JOURNAL OF BA~~~~OLOGY, Oct. 1967, p. 1112-1123
Copyright @ 1967  American Society for Microbiology

Vol. 94, No. 4
Printed In U.S.A.

Genetics and Physiology of Colicin-tolerant Mutants
         of Escherichia coli

           ROSA NAGEL DE ZWAIG AND S. E. LURIA
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

            Received for publication 10 July 1967

A series of colicin-tolerant (tol) mutants of Escherichiu co/i K-12, which ad-
sorbed colicins but were not killed by them, were isolated and studied genetically
and physiologically. Three major classes of mutants were found: tol 11, tolerant
to colicins A, El, E2, E3, and K; tof ZZI, tolerant to A, E2, E3, and K; and tof VIII,
tolerant to El only. The sites of to1 II and to1 III mutations mapped near the gul
region (gene order: to/-gal&o) and were cotransduced with gal by Pl. In hetero-
zygous diploids, tol+ was dominant over tol; to1 II and to1 III gave full comple-
mcntation. All the tol mutations that mapped near gal rendered the bacteria more
fragile during growth and hypersensitive to deoxycholate and to ethylenediamine-
tetraacetic acid. The tol VIII mutation mapped between str and his. These mutants
were extremely sensitive to deoxycholate and were also hypersensitive to methylene
blue, acridines, and various other compounds. The sensitivity is attributed to
increased uptake due to selective alteration of the permeability barrier. The colicin-
tolerant mutations are interpreted as affecting some components of the cytoplas-
mic membrane which mediate between the adsorbed colicin molecules and the
target sites of their biochemical effects in the bacterial cell.

The cytoplasmic membrane of a bacterium
plays a major role in the functional organization
of the bacterial cell. Not only is it the known or
reputed site of many enzymatic activities-hy-
drogen transport,  oxidative phosphorylation,
lipopolysaccharide and murein biosynthesis (24)
-but it has also been variously implicated in
deoxyribonucleic acid (DNA) synthesis, protein
synthesis, as well as in cell division and the con-
comitant orderly distribution of new copies of
genetic material to daughter cells (9). Yet, the
analytical study of the cytoplasmic membrane
has barely started. By analogy with other prob-
lems of the functional organization of cells, a
critical step in opening up the study of the cyto-
plasmic membrane may be a genetic approach,
providing functional anomalies that can be
traced to alterations of specific genes, of their
products, and of the interactions of these products
with other cellular components.

on specific cellular functions while remaining on
the surface of the bacterial cell, and that at least
the primary inhibitory functions are reversible
by removal of colicin with proteolytic enzymes.
The obvious candidate as the site of the inhibitory
actions is the cytoplasmic membrane; the
existence of mechanisms for generalization of the
effects of a single colicin molecule to the mem-
brane as a whole has been suggested to account
for the one-hit kinetics of colicin action (13, 18).

Different colicins have different receptor sites
on the cell wall, whose mutations render the bac-
teria resistant to specific colicins (3) ; for example,
colicins El, E2, E3 (and phage BF23) share
identical or closely overlapping receptor sites.
Different colicins also differ in their modes of
action (18); colicins El and K arrest macro-
molecular syntheses and active transport, colicin
E2 causes DNA breakdown, and colicin E3 in-
hibits protein synthesis.

The work reported in this paper was initiated   For the purpose of the present work, mutants
with the purpose of finding bacterial mutants  were sought that became tolerant (tol) to one or
that may be specifically altered in the functional  more colicins which they could still adsorb. The
organization of the cytoplasmic membrane. The  existence of such mutants was first discovered by
system chosen was the resistance of Escherichiu  Nomura (19). Among these mutants, one may
coii to colicins. The rationale for the approach is  expect to find "membrane" mutants, altered in
provided by recent work, especially by Nomura  the response to colicins because of alterations in
(18; Ann. Rev. Microbial., in press), which sug-  specific membrane constituents. The initial search
gests that the colicins exert their inhibitory role  was made for mutants doubly tolerant to two

Ill2


VOL. 94, 1967        COLICIN-TOLERANT MUTANTS OF E. COLI            1113

colicins, El and K, with different receptors but
similar mode of action. This approach proved
unnecessarily sophisticated because our own
work, as well as reports from other laboratories
(1, 19,19u, 21), revealed that a substantial propor-
tion of the colicin-resisting mutants were of the
tolerant type, which adsorbs colicins but fails
more or less completely to be inhibited by them.

The present paper describes some cohcin-
tolerant mutants of E. coli K-12 whose physio-
logical characteristics indicate that these mutants
have defects in some surface components which
are recognizable independently of the tolerance
to the colicin. Some of these mutants are similar
to others described by Nomura (19), Clowes (l),
and Reeves (21), and, in greater detail, by
Nomura and Witten in the accompanying paper
(19a).

