Classical papers

1. VIROLOGY 9, 31g331 (1959)
Determination of the Order of Mutational Sites Gov-
erning L-Arabinose Utilization in Escherichia coli B/r
by Transduction with Phage Plbt”*
J. <:mss3 and $1. ENGLESBERG~
Depurlnw~t of Biologicul Sciences, LTniversity of Pittsburgh, Pittsburgh, Penn-
sylmnin, and Department OJ” Genetics, Carnegie Institution of Washington,
Cold Spring Harbor, New York
Accepted JIL~!J 16, 1959
The order of seventeen I,-arabinose mutants of Escherichia coli B/r has
been determined by three- and four-factor transduction experiments with
phage Plbt. The assortment of the markers thr and leu, located on either side
of the arabinose cluster, was observed among recombinants between pairs of
arabinose mutants. The sit.es of the arabinose mutants are arranged in a
linear sequence. The arabinose mutants are divided into three groups by
biochemical analysis and the application of the abortive transduction test
for complementat,ion. (iroup A (four mutants) accumulates trace amounts
of a keto sugar and is inhibit,ed by L-arabinose; group B (eight mutants)
accumulatjes large quantities of the keto sugar, ribulose, and is inhibited by
I,-arabinose; group C (five mutants) accumulates small amounts of keto
sugar (more than group A) and is resistant to L-arabinose. Group C mutants
are distinguished from mutant,s in groups A and B by abortive transduction
test,s. Comparison of t,he order of the mutant sites with the grouping by
funct,ional crit’cria demonstrat,es complete correspondence between the order
of the miltants and the groups t,o which t,hey belong. The transduction ex-
i This investigation was supported in part by a contract from the Office of
Naval Research and t)he University of Pittsburgh (NH. 103.429), and by research
grant.s from the National Science Foundation (G 4979) and from the National
Institute of Allergy and Infectious IXsease, United States Public Health Service
(E 2341). Reproduction in whole or in part is permitted for any purpose of the
United States Government.
2 Brief summaries of this work have appeared in Bacteriological Proceedings,
(Society of American Bacteriologists) 1959, p. 37.
3 Present address: M.R.C. Unit for Microbial Genetics Research, Hammer-
smith Hospital, I>ucane Road, London.
4 Present address: Department of Biological Sciences, University of Pittsburgh,
Pit,tsburgh 13, Pennsylvania.
314

2. ORDER OF L-ARABINOSE MUTANTS OF E. COLI B/r 315
periments involving pairs of arabinose mutants were characterized by the
presence of marked negative interference.
INTRODUCTION
In the work of Demerec et al. (1955), Demerec and Hartman (1956),
and Hartman (1956) it has been shown that auxotrophic mutants of
Salmonella typhimurium deficient in the biosynthesis of tryptophan and
histidine may be divided into distinct groups on the basis of the par-
ticular blocks in the biosynthesis of these amino acids. These different
functional groups were distinguishable from each other in several cases
by abortive transduction tests for complementation (Ozeki, 1956). Mu-
tants belonging to different groups were often found linked in transduc-
tion experiments. Mutants belonging to the same group were found for
the most part genetically different, as shown by recombination analysis,
and on the average were more closely linked to each other than mutants
belonging to different groups. This has been interpreted as indicating
that the grouping of mutant sites on the chromosome corresponds
closely to their grouping by functional criteria. It appeared, furthermore,
in the above cases, that the order of the groups on the chromosome was
the same as that of the sequence of biochemical reactions which they
controlled. However, except for the ordering of certain tryptophan sites
by three-factor crosses (Demerec and Hartman, 1956), the precise order
of the great majority of mutant sites has not been determined, nor has
the linearity of the arrangement of these sites been demonstrated.
Lennox (1955) has extended observations of transduction to strains
of Escherichia coli and Shigella, and has demonstrated that characters
found to be closely linked in sexual recombination could be jointly
transduced, and that the frequency of joint transduction was greater,
the closer the linkage between markers in sexual recombination. Certain
threonine (thr), leucine (Zeu), and L-arabinose (ara) markers of strain
W945 of Escherichia coli K12 could be jointly transduced by phage
Plkc, ara being located between thr and leu. Because of this linkage
relationship it appeared that it might be possible to determine the
precise order of a number of arabinose-negative mutants by means of
transduction experiments employing thr and leu as unselected markers.
In this paper we report experiments designed to establish the order of
seventeen independently isolated mutants of Escherichia coli B/r unable
to utilize L-arabinose, and the correlation between the order obt,ained
and the grouping by functional criteria.

