic exchange between these phage races is beginning to be limited.
I am deeply indebted to R. S. EDGAR for exemplary guidance during the course of this work, and to F. W. STAHL for sharing both information and mutants. J. J. WEIGLE, R. J.
J. A. KING, and J. E. FLATGAARD provided many helpful discussions, and MRS. LAURA YOUNG provided able technical assistance during some of the later phases of the work. Copious amounts of materials were provided by BERTHA JONES, JEANETTE NAVEST, and IRENE VON HARTMANN,
to whom I am most grateful.
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