MATERIALS AND METHODS

Media. The following media were used: LB (tryp-
tone-yeast extract) broth and agar (14); LC (LB broth
and agar plus 2.5 IIIM CaC&); A broth and X agar
(tryptone, 10 g; NaCl, 2.5 g per liter); Penassay Broth
(Difco); 63 minimal medium and agar [K&PO~,
5.3 g; K,HPO,, 10.6 g; (NH3804, 2 g; 1 N KOH,
3 ml; distilled water, 1 liter; MgS04, added to a final
concentration of 1 mu]; O&i minimal medium and
agar (K,HPG,, 10.5 g; KHZPO, 4.5 g; (NH4)1S04,
1 g; sodium citrate, 0.47 g; MgS04, 50 mg; distilled
water, 1 liter]. Glucose or succinate was added at
0.4y0; vitamin B, at 1 +g/ml; L-amino acids at 20 fig/
ml; and streptomycin at a final concentration of
200 pg/rnl. EMB-agar contained (per liter) : tryptone,
8 g; yeast extract, 1 g; NaCI, 5 g; agar, 15 g; K*HPO,,
2 g; sugar, when present, 10 g; eosin Y, 300 mg; and
methylene blue 50 mg. BTB-agar was the same as
EMB but with 24 mg of bromothymol blue instead
of eosin and methylene blue. Phosphate buffer was
composed of KH2P04, 3 g; NaHPO,, 6 g; distilled
water, 1 liter.

So/-agar test of response to colicins. A colicinogenic
strain was stabbed in an LB agar plate, which was
then incubated overnight. The plates were sterilized by
chloroform vapors, and a layer of melted top agar,
inoculated with the strain to be tested, was poured on
the plate surface (3). Resistant colonies were readily
isolated from within the sterile halo formed by the
action of the colicin on the sensitive strain.

Preparation of colicins. Colicinogenic bacteria were
grown in 200 ml of LB broth to about 2 X 1oB cells/
ml; the cells were collected, suspended in 10 ml of
saline, and irradiated in a petri dish for 60 set (Gen-
eral Electric Germicidal lamp, IS-w, 50cm distance).
The cells were transferred to 400 ml of 63 minimal
medium plus 10 IIIM MgSO, plus 0.5% tryptose
(Difco), and incubated with shaking for 3 hr at 37 C.
Cells were collected, suspended in 3 ml of buffer, and
treated for 2 to 3 mm in an MSE sonic oscillator. The
sonic-treated suspension was centrifuged to remove
whole cells and debris, and the supernatant fluid was

frozen. The colicin titers were about 10L1 killing
particles/ml.

Mating procedures. The techniques of Jacob and
Wollman (10) were followed. Platings on selective
media were done after 2 hr of incubation at 37 C. The
frequency of unselected markers among the recombi-
nants was determined by transferring the recombinant
colonies to plates of the same selective media and
replica-plating onto appropriate media. For the test
of colicin sensitivity or resistance, the replication was
done on plates prepared by seeding with a colicino-
genie strain in a soft-agar layer. After overnight
growth, the soft layer was scraped off and the plates
were sterilized with chloroform.

PI transduction. Pl lysates were prepared by the
method of Lennox (12). For Pl transduction, the
procedure of Rothman (23) was followed.
@-Gafactosidase assay. Free @-galactosidase was
assayed by using o-nitrophenyl-@galactoside (ONPG)
as described by Revel, Luria, and Rotman (22). To
test for ONPG hydrolysis by whole cells, the bacteria
were grown in X broth to an optical density (OD)
value (420 m) of 0.4 to 0.5; 1 ml of culture in a
spectrophotometric cuvette was mixed with 0.3 ml
of 0.013 M ONPG in 0.25 M sodium phosphate buffer
(PH 7.0), and the rate of formation of o-nitrophenol
was measured at 420 rn* for 30 min.
F curing. For `curing" F' strains by acridine
orange, the method of Hirota (6) was followed.
Bacterial and phage strains. Bacterial strains used
are listed in Table 1. Phage strains used are from the
collection of this laboratory.

TABLE 1. Bocteriai strains"

Strain

Escherichio co/i K- I2
  derivatives
C600

w602
W4032

PA309
AB1122-1
LA348
w3101
P678
P678 sue-
Y20
Other strains
E. co/i K235
E. coli CA38
E. coli06:Hl6,23
Salmonella ryphi-
  murium (cys-36)

1

Relevant characteristics

F- thr- leu- thi- Iac- (i+ z+ y-) m-s

TI, TS-r

F- Iru- thi- bio- gal- str-r
Hfr Cavalli pro- mrr- 51,.s T6-r; po-
larity of transfer: leu rhr arg his
rrp gal

F- thr- leu- fhl- trp- his- arg- stw
F- 511-r

F- &r-l T6-r K-r
F' 811-s (F-sue+ gal+ bio+)
F- rhr- Icu- rhi- WC+
F- rhr- Icu- thi- suc-
F- rhr- Ieu- fhl- (ColE,-K30)

(`2X)
(ColE3. Coil)
(alA)
(ColEZ-P9)

a Symbols: fhr - threoninc; ku = leuciae; fhi = thiamine;
IrP  - tryptophan; his - histidine; arg = arginine; pro =
proline; met = methionine; w = streptomycin; lot = lactose;
gal - palactose; SUE = succinate; bio = biotin; TI. T-5. T6 =
phages Tl, T5, T6; K = colicin K; r = resistance; s = sensi-
tivity; Co1 = colicinogenic. Most bacterial strains used were
from the collection of this laboratory. Strain W3101 was re-
ceived from E. L. Wollman.