3. 316 GROSS AND ENGLESBERG
MATERIALS AXD METHODS
illedia. The following media were employed: L broth (Lennox, 1955);
EMB complete agar (Difco formula with 1% arabinose as sole carbo-
hydrate); minimal tris medium (Hershey, 1955); minimal medium (Davis
and Mingioli, 1950) with L-arabinose or glucose as carbon source, with
or without the addition of t,hreonine and/or leucine; casein hydrolyzate
mineral medium and casein hydrolyzate mineral arabinose medium:
KHJ’O4-K2HPO4, pH 7.0,1%; MgSO4.7H,O, 0.01 %; (NH&SO,, 0.1%;
cascin hydrolyzate (Difco), 0.05%; and L-arabinose when employed, 0.2%.
Phage. The phage Plbt. employed in this study is related to the tem-
perate phage PI of Bert’ani (1951). It was derived by picking a single
t,urbid plaque from a lysat’e obtained from P. D. Skaar. Lysates were
prepared by a modification of the soft-agar layer technique of Swan-
strom and Adams (1951) using minimal tris glucose agar medium
supplemented where necessary with threonine and/or leucine. These
lysates usually had titers between 5 X 10yand 5 X 10’” plaque-forming
part’icles per millilit,er. The phage suspensions were preserved over
chloroform and are generally st’able after an initial drop in titer during
the first few days of storage.
Bacterial strains. Escherichia coli B/r, a threonine-requiring mut,ant
(thr), and a leucine-requiring mutant, (Zeu) were obtained from M.
Demerec (mutants thr-1 and ZCU-1in his collection).
Construction of thr leu strain. The thr leu strain from which all the
seventeen arabinose mutants were derived was constructed in the
following manner. A culture of the th,r mutant was irradiat’ed and an
ara mutant isolated as described below. Phage was grown on the 1~7~
mutant and used to transduce this thr ara strain, selection being carried
out for aru+ transductants. Several of these were purified, and one,
which was nonlysogenic and had retained the threonine requirement
and in addition acquired the leucine requirement of the donor by linked
transduction, served as the strain from which all ara mutants described
were derived.
Isolation of ara mutants. An overnight, culture of thr leu was centri-
fuged, washed, and resuspended in buffer consisting of KZHP04, 0.7 %;
KHZI’04, 0.2 %; MgS04, 0.012 %; and distilled water. Five-milliliter
samples were irradiated in petri dishes with a dose of ultraviolet (W)
irradiation giving a survival of about 1OW. Aliquots of the irradiat,ed
suspension were plated onto EMB plates without dilution. After 21
hours’ incubat,ion several hundred colonies appeared on each plate and

4. ORDER OF L-ARABINOSE MUTASTS OF E. COLI B/r 317
those which appeared to be arabinose negative were purified and tested.
Isolates which were able to grow with glucose as sole carbon source,
but not with n-arabinose, and which required threonine and leucine,
were employed in the study. Ara mutants were numbered in the order
in which they were isolated.
Production of thrf ara lea+ strazns. Each of the thr ara leu mutants
served as recipient for phage grown on wild-type bacteria and thrf leuf
transductants were selected on minimal medium with glucose as carbon
source. By streaking on EMB arabinose plates it was found in each case
that about 20% of these transductants were arabinose negative. Thr+
ara leu+ were purified, and nonlysogenic isolates were preserved on
nutrient agar slants. In this way a thrf ara leuf strain corresponding to
each thr ara leu strain was obtained.
Procedure for transduction. Recipient bacteria were grown to satura-
tion in L broth at 37”, mixed with an equal volume of the appropri-
ate phage lysate diluted as required, and CaClz added to 2.5 X 10e3M.
The mixture was incubated for 30 minutes in a water bath at 37”. At
least 80% of the phage are absorbed under these conditions and about
50% of the infected bacteria survive. Two controls were employed:
(1) recipient bacteria were infected with phage grown on the recipient
strain itself (this serves as a control for spontaneous mutants) ; and
(2) lysates were spotted on nutrient agar for sterility tests.
Selection of transductants. Araf transductants from thr ara leu strains
were selected on minimal medium supplemented with threonine and
leucine and containing arabinose as sole source of carbon. The ara+
transductants were scored for threonine and/or leucine independence
either by streaking or by replica plating onto plates lacking the threonine
or leucine supplement, respectively. Thr+ and Zeu+ transductants were
selected on plates containing glucose as carbon source and appropri-
ately supplemented.
Test for lysogeny. The spot test method of Bertani (1951) was em-
ployed. The use of chloroform to sterilize the bacteria in the cultures
to be tested for free phage was unnecessary, since clear halos of lysis
were found without its use.
Growth experiments. The growth from an overnight casein hydrolyzate
mineral glucose slant culture was washed off with 1 ml of saline and
transferred to a centrifuge cup. An additional 5 ml of saline was added
to the suspension and the cells were spun down and resuspended in
saline to a turbidity of 85 (see below). A 0.2-ml sample of this suspension