1114                NAGEL DE ZWAIG AND LURIA            J. BACTERIOL.

RESULTS

Mutants resistant to colicins were isolated
from E. coli K-12 strains C600, W4O32, and W602
without mutagenic treatment. Selection was done
either with single colicins (A, or El, or K) or
with colicins El and K simultaneously by using
the soft-agar test and the appropriate colicino-
genie strains (Table 1). After purification by re-
streaking, the mutants were tested for sensitivity
to a series of colicins and phages. The mutants
that were studied further are listed in Table 2.
In addition to the classical mutants, resistant only
to colicin A, or to colicin K and to phage T6, or
to colicins El, E2, E3, and to phage BF23 (note
that this type of mutant is resistant also to colicin
A), there were several classes of mutants with
other patterns of resistance. Five classes were
recognized and named as follows: to1 II, tolerant
for A, El, E2, E3, and K; to/ Zfu, partially toler-
ant for the same colicins; to1 III, tolerant for A,
E2, E3, and K; to1 Illa, tolerant for A, E2, E3,
and partially tolerant for K; and to1 VZZZ, tolerant
for colicin El only. [The designations of various
classes of mutants are provisional names selected
to agree with those used in the accompanying

TABLE 2. Patterns of colicin resistance of spontaneous
mutants isolated from Escherichia coli K-Z2 strains"

Class

Id+
lOI II
ml III
to1 IlIa
ml vmb
E-r
BF23-r
A-r
T6-r K-r

_-

-

E3
-

-
l-6

D Symbols: s = sensitive; r = resistant; pr - partially re
s&taut. Italicized symbols correspond to the colicins used for
the selectiott of a given mutant. The strain used as producer
of colicin E3 also produced colicin I (see Table 1). Distinction
between the two colicins was readily made on plates, since
colicin E3 produced a much larger inhibition zone than colicin
I; I-s E3-r strains showed only the small zotte of inhibition
by colicin 1. All the tol mutants were sensitive to colicin V
(from E. toll K94), B (from E. coli CL139). H (from E. coil
CLlO), D (from E. co/f CA23), and I (from 0500 ColW9).

bB~ the soft-a&w method, the rol VIII mutants showed a
decreased sensitivity to colidns A and K.

article by Nomura and Witten (1%). The roman
numerals are used to designate specitic patterns
of colicin tolerance.] Many of these tol mutants
appear to arise by point mutations, since they
revert at rather high frequency to colicin sensi-
tivity. Multiple tolerance reverts in a single step.
Most of these mutant types have their counter-
parts among the mutants reported by Clowes (l),
Reeves (Zl), Nomura (19), and Nomura and
Witten (1%).

The to1 mutants were tested also for sensitivity
to phages TZ, T3, T4, T5, T6, T7, h, and MS2.
They showed the same patterns of sensitivity as
their respective parent strains. Additional types
with partial tolerance for some colicins have been
found but have not yet been studied.

Adsorption of colicins. Selected mutant strains
derived from 0500 were tested for ability to ad-
sorb colicins El and K. Representative results
are shown in Table 3. The mutants of types to1
II, to1 III, to1 ZIZa, and tol VIII adsorbed cohcins
about as effectively as did the parent strains.
(Mutants of type to1 ZZu were not tested because
they were not available in any str-s strains, as
needed for our colicin-adsorption test. No sig-
nificant results could be obtained with colicin A
because of poor adsorption in liquid media.) The
classical resistant types did not adsorb colicin
to any measurable extent.

Genetic analysis of the mutants. Bacterial mat-
ings and transduction with phage Pl were used
to map the various mutant types and showed that
the toi II, to1 ZZa, tol III, and to1 ZZZa mutant sites
map in a narrow region near the gal genes,
whereas to1 VZZZ maps not far from the his region
of the E. coli chromosome.

Mapping of to1 VIII. Crosses between strains
W4032 Hfr to1 VIII SJT-s and PA309 F tolt str-r
were performed and recombinants were selected
and tested by replica-plating for various non-
selected markers, including tolerance to colicin
El. Results of two typical crosses with a single
to1 VZZZ mutant donor are presented in Table 4
and indicate for this to1 VZZZ mutation a location
between the his and str loci. A similar location
was reported for analogous El-tolerant mutants
by Clowes (1).

Mapping of to1 ZZ, tol ZZa, tot ZZZ, and tol ZZZa. Pre-
liminary mapping by crossing W4032 to1 II with
PA309 rol+ indicated that the zol ZZ site was prob-
ably linked to gaZ. Linkage between gal and a
multiple-colicin-resistance site had been en-
countered previously by Anton (Ph.D. Thesis,
University of Buenos Aires, 1963) and Gratis
(4). Pl transduction experiments (Table 5) were
performed with lysates grown on gal+ bio+ to1
strains to transduce gal+ to gaZ- bio- toi+ re-
cipients. The gal+ transductants were tested for


VOL. 94, 1967

COLICIN-TOLERANT MUTANTS OF E. COLI

TABLE 3. Adsorption tests for colicins El and ICa

1115

Erpt 1                   Erpt 2

   Colicin El cuncnb        / Colicin El c,oncnb

Strain used for
preadsorptioo

a00 toi+. .......
C@lO E-riBF23-r .
c#o to1 II .......
None. ...........

            Strain used for
1.4 x I I
      10-a     preadsorption
lo- '    1 i
                        102   2.5 x
                                10-4

--___        ~-

328   428   cm3 fol+. . .  385   730
18    47   cm to1 II..  595   700
388    457   cm to1 VIII....  643   714
14    95   C6fXl E-r BF23-r.   37    92
          None..   30    59

Expt 3

I Colicin K concnh

Strain used for
preadsorption I I
            lcr'    2.5 x
                   10-4

I -
-/

c600 tol+ .
cao to1 II..
c600 tol III..
C%00 to1 IIIa..
CEO0 K-r T6-r .
None.

;:
E!i
0
4

236
248
248
236
24
45

0 An 0.2-ml sample of a log-phase culture in LB broth (2 X lo* to 5 X IO* cells/ml) of the (strepto-
mycin-sensitive) strain to be tested was mixed with O.l-ml colicin dilutions and incubated at 37 C.
After 20 to 30 min of incubation, an 0.2-ml sample containing 200 to 600 cells of a str-r colicin-sensitive
indicator strain was added to the mixture, which was further incubated for 10 min. The mixture then
received 2.5 ml of melted soft agar with 200 Gg of streptomycin per ml and was poured on streptomycin-
agar plates. The colonies of str-r indicator strain were counted after incubation at 37 C. For colicin
El (experiments 1 and 2), the indicator strain used was LA348; for colicin K (experiment 3), the indi-
cator was AB1122-1. Thus, the results of experiments 1 and 2 are expressed as numbers of str-r survivors
after test with colicin El; those of experiment 3, after test with colicin K.

b Inverse of dilution factor from stock. When no colicin was added, experiment 1 yielded 460 str-r
colonies; experiment 2 yielded 730; experiment 3 yielded 240.

TABLE 4. Mapping of to1 VIII mutants in the cross Hfr W4032 pro- met- to1 VIII X PA309 thr- leu- arg-
                     his trp- str-r tol+

ht        Selected markers

1       thr+ leu+ str-r
     arg+ str-r
       his+ str-r
     trp+ str-r

2      his+ pro+ met+
      trp+ pro+ met+

Q Resistance to colicin El.

Per cent of unselected markers

  No. of
colonies scored
          tb?+ led     am+      his+
1:            31.3
          60      3.8 1.9
  106      50     30
  103       71     39      34


           e-3       101 VIII'

1rp+     101 VIII'


2.5     1.3
2;::  17.0 3.7
       4.9

his+  I   1ry+

to1 and bio. Reverse transductions were done with
lysates from gal+ bio+ tol+ strains and gal- bio-
to1 recipients. One isolate of each of the strains
mentioned in Table 5 was mapped in this manner.
The results indicate the order to1 - gal - bio
for all four mutations to1 ZZ, to1 ZIa, to1 ZZZ, and
to1 ZZZa. The data do not permit us to establish a
relative order among the various tol mutant sites.

Pl lysates grown of one isolate of C600 to1 II
were used to transduce gal+ to W602 to1 ZZ, to1
ZIa, and to1 ZZZ (one isolate each), and the gal+
transductants were analyzed. We found that tolt
(colicin-sensitive) recombinants were produced
in the to1 ZZZ X to1 ZZ and to1 ZZa x to1 ZZ tests,
but not in the to1 ZZ x to1 ZZ (O/160 tested) cross.

Close linkage of several to1 mutants with gal
has been reported also by Reeves (21) and by
Nomura and Witten (19a).

Dominance and complementation analysis. The
linkage with the gal region made possible the
testing of several to1 mutants in diploid condition
for dominance and complementation. The F-gal
factor present in strain W3101 (F-gao carries the
sue, gal, bio loci and the X attachment site. This
F-gal was transferred to to1 ZZ, to1 ZZa, and tol ZIZ
derivatives of W602, and gal+ colonies were se-
lected on EMB-galactose-streptomycin plates.
Most of these gal+ strains proved to be diploids
gal-/galal+ and sensitive to the colicins. Their gal-
segregants were again tolerant. Also, when these


1116                NAGEL DE ZWAIG AND LURIA             J. BACTERIOL.

   TABLE 5. Mapping oj'tot II, rol Ha, tot III, and rot IIIa muta:ions by PI transductiona

Pl grown on            Recipient         t

c600 to1 II      W6O2 gal- bio- tol+
0x0 to1 III     W6Q2 gat bio- tol+
C600 to1 IIIa     W6Q2 gat bio- tol+

c&o rol+      W&32 gal- bio- tol II
c600 tolf      W6Q2 gat bio- toi IIa
cfm tol+      W602 gal- bio- toi III

95
105
102








30
28
112

5





Per cent of unselected markers

K-r



52
58
67




K-S



56.8

bid



40
47
50




bid



70
75
55

   K-r bio+


Observed  Erpected


18     20.8
27.6    21.3
30.4    33.5


   K-s bio+


Observed  Expected

40     39.8
35.6    35
33     34.4

a The gal+ transductants from EMB plates were picked and purified by restreaking; their bio+ and
colicin resistance characteristics were then determined. Expected values were calculated from the as-
sumption of a map order rof - gal - bio and of integration of markers into the genome of gal+ trans-
ductants without interference. A representative sample of the K-sensitive recombinants were tested
against the other relevant colicins and found to be sensitive to them as well.

colicin-sensitive diploid strains were tested for
colicin sensitivity, the inhibition areas exhibited
many little colonies which proved to be mostly
gal- to1 segregants along with gal+ tol recom-
binants. The dominance of toi+ has also been
demonstrated by Nomura and Witten (19a) for
several rol mutations in the gal region.

An F-gal factor incorporating the tol II gene
was obtained from a C600 to1 ZZ (F-gal) strain by
plating on colicin K, picking colicin-tolerant
colonies, and testing them for their ability to
transfer gulf at high frequency. A strain desig-
nated C600 tol II (F-gal to1 Ii) was used as donor
of F-gal rol II to various derivatives of W602
(Table 6). It appears that there is full complemen-
tation between to1 III and tol II, but no com-
plementation between two tof II mutants (of in-
dependent origin). The mutations to1 I1 and tol
ZZu, which confer different levels of tolerance to
the same colicins, give partial complementation,
possibly of the intracistronic type. With another
mutant similar to tol Ila but somewhat more re-
sistant to the colicins, no sensitivity was found in
a partial diploid carrying F-gal to1 II.