5. 318 GROSS .4ND ENGLESBEItG
was used to inoculate 4.8 ml of medium in optically tared test tubes.
The tubes were incubated at 37” on their sides with a 5’ angle from the
horizontal on a shaker which rotated at a speed of 115 rpm with an
eccentric of 9 mm in radius. The tubes were removed at intervals, and
turbidity was measured directly, using the Fisher Electrophotometer
modified to accept the growth tubes. A 425-rnp filter was employed
with t,he uninoculated medium as the blank. Results are recorded in
units of optical density (O.D.) X 100, and are based on the average of
duplicate tubes. With an exponentially growing culture of B/r, 1 unit
on the photometer scale equals 1.2 X 10’ viable cells.
Keto sugar analysis. Analysis for keto sugars was carried out employ-
ing the cysteine-carbazole test (Dische and Borenfreund, 1951) and
paper chromatography (Englesberg, 1957). The color produced in the
cysteine-carbazole test was determined 1 hour after the addition of
reagents, using a Klett, Summerson Colorimeter with a No. 54 filter.
Ribulose o-nitrophenylhydrazone was employed as a standard. A Beck-
man DU Spectrophotometer with cells 1 cm in light path was employed
in determining the absorption spectrum of the chromogen produced in
the cysteine-cnrbazole test.
RESULTS
Position of ara 111&ants in 1Zelation to thr and leu
Lennox (1955) determined the frequency of joint transduction by
phage I’1 kc of markers in the thr leu region of E. coli K12 and presented
evidence that the ara marker of strain W945 is located between thr and
leu, close to Zeu and far from thr. This result is in agreement with infor-
mation available from sexual crosses (Cavalli and Jinks, 1956). Table 1
presents data from an analysis of joint transduction involving the
markers thr ara-2 ara-3 and Zeuof E. coli B/r. The results demonstrate
close homology between the corresponding regions of strains B/r and
K12 of E. coli (Lennox, 1955). It will be seen that both ara-2 and ara-3,
which are located at opposite ends of the arabinose region (see Fig. 2),
are closely linked to Zeu and weakly linked to thr, as estimated by
frequencies of joint transduction.
The evidence that the order of the markers is thr ara leu, not thr leu
am, may be summarized as follows:
1. Six per cent of thrf transduct,ants are ara+, whereas only 4 % are
1cu+.
2. Eighty per cent of thr+ le7rf transductants are ara+, whereas only
55.4 % of ZezL+transduct’ants are ara+ (ara-3).

6. ORDER OF L-ARABINOSE MUTANTS OF E. COLZ B/l. 319
TABLE 1
JOINT TRANSDUCTION IN THE th, axz leu I~WION OF E. coli U/n=
Recipient
thr an-3 leu
Number of trans- % Selected colonies containing the
Selected marker du$y&.r Pl unselected marker
led fhr+ araf
thr+ 2.5 X 1O-5 4.1 - 6.7
leu+ 5.0 x 10-S 1.9 55.4
thr+ leu+ 1.0 x 10-r 80.0
ara+ 3.5 x 10-S 72.G 4.3
thr ara-2 letA ara+ 3.5 x LO-” 59.B 5.2
(1The donor strain is wild type B/r. Colonies from the transduction plat,es were
replicated, or streaked with a fine loop, onto the appropriate plates for scoring
unselected markers. Colonies were replicated to determine the rare frequencies of
joint transduction and streaked individually to obtain the higher frequencies.
Each datum was obtained from a t,otal of several hundred colonies.
3. Ara-2 shows stronger linkage to thr than does ara-3, but is more
weakly linked to leu than is ara-3. Thus 5.2 % of ara-2f transductants
are thr+ and 59.6 % are leu+; whereas 4.3 % of ara-3+ transductants are
thr+ and 72.6 % are leu+.
4. It will be seen below (Table 2) that thr and Zeubehave in a com-
plementary manner in the four-factor crosses; i.e., if in a given experi-
ment a high proportion of the ara+ transductants are leuf, then a low
proportion are thr+, and vice versa. Reference to Fig. 1 should make it
clear that this behavior can be expected only if thr and leu are on either
side of the arabinose region.
Recombination between ara Mutants
A cross is made between two nonidentical ara mutants with the
recipient thr leu and the donor thr+ leu+. Selection is carried out for
thr+, Zeuf, and ura+ transductants, each on separate plates. The numbers
of thr+ and Zeu+transductants found are roughly the same as the num-
bers obtained with wild type as donor, but the number of uru+ trans-
ductants is lower. An estimate of the distance between two ara sites may
be obtained from the ratio of the yields of araf to leu+ transductants
in the same experiment. This ratio is approximately 70% with any ara
mutant as recipient and wild t,ype donor, and varies from about 0.6%
for the distance between ara-3 and ara-5 to 16.9% for the distance
between ara-2 and ara-21 (see Fig. 2). A later publication will describe
the results of mapping experiments carried out by this method. The