All diploid strains shown in Table 6 segregate
out gal- cells with the colicin sensitivity pattern
of the endogenote and also gal+ to1 recombinants.
The complementation studies suggest that mu-
tations tol ZI, rol Ila, and to1 III involve at least
two different cistrons.

Kinetics of killing of partially tolerant strains
by colicins. It is known from Fredericq's (3) and
Nomura's (19) work that the immunity of coli-
cinogenic strains to colicin is not absolute since

TABLE 6. Properties of strains diploid for toi genes

Diploid

W602 gal- tolf (F-gal+ to! II)    +       -
W602 gal- to1 II (F-gal+       -      +
to1 II)
W602 gal- to! IIa (F-gal+    +       f
tol II)

W602 gal- to1 IIa (F-gal+    + -
tol II)

a See Table 9; f, partial sensitivity (light
growth at DOC concentrations of 0.5 and 1%).

it can be overcome by large concentrations of
colicins. As pointed out by Nomura (19), the
need for high colicin levels might reflect either a
reduced efficiency of colicin action at individual
sites or a requirement for cooperative effects at
many sites (as would be expected, for example, if
immunity resulted only from failure of a "spread-
ing action" needed to generalize the effects of a
local stimulus). Using the partially tolerant mu-
tants to1 ZIa and toi IIIa, we found it possible to
make a choice between the two alternatives.
Figure 1 presents survival curves of various bac-
terial strains as a function of the concentration
of colicins El or K acting on them for 10 min. It
is clear that the killing kinetics, both for sensitive
and tolerant strains, are of the one-hit type. The
differences in slope of the killing curves corre-
spond to factors of about 10 to 15 for colicin


VOL. 94, 1967        COLICIN-TOLERANT MUTANTS OF E. COLI            1117

r

I            I

A

I            I
50          100
               a

Colicin concentration

Fta. 1. Kinetics of kiliing of to1 IIa and to1 IIIa
mutants by colicins El and K. Mixtures containing 0.4
ml of bacterial ctdtures (2 X I@ to 4 X IOs cells/ml)
and 0.1 ml of various colicin dilutions were incubated
at 37 C. After IO mitt, a I:100 dilution was made in
ice-cold broth. Samples were further diluted and plated
for colony counts. (A) Colicin K; (B) coiicin El. * ,
W602; 0, W602 to1 Zfa; 0, C600 tol+; 0, C600
to1 Illa. Note: in Fig. IB the curve for strain W602
tol IIa reaches a plateau for colicin El concentrations
about 20 times higher than that corresponding to 1 kih-
ing unit for strain W602 toI+. If the plateau is reached
because of saturation of receptors, this means that there
are about 20 times as many receptors as there are mole-
cules of colicin EI in 1 killing unit.

El and at least 20 for colicin K. We conclude
that, in the partially tolerant mutants, there is a
reduced average probability of a killing response
to individual colicin molecules.

Physiological properties of tolerant mutants. A
number of growth characteristics of mutants of
types to1 II, to1 Ha, to1 III, and to1 IIIa were
indicative of some abnormalities in the stability
of cellular organization in these mutants. Further
study revealed an unusual type of fragility which
suggested a defect in some surface structure of
the cells. At the same time, the similarity of our
tof VIII mutants to the El-tolerant mutants of
Clowes (1) prompted us to reinvestigate the
sensitivity of these mutants to dyes and led us to

s
4
Ll
300

reinterpret the to1 VIII defects in a way that also
assumes a surface alteration.

Fragility of mutants to1 II and to1 III. All
to1 II and to1 III mutants grew more slowly than
did their parent strains. C600 to1 II was studied
in detail; it grew more slowly than C600 in a
complex medium at all temperatures tested, but
especially at 45 C (Fig. 2). Sucrose (0.5 M) gave
only a slight protection at 42 C. In phosphate-
glucose-ammonia minimal medium (with or
without added citrate), 0500 to1 II grew very
poorly; aggregates of partially lysed cells were
visible in the medium.

As a measure of spontaneous cell disruption,
the amount of &galactosidase was determined
in the supernatant fluid of cultures grown in a
complex medium containing 5 x 10-' M
isopropyl-fi-D-thiogalactoside (IPTG) as in-
ducer of the lac operon. A much greater propor-
tion of enzyme was released by C600 to1 II
than by C600 at all temperatures, but especially
at 45 C (Table 7).