7. 320 GROSS Alr;D ER'GLESBERG
present communication is restricted to observations regarding the segre-
gation of thr and Iell. in t,hree- and four-factor crosses.
Order qf ara Sites
The general methodology which has been employed in ordering all
seventeen ara sit,es with respect to each other and to thr and leu is
based upon “reciprocal” crosses involving pairs of ara mutants. The
recipient in each case is thr leu and the donor thr+ leTif; each ara site is
employed as donor in one cross and recipient in t,he other. As shown in
Fig. 1 cross A, thr+ ma-2 lelrf as donor is crossed with a recipient which
is thr ara-3 leu and selection is carried out for arabinose-positive trans-
ductants by plating on mineral arabinose medium containing threonine
alld leucine. The am+ t,ransductjants arc t’hen scored for thr+ and Zeu+.
I:ollowing this the (so-called) reciprocal cross is carried out (Fig. 1,
cross B) in which thr+ am-3 leu+ is the donor while thr am-2 lcu is the
CROSS A
DONOR{ Ihr+le”+ (1~0 -2 ) + ara-2 + +
/- --
RECIPIENT( thr- leu- ara-3 )
l 2 3/ 4 5
-.J
thr- + ora- let
CROSS B
DONOR (thr+leu +oro-31
RECIPIENT( thr- leu-m-2)
+ + aro-3 +
-- -l
I 2
3
4 5
L.,---
thr- ora- + IeU-
FIG. 1. Partial, schematic, represent,ation of crossing over responsible for
ara+ recombinant,s in “reciprocal crosses” between ~a-2 and ax-3. Upper lines
represent the chromosomal segment introduced by the phage; lower lines the
bacterial chromosome. Only the crossover in region 3, which is required in order
t,o give an ura+ recombinant is diagrammed. This crossover is not sufficient, since
only recombinants resulting from an even number of crossovers are recovered.
In cross A a recombinant will be recovered if a second crossover occurs either
in region 4, giving rise to the genotype Ihr ara + 1~71,or in region 5, giving thr aru+
leu+. The recombinant will only include the thr+ marker as a result of a quadruple
crossover. In cross B the reverse is true; the thr+ marker is incorporat,ed into
ara+ recombinants as a result of a double crossover event, whereas t,he incorpora-
tion of the leu+ marker requires a quadruple crossover.

8. ORDER OF L-ARABINOSE MUTANTS OF E. COLI B/I’ 321
recipient. If ara-2 is to the “left” of ara-3, i.e., closer to thr, when ara-2
is the donor one would expect more ara+ led transductants than when
ara-2 is in the recipient genome. This is so, since when ara-2 is the
donor, ara+ leu+ transductants are the result of a double crossover,
whereas when ara-2 is the recipient, ara+ led transductants are the
result of the rare quadruple crossover. By similar reasoning one would
expect less ara+ thr+ when ara-2 is the donor than when ara-2 is the
recipient. Alternatively if ara-2 were to the “right” of ara-3, i.e., closer
to leu, one would expect a smaller percentage of leu+ araf when ara-2 is
the donor as compared to when it is the recipient. The opposite would be
expected with regard to the ara+ thr+ recombinants. The results of such
an experiment are shown in Table 2 and demonstrate that ara-2 is
closer to thr than ara-3 (ara-2 is to the left of ara-3). On the same basis
it is inferred that the order of the other mutant sites is thr ara-2 ara-1
ara-3 leu.
One of the main features of the results presented iu Table 2 is the
complementary behavior of the thr and lelh markers in the four-point
tests; the same order may be derived from consideration of the assort-
ment of either the thr or of the leu marker alone. Advantage has been
taken of this fact in further experiments in which the assortment of the
leu marker only has been observed. Use of the thr marker is laborious,
TABLE 2
FOUR-FACTOR CROSSES BETWEEN ara-1, ara-2, and ara-3a
Recipient
ara-I
ara-2
Donor
ara-2
ara-1
ara+ leu+ ara+ thr+
x 100 x 100
total ara+ total ara+
64.4 1.2
17.4 7.4
ara-l aTa- 26.1 6.4
ara-3 ara-1 52.4 2.4
ara-2 ara-3 14.3 9.5
ara-3 ara-2 65.8 2.8
a The recipient was in every case thr leu and the donor thr+ Zeu+. Selection
was carried out for ara transductants, and some of the colonies obtained were
replicated to score for thr and others were streaked individually to score for leu.
Each datum was obtained from a total of several hundred colonies from several
independent experiments.