Advantage was taken of the fact that the Iac-
strain C600 is a fi-galactosidase-positive (z+),
galactoside permease-negative (y-) strain to
differentiate tol+ from to1 mutants on agar plates
in the following way. When 5 X 10-d M IPTG
was added to EMB-lactose-agar plates, the tol
mutants were almost as dark red as la& colonies,
whereas the toti strains gave pink colonies with
or without IPTG. The lac+ phenotype is in-
terpreted as being due to the fact that the
fragility of the to1 ceil causes a release into the
surrounding medium of some galactosidase
(which in zf y- cells is induced by 5 x 10-4 M
IPTG but not by 3 X 101 M lactose). Another
difference was noted on minimal lactose-agar
with IFTG added; after 48 hr of incubation
at 37 C, C600 to1 mutants formed large, rather
mucoid colonies, whereas C600 tol+ cells formed
tiny colonies only, a difference probably due to
release of /3-galactosidase into the medium.
Neither the release of /?-galactosidase into liquid
media nor the appearance of colonies on EMB-
lactose-IPTG-agar was significantly altered by
the addition of 0.5 M sucrose. Even when plated
on EMB-agar without sugar, mutants of the
to1 II, to1 III, and to1 IIIa types from C600
were of a darker color than C600 tolt colonies,

The to1 II, to1 IIa, and to1 III mutants derived
from W602, which is lac+, could also be dis-
tinguished from W600 tol+ by their smaller
size and darker color on EMB-agar without
added sugars. The classical colicin-resistant
mutants were indistinguishable from sensitive
strains in any of these tests.

The colonial appearance of these to1 mutants


1118                NAGEL DE ZWAIG AND LURJA             J. BA~~~IOL.

2       4

2       4

hours

FIG. 2. Growth of C600 toi+ and C600 to1 II at various tempemtures. (A) Overnight cultures were inoculated
into A broth plus yeast extmct (O.l~o) and IPTG (5 X IO-' M). When an OD (550 mu) vaIue of 0.7 was reached,
the cells were collected by centrifugation, resuspended, diluted in the same medium (prewarmed at the desired
tempemtures), and incubated with shaking. Samples were withdrawn at intervals for OD readings and @galactosi-
&e assay. After 2 hr, the cultures were diluted I:5 with prewarmed medium; the 4-hr readings were corrected
accordingly. (B) Effect of sucrose on growth at 42 C. Same conditions as in A, except that pamIiei ctdtures were
incubated in media with or without 0.5 Y sucrose. The bacteria for the sucrose cultures had been pregrown in
sucrose medium. 0, C600 tolf; A, C600 toi II; o , C600 toi+, sucrose; A, C600 tol II, sucrose.

makes it possible to start experimental cultures
from single mutant colonies, thereby avoiding
accumulation of revertant types.

Dye sensitivity of tol VIII mutants. The to1
VIII mutants did not show the increased fragility
exhibited by the other to1 mutants described
above. Instead, as expected from the report by
Clowes (l), to1 VIIZ mutants proved to be hyper-
sensitive to a series of dyes (Table 8). Experi-
ments on the binding of methylene blue, acridine
orange, and acriflavine were done in collabora-
tion with R. Arditti, and revealed a much higher
rate of binding by tol VIII than by tol+ cells
(Fig. 3). The binding of methylene blue was not

eliminated but rather increased by KCN (Fig.
3A), an indication of a passive reaction.

The sensitivity of tol VIII mutants to acridine
dyes was also tested by the effect of acridine
orange on mutants carrying an F-k factor.
An F-lac factor was transferred to C600 toi+
and C600 to1 VIII, which are lac-. When the
resulting luc+ strains were treated with acridine
orange, comparable curing effects were obtained
with 2 and 5 ug/ml for C600 to1 VIIZ F-lac
and with 40 to 60 pg/ml for 0500 tolt(F-Iac).
These findings are consistent with the hypothesis
that tci VIII mutants have an increased per-
meability to dyes. Such increased permeability


VOL. 94, 1967        COLICIN-TOLERANT MUTANTS OF E. COLI            1119

of to1 VIII mutants to dyes is not an instance of a
generalized permeability for all substrates. For
example, the rate of ONPG hydrolysis by whole
cells of C600 rolt and C600 to1 VZZZ is the same.
Since in these z+ y- bacteria the rate of ONPG
hydrolysis is limited by the rate of ONPG entry,
an increased permeability should increase the rate
of hydrolysis. Also, the tol VZZZ mutants are as
resistant to actinomycin as is the parent strain.

Clowes (1) and Clowes and Moody (2) sug-
gested that the sensitivity of El-tolerant mutants
to oxidation-reduction dyes is due to inability
to reduce the dyes, possibly because of a defect
in the succinate dehydrogenase. The tol VZZZ
mutants described here grow on succinate as
sole carbon source at rates not significantly
different from those of their parent strains.
A succinate dehydrogenase mutant (P678 SW-;
7), which does not grow aerobically on succinate
as sole carbon source, proved fully sensitive to
colicin El and as resistant to methylene blue
(and to deoxycholate; see below) as its sue+
parent strain.

A study of reduction of triphenyl tetrazolium
chloride (TTC) revealed further aspects of the
dye sensitivity of to1 VZZZ mutants. Reduction
of TTC by intact cells leads to a precipitation of
the red forrnazan, revealed by the color of a cell
suspension or microscopically by the appearance
of large red intracellular granules (11). Various
concentrations of TTC were added to growing
aerated cultures of C600 tot+ and C600 to1 VIII,
aeration was stopped, and incubation was con-
tinued at 37 C for 15 to 30 min. At 0.005%
TTC, to1 VZZZ cells reduced the dye, but tol+
cells did not; at 0.05% TX, both tof+ and tof
VIII gave a positive red reaction; at 0.1% TTC,

TABLE 7. Release of a-galactosidase during growth
  of C600 toi+ and C&W to1 II at different
            temperatureP

Time

hr

0

Strain

c600 toI+
c600 tot II

aa0 101+
cbal 101 II

c600 rof+
a00 rol II

-





-.