9. 322 GROSS AND ENGLESBERG
owing to its loose linkage to the ara mutants, and would be difficult
with certain pairs of mutants which give low yields of araf recombinants.
It should be emphasized that] in no case where both markers have been
used has an exception to the complement,ary segregat,ion been observed.
The experiments were carried out in a manner identical to t’he four-point
tests, the only difference being that the ara+ transduct’ant’s are not scored
for thr/thr+.
It is, of course, not necessary t,o carry out, all possible crosses between
pairs of mut,ant,s in order to derive t’he completje order. In the present,
TADLE 3
THREE-FACTOR CROSSES BETWEEN ALLIACENT am SITES”
ara+ leu+ I
, cross
2 x 13 (1) 15/62 24.2
13x 2 (2) 104/218 47.7
13x 7 (2) 69/216 31.9
7 x 13 (2) 113/199 56.8
7x 4 (1) 34/141 24.1
4x 7 (I)* 74/160 4G 3
4 x 16 (1) 30/104 28.9
16X 4 (1) 67/128 52.3
16X 6 (1) 9/33 27.3
6 x 1G (2,* M/l64 40.2
G X 23 (1) 15/80 18.8
23X 6 (2) 54/100 54.0
23 X 14 (2) 29/140 20.7
14 X 23 (3) GO/163 42.3
14 x 15 (2) 21/M 23.9
15 x 14 (2) 43/100 43.0
I-
I-
-______
15x 1 (3) 20195 21.1
1 x 15 (3) 132/243 50.G
1 x 24 (3) 29/129 22.5
24x 1 (21 55/104 52 .9
24X 8 (3) 351148 23.6
8 x 24 (3) u/149 54.4
8 x 12 (2) 29/111 26.1
12x 8 (1) 46191 50.5
12x 5 (2) 10157 17.5
5 x 12 (3) 206/339 GO.8
5x 3 (5) 202/7OG 28.6
3x 5 (4) 1721380 45.3
3 x 21 (3) 89/297 30.0
21X 3 (3) 133/323 41.2
21 x 19 (3) 0 0
19 X 21 (3) 0 0
“ In each cross the recipient is listed first and the donor second. The recipient
was in every case fhr Zeu and the donor thr+ leu+. Am+ transductjants were selected
and scored in the usual manner for Zeu+. The numbers in parentheses indicate t,he
number of separate experiments from which the data are derived. The result,s in
the crosses marked with an asterisk were obtained by replica plating.

10. ORDER OF L-ARABINOSE MUTANTS OF E. COLI B/r 323
work mutants numbers 1, 2, and 3, were chosen as reference sites, and
the remaining mutants were divided into groups depending upon whether
they were to the left or right of these reference sites. Mutants belonging
to the same group were then crossed with each other, and the complete
order obtained.
Table 3 contains the results of three-point tests between mutants
found to be adjacent, and Fig. 2 depicts the order derived from the
I I I
I I i I
A I B I C I
thr--wm----/ i i3 i 4 1166 23 14 15 1 24 81 12 5 3 19: ‘----leu1
I I
I *’ i
I I I I
FIG. 2. Order of sevent,een a7.a mutants wit,h respect, to thr and lee. The vertical
lines indicate the boundaries of the functional groups A, B, and C. No attempt
is made to indicate the relative dislances between the mutant sites.
TABLE 4
THREE-FACTOR CROSSES RETWEEN NONADJAWNT PAIRS OF MUTANTS”
am+ led
am+
?l’ Cross
2x 7 (1) 13/(iO 21.7 / 14 X 8 (2) 26/l 27 20.5
7x 2 (2) 48/Q:! 52.2 8 x 14 (1) 45/80 56.2
16 X 23 (1) IO/40 25.0 23X 8 (1) 13/48 27.1
23 X 16 (1) 3(i/58 ti2.1 1 8 x 23 (1) fi(i/127 52.0
--~---------------~ ~~~~----------------
23X 1 (2) 31/Q7 32.0 1tix 1 (2) 491174 28.2
1 X 23 (1) 32/63 50.8 1 X 16 (1) 104/179 58.1
14x 1 (2) ati/ 29.2 16X 8 (1) 16/52 30.8
1 x 14 (1) lti/27 ““:“L”“j_2’~YY”” 66 3
__.__ 1
1x 8 44/145 30.3 5 x 21 (3) 91/379 24.0
8X 1 Qti/200 49.0 21 x 5 (3) 93/248 37.5
16 x 14 (1) 4/37 10.8
14 x 16 (1) 52/90 57.8
a In each cross the recipient is listed first and the donor second. The recipient
was thr leu and the donor thr+ leu+. dra+ transductants were selected and scored
in the usual manner for Zeu+. The number in parentheses indicates the number of
separate experiments from which the data are derived.

11. 324 GROSS AND ENGLESBERG
TABLE 5
THREE-FACTOR CROSSES OF ara-3 WITH OTHER ara MUTANTS AND WITH arafa
Site am+leu+/ara+ (individual experiments)
ara+ donor
ara-2 donor
ara-2 recipient,
ara-1 donor I-
ara-1 recipient
119/157;117/lGS;lOG/134;148/216 490/675
75.8 G9.G 79.1 68.6 72.6
94/139; 52/74; 85/138 231/351
67.6 70.3 Gl.6 65.8
__--___---_---------- ----
331220 ; 5G/402 891622
15.0 13.9 14.3
57/109; 83/158 140/267
52.3 52.5 52.4
59/209; lG/78 75/287
28.2 20.5 26.1
32/Gl; 91/M ; 45/99 168/341
52.5 50.3 45.4 49.2
23/G7 ; 17/85 40/152
34.3 20.0 2G.3
36/77; 11/26; 85/198; 40/79 172/380
4G.8 42.3 42.9 50.7 45.3
71/236; 15/49; 13/32; 22/62; 91/327 202/706
30.1 30.6 40.6 35.5 27.8 28.D
,-
ara-12 donor
ara-12 recipient
I-
ara-5 donor
ara-5 recipient
With leu marker
reversed
ara+ donor
arat leu/ara’ (individual experiments) Pooled ratio
12G/163; 110/156 236/319
77.3 70.5 74.0
ara-5 donor I- 5G/120; G2/148 118/268
46.7 41.9 44.0
Pooled Differ- Dis-
ratio ence tance
13.06
51.5 ---
13.7
3.17
2G.3 --
3.95
--- ---
1.30
23.0 ---
0.64
--- --
0.77
16.7---
0.42
a Selection was carried out for araf and leu+ transductants on separate plates,
with appropriate dilutions of the phage-bacterium mixtures, and the ara+ trans-
ductants were streaked individually to determine the proportion which were
le&. This proportion is given for each of the experiments, the upper line giving
the actual data and the lower line, the data expressed as a per cent. The distances
in the column headed “Distance” are derived from the average of the absolute

12. ORDER OF L-ARABINOSE MUTANTS OF E. COLI R,/l- 325
data. Araf recombinanta were obtained in crosses of all pairs of mu-
tants except ~~a-19 and ~a-21 (which must therefore be either identical
mutations at the same site or at overlapping sites). The proportion of
Zeu+transductants in one member of each pair of crosses generally falls
between 20 % and 30 %, and that in the other member is usually be-
t’ween 40 % and 55 70; the mean difference between members of pairs is
24.6 %, and the smallest difference is 11.2 %. There is therefore no am-
biguity in deriving the order from these results. Some idea of the error
involved in these estimates may be obtained from Table 5, which gives
data from a series of independent experiments involving several pairs of
mutants. It will be seen that the variation in the estimates is consider-
ably less than the differences between members of pairs of mutants.
Table 4 presents the results of a few crosses between nonadjacent
sites. About half the possible 289 pairwise crosses between the seventeen
mutants have been performed and the results obtained were in every
case in agreement with the order given in Fig. 2. This agreement estab-
lishes beyond doubt the linearity of the mutant sites.
Negative Interference
A comparison of the differences in the proportion of leu+ transductants
between pairs of adjacent and nonadjacent markers shows that the
differences are on the average somewhat larger in the latter group of
crosses; the mean difference in Table 8 is 24.6 % and in Table 4 is 29.3 %.
This suggests that the magnitude of the difference between members of
a pair of crosses may be less t,he closer the uru sit,esinvolved, and indi-
cates the presence of negative interference. Table 5, which presents the
results of crosses between ~a-3 and a series of mutants at various
distances from ara-3, shows this effect clearly. As the distance between
the aru sites decreases, the proportion of leuf transductants in the “high”
member of each pair decreases, and in the “low” member, increases;
hence, the differences between members of the pairs decreases markedly.
Also included in Table 5 are the results of crosses in which the Zeu
marker was reversed, the recipient being thr+ leu+ and the donor thr leu.
yields of ara+ and leu+ transductants in each experiment and are expressed as
ara+/Zeu+ X 100. The first column gives the ara site other than ~~a-3 involved in
each cross. For example, “uru-2 recipient” refers to an experiment with ara-2 as
recipient and ara-3 as donor. With t,he exception of the last two crosses the recipi-
ents were thr Zeu and t.he donors thr+ leu+. In the last two crosses the recipients
were thr+ leu+ and the donors thr leu. The column “Difference” gives the differ-
ence between t,he pooled percentage Zeu+nra+/nru+ for the two crosses in each pair.