-

Free &galactosidase (% of total)

30C     31 c
--

0.28

1.1

0.39   0.08
3.1 1.4

0.06    0.05
0.6 1.5

4s c

0.19
5.6

0.4
16.1

0 The conditions of growth correspond to those
described in Fig. 1.

TABLE 8. Sensitivity of to1 VIZI mutants to dyep

Bacterial strains

c600 Id', w4032 to/+. .......
c600 Id VIII ................
W4032 ml VIII. ..............

C600 ml+, W4032 ml+. . . . .
C600 ml VIII, W4032 rol VIII..

C600 ml+, W4032 ml+. .
0500 to1 VIII, W4032 id VIII.

C600 d+. ..................
c600 ml VIII.. ..............

C600 toP. .................
c600 ml VIII.. ..............

-

-

100
-

+



-

Metbylene blue
(rg/ml in A broth)

25 10 5 1 0
-----

+ + + + +
- - - f +
- -  - + +

Acridine orange
L&ml in A broth)

2s    10    5    1
-~--

- + +
- - -  :


Eosin Y (mg/ml in A broth)

Triphenyl tetrarolium chloride
(mg/ml in Penassay Broth)

5   1  0.5  0.1  0.05  0
-----_

-+++++
----*+

a A 1: 100 dilution of an overnight broth culture of the
strain war made in 2 ml of the medium indicated and incubated
at 37 C with shaking. Full growth (+). absence of visible
growth (-). of Ii& growth (a) WBI recorded after 4 hr of
incubation.

the tol+ cells reduced the dye more effectively
than did the tol VIII mutants; and at 0.5 or l%,
neither cell type could reduce TTC. We interpret
these results as indicating that the mutant, being
more permeable to TTC, can give a greater TTC
reduction at lower concentration but is more
effectively inhibited by concentrations that do
not inhibit the tof+ cells; the latter are themselves
subject to inhibition by still higher levels of the
dye.

In conclusion, all behavior of to1 VZZZ mutants
towards dyes appears to be better explainable
by an enhanced penetrability by the dyes rather
than by any specific disturbances in metabolic
processes.


1120

NAGEL DE ZWAIG AND LURIA             J. BACTERIOL.

0.8

  0.6
s
g 0.4

I        I       I
IO 20 30

minutes

0.8

0.6
s:
Q-
8 0.4,

8

1

L
      minutes

FIG. 3. Binding of methylene blue and acridine orange to bacterial cells. (A) CuItures of C600 toi+ and C600
to1 VIII were grown in minimal medium plus 0.40/, glucose and 0.25y0 Casamino Acids to approximately 5 X IO'
cells per ml. Methylene blue was added at a final concentration of IO @g/ml and the cultures were aerated at
room temperature. At intervals, 2-m/ samples were jiltered through Millipore membranes (0.45-p pore diameter)
on jilter pads. The membranes were transferred to I ml of buffer, the bacteria were resuspended, and 0.25 ml of
a 25yo solution of sodium dodecyi &fate was added. The sampIes were incubated at 45 C until fysis was complete
and then centrifuged to remove debris. The supernatant ~7uids were read in a Zeiss spectrophotometer at 660 rnp.
KCN (Iv M), when present, was added 3 min before methylene blue. (B) Same procedure, with acridine orange
(100 gx/rnI). Readings were done at 450 mp. 0. C600 toP; o , C600 tol+ plus KCN; A, C600 to1 VIII; A,

&k ;dr ViII plus K-CN.

Itikawa (8) reported that a group of El-
tolerant mutants similar to group VIII has in-
creased sensitivity to 2,4-dinitrophenol and to
chloramphenicol. This was found to be true
also of the group VIII mutants described here.

A class of E. coli K-12 derivatives hypersensi-
tive to basic dyes, apparently because of an
enhanced uptake, has been described by Naka-
mura (16, 17) and Sugino (25). The correspond-
ing gene maps near the lac region. These mutants
are also more readily curable of an F factor by
acridine orange.

Strain C600 derivatives carrying a Co1 El
factor (El-K30) and therefore immune to the
colicin El do not show hypersensitivity to
methylene blue. The to1 II and to1 III mutants
do not show increased sensitivity to dyes, but are
slightly more sensitive to penicillin.

Deoxycholate and EDTA sensitivity. When
sodium deoxycholate (DOC), a mild surface-
active substance, was added to growth media,
all to1 mutants proved to have heightened sensi-
tivity (Table 9). This was especially true of
to1 VZIZ mutants, but all other tol mutants were

more than 10 times more sensitive than to/+
cells. This effect of DOC was not significantly
counteracted by addition of 0.5 M sucrose. Mutant
to1 VIII was very effectively killed in 30 min
by treatment with 0.5% DOC (survival <10-j)
under nongrowing conditions, that is, in the
absence of a carbon source and in the presence of
chloramphenicol, with or without 0.5 M sucrose;
under these conditions the to1 II mutant was
not more effectively killed than tof+.

The growth of tol II, to1 Ha, and to1 III mutants
was completely inhibited by addition of 1O-3
M ethylenediaminetetraacetate (EDTA) to LB
medium, whereas top or tol VIII mutants can
grow in the presence of 5 X 10-s M EDTA
(Table 9). Attempts to counteract EDTA effects
with various concentrations of divalent cations
have given no clue to any specificity of action.
On the other hand, strains C600 (ColEl-K30) and
C600 (ColE2-P9), and the resistant (nonad-
sorbing) mutants, did not show this increased
sensitivity to DOC or EDTA.