13. 326 GROSS AND ENGLESBERG
In these crosses the proportion of leucine requirers among the ara+
transductants is scored. It can be seen that in the crosses with ara+ and
ara-5 as donors and the thr and leu markers in the usual arrangement,
72.6 % and 45.3 %, respectively, of the ara+ transductants were leuf. In
the comparable crosses with the markers reversed, 74% and 44 o/o,
respectively, of the ara+ transductants were leucine requirers. The fact
that, similar results were obtained independently of whether leu+ or leu
was being introduced shows that the negative interference cannot be an
artifact due to impurity of the transduction colonies with respect to the
unselected marker. Such impurity would, in any case, always increase
the estimate of the proportion of Zeu+transductants in each cross, and
is therefore not applicable here since one cross in each pair is character-
ized by decreasing numbers of leu +. These results also exclude the possi-
bility of significant selection on the transduction plates in favor of lel&+
transductants. The question of purity of transduction colonies has aiso
been approached in a more straightforward mtLnner by tcsbing directly
for heterogeneity; 140 thrf leu+ and 130 leu+ transductants were tested
for impurity with respect to arabinose genotype, and only five mixed
colonies were found. In addition 70 araf t,ransductants, some from crosses
with ara+ as donor and some resulting from recombination between ara
mutants, were examined for impurit#y with respect’ to ZPUgenotype, and
only one mixed colony was found. These observations on the purity of
t,ransduction colonies arc markedly different. from those obtained in
similar tests by Lennox (1955) with the K12-Plkc system.
Division of the 17 Arabinose Nonutilizing Mutants into Three Groups
(A, B, and C) on the Basis of Physiological Differences
All seventeen ara mutants, as well as the ara+ strain, were compared
in quantitative growth experiments in a casein hydrolyzate medium
with and without L-arabinose. Subsequent to full growth (4 hours of
incubation) the cultures were assayed for keto sugar accumulation by
the cysteine-carbazole test. Figure 3 shows the results of such an experi-
ment with ara+ and with ara-1, ara-2, and ara-3, each of the latter
demonstrating one of the characteristic responses shown by the remain-
ing mutants. Growth of ara-2 in casein hydrolyzate is inhibited by
L-arabinose and only t’race quantities of a keto sugar were detected in
the growth medium. Mutants ara-13, -17, and -4 give similar results to
ara-2; these four will be referred to as group A mut,ants. Ara-3 is com-
pletely resistant to the L-arabinose inhibition and accumulates small

14. ORDER OF L-ARABINOSE MUTANTS OF E. COLI B/F 327
0 I 2 3 4
TIME,HOURS
CH + L-arobinoae
I
0 I 2 3 4
TIME.HO”RS
I I
0 I 2 3 4
TIME.HOURS
I 2 3
TIME .HO”RS
FIG. 3. Comparison of the growth (solid lines) and keto-sugar production
(black bars) of Escherichia coli B/r and arabinose-negative mutants in group
A (ara-2), group B (ara-1), and group C (ara-3) in a mineral 0.05 $& casein
hydrolyzate (CH) medium in the presence and absence of 0.2 y0 L-arabinose.
amounts of a keto sugar (more than group A). Mutants ara-12, -5, -19,
and -21 are similar to ~~a-3 and are placed in group C. Growth of ~a-1
on casein hydrolyzate is inhibited by L-arabinose, as is the case with
group A mutants; however, ~a-1 accumulates large quantities of keto

15. 328 GROSS .4ND ENGLESl3ERG
sugar. Growth of mutants ara-16, 4, -23, -11, -15, -24, and -8 is exactly
like that of ~a-1 and each mutant accumulates an amount of keto
sugar significantly larger than mut,ants in groups A or C. These eight
mutants are placed in group B.
An analysis of the casein hydrolyzate arabinose cultures of the group
H mutants has demonstrated that each of t’hese mutants accumulates
one and t’he same keto sugar, L-ribulose. This conclusion is based upon
t,he following observations: The keto sugar in the various supernatants
has the same characterist’ics in paper chromatographic analysis as
rihulose. The time for full-color production (12Z-16minut,es) in the cys-
t.rine-carbazole test, and the absorption spectrum of the chromogen
produced (maximum at 540 mp) was t.he same for each mutant and
identical t,o that for ribulose. No att.empts were made to identify the
small amounts of kcto sugar accumulatjed by group A or group C
mutants.
Itlentijication of Group C Mutants ~IJAbortive Transduction
When group C mutants are used as recipients with wild t’ype as donor
and selection is carried out for ara+ transductants, numerous minute
colonies, invisible to the naked eye, are present in addit,ion to the normal-
sized araf transductants. The appearance and number of these minute
colonies is unaffected by the arrangement of the thr and Zeu markers.
The minute colonies are also observed when both donor and recipient
are thr+ Zeu+.So A or B group mutants give minute colonies with any
wild type or mutant donor tested. The C group mut’ants produce
minute colonies also when infected with phage grown on any A or B
group mutant, but do not give them with phage grown on any group C
mut,ants. The ratio of minute colonies tJo am+ transductants is about
G:1 with wild type donor. The ratio increases with phage grown on A
and R group mutants, as t,he distsancc bet’ween sites in donor and re-
cipient decreases. Thus with am-2 as donor the ratio is about 50: 1, and
with ~a-1 as donor it) is about 250: I. The appearance of these minute
colonies identifies t,hem with the abortive transduction colonies de-
scribed by Ozeki (1956). Since the A and R group mutants do not give
rise t.o any observable abortive transductants, no test, of complementn-
tion can be applied to t,hem. The difference bet,ween the C group mu-
tants and the A and B groups in this respect may be relat,ed to the fact
that the former are resistant t,o inhibition by arabinose, whereas the
latter are sensitive.

16. ORDER OF L-ARABINOSE MUTANTS OF E. COLI B/I‘ 329
DISCUSSION
The results reported demonstrate that a series of closely linked mu-
tant sites involved in the control of L-arabinose utilization are arranged
in a linear order. This order was demonstrated by transduction experi-
ments in which the assortment of unselected linked markers was ob-
served among recombinant#s between nonidentical arabinose mutants.
The same method was used in experiments by Demerec and Hartman
(1956), but no systematic attempt was made by them to determine the
order of a sizable number of sites. Approximations to such an order,
based upon the absolute yields of recombinants in crosses between
mutants, have been made, but the considerable variation from experi-
ment to experiment, the small differences involved, and other factors
make it very difficult, if not impossible, to construct a detailed map by
such procedures. The advantage of the method employed in the present
work is that the results are independent of variations in yields of re-
combinants and depend not on small differences in yield, but on reln-
tively large differences in the proportion of recombinants carrying un-
selected markers. Perhaps most important, in this connection is the fact
that this method is not affected by the very common but unexplained
discrepancy between the absolute yields of recombinants in reciprocal
crosses.
It has been shown in the case of Aerobacter aerogenes (Simpson et al.,
1958; Simpson and Wood, 1958) and Lactobacillus plantarum (Heath
et al., 1958; Burma and Horecker, 1958) that L-arabinose is first con-
verted into L-ribulose b.y the enzyme L-arabinose isomerase. The enzyme
L-ribulokinase, in turn, catalyzes the conversion of L-ribulose to ribulose
5-phosphate. The accumulation of L-ribulose by group B arabinose
negative mutants of E. coli suggests that these mutants are deficient in
the enzyme ribulokinase.5 It is interesting to noOe that the L-arabinose
inhibitory effect, as detected with group A and group B mutants, is
similar to the naturally occurring sensitivity to pentose and methyl
pentose inhibition in wild-type strains of Salmonella typhosa (Barkulis,
1949; Englesberg and Baron, 1959).
5 Subsequent experiments (Englesberg and Killeen, 1959), which will be de-
scribed in detail elsewhere, demonst,rate that group A mutants lack r,-arabinose
isomerase, group B mutants lack ribulokinase; each mutant in this group has a
characteristic level of L-arabinose isomerase activity, varying from one-tenth
to four times the activity of the prototroph. All mutants in group C are deficient
in both kinase and isomerase.

17. 330 GROSS AKD ENGLESBERG
Comparison of t,he order obtained for t’he seventeen mutant sites with
the grouping of tOhemut’ants by functional criteria reveals exact cor-
respondence between the locations of the sites and the groups to which
they belong; mutants belonging to the same group are located next to
one another. This correspondence shows that it is meaningful to speak
of the order of t,he groups of functionally similar mubants, or loci, and
underlines the importance of the existence in bacteria of the close
linkage bet’ween loci involved in successive steps of metabolic pathways
(Demerec and Hartman, 1956; Hartman, 1956). The work of Crawford
and Yanofsky (1958) provides an indicat,ion of one way such linked loci
may cooperate in enzyme formation. Work designed t,o detect possible
intcract,ions betweeu t,he functjions of t,he different loci in the arabinose
clust,er is in progress.
The marked ncgat,ive interference observed in the present series of
crosses has been reported in Aspergillus (Pritchard, 1955) and in phage
(see Edgar, 1956; Chase and Doermann, 1958; and Hershey, 1958). It is
noteworthy that in spite of this negative interference it was possible to
obtain the order of even t,he closest of the ara sit,es. In several cases in
t,he other organisms where negat,ive int’erference has been encountered,
it was not possible t’o determine the order of sit’es due to the equilibrium
wit.h respect to close outside markers produced by the interference.
The authors would like to thank Mrs. ?;. Killeen for her able assistance in
many of the experiments.
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