The actions of DOC and EDTA on bacterial
surface layers are not well enough understood to


VOL. 94, 1967

COLICIN-TOLERANT MUTANTS OF E. COLI

TABLE 9. Sensitivity of to1 mutants to DOC and EDTAO

1121

Bacterial strains

C&X tol+; W4032 to/+; W602 tof+
C%OO to1 II, to1 III, to1 Illa; W4032
to1 II; W6Q2 to1 II, to1 110, to1 III
C600 to1 VIII; W4032 to1 VIII

a00 to/+; w602 tol+
c600 to1 II, to1 III, to1 Illa; w602
to1 II, tollla, to1 III
c600 to1 VIII

0 Procedure as in Table 8.

+
-

DOC (% in LB broth)

0.5  1   0.2  )   0.1  1   0.05  1   0.01  I   0

+     +



                    +     +

  EDTA (molar concn in LB broth)

5 x 1P  1 2.5x10-J 1 10-l

                          5 x 10-r       0


+       +       +              +
-         -         -   T-       +

+       +       +       +       +

permit specific interpretations, but the findings
are in keeping with the conclusion that the to1
mutants are altered in some surface components.
DOC can presumably disrupt bonds between
phospholipids and other membrane components.
Increased sensitivity to DOC is found in Sal-
monella typhimurium mutants lacking the heptose
as well as almost all other sugar components of
the lipopolysaccharide core (M. J. Osborn and
B. A. D. Stocker, personal communications),
possibly because of a higher accessibility of
lipoproteins to the reagent. The EDTA effect
may bespeak the well-known (but poorly under-
stood) role of divalent cations in membrane
integrity and a greater sensitivity of some to1
mutants to removal of such ions or a greater
availability of them to the chelating agent.

Preliminary chemical studies on to1 mutants.
Through the courtesy of P. W. Robbins, some
comparative analytical data were obtained on
rol+ and rol bacteria. The major phospholipid
components of C600 tol II and W4032 to1 II
were analyzed by gas chromatography, and no
differences were found from the analyses of the
parent tolf strain. A set of to1 II to to1 VIII
mutants of C600 was used to extract the lipo-
polysaccharide fraction, which after hydrolysis
was tested for heptose. No differences were
found between the mutants and C600 tol+.

DISCUSSION

The attachment of a colicin to its cell-wall
receptor triggers a series of events that affect a
specific biochemical target, leading to cell
death. On the one hand, colicins with apparenty
common receptors, such as El, E2, and E3,
have different modes of action, hence different

targets. On the other hand, some colicins with
different receptors, such as El and K, apparently
act on the same target.

Mutations to tolerance reduce the probability
of successful action of a colicin on its specific
target. Again, the specificity of the to1 muta-
tions cuts across the patterns of specificity of
cell wall receptors and biochemical targets.
to1 VIII mutants are insensitive to El but not to
the other E colicins nor to colicin K; the other to1
mutants are insensitive to many colicins with
diverse receptors and modes of actions. It seems
reasonable to conclude that the to1 mutations
reveal another stage in the process of colicin
action, presumably one that involves cellular
components which mediate between the receptor-
bound colicin molecules and the enzymatic
constituents of the biochemical targets. The most
likely candidates for the roles of mediators are
components of the cytoplasmic membrane.

The present study of tolerant mutants has
revealed an association between tolerance and
alterations of certain cellular properties related
to membrane functions. Thus, the to1 II and
to1 III groups of mutants exhibit a definite
cellular fragility and an increased sensitivity to
DOC and to EDTA. The to1 VII1 mutants, be-
sides being extremely sensitive to DOC, are
abnormally permeable to a series of dyes. These
observations are readily understood by postu-
lating that the membrane components which
mediate between the colicins and their ultimate
biochemical targets also play roles in determining
other membrane properties: stability, permea-
bility, and sensitivity to reagents that affect the
interactions among membrane components.

Our findings and their interpretation are in


1122              NAGEL DE ZWAIG AND LURIA            J. BACTERIOL.

line with the hypothesis first advanced by
Nomura (18) that colicins inhibit cellular
functions by interacting with specific constituents
of the cytoplasmic membrane. The existence of
mutants with temperature-dependent tolerance
(19u) suggests that the relevant membrane
components may be proteins.

An alternative explanation, which cannot be
excluded at the present time, is that the mutations
to tolerance alter the bacterial envelope in such a
way as to activate mechanisms that destroy
specific sets of colicins.

The recessive character of the to1 mutations
(at least those that map near the gal region)
can be interpreted in a variety of ways. There
might be a preferential incorporation of the wild-
type product, or the mutant to1 genes might
make no utilizable products at all (in which
case such products would have to be dispensable
cell constituents), or a mosaic of tol+ and tol
gene products might be adequate for normal
functions.

It is worth noting that tolerance to colicins
differs from the immunity generated by colicino-
genie factors. Immunity is epistatic to sensitivity,
whereas tolerance is recessive. The specificity
of immunity contrasts with the range of tolerance
conferred by some of the to1 mutations. Also,
colicinogenic bacteria do not exhibit the sensi-
tivities to methylene blue or DOC found in
tolerant mutants.

Yet, it is reasonable to think that tolerance
and immunity to colicins alter some cellular
components that play specific roles in membrane
organization. Some recent findings are relevant
in this connection. Puig et al. (20; personal
communication) described in E. coli, as well as
in other bacteria, pleiotropic mutants that af-
fect the function of several enzymes involved
in anaerobic catabolism: formic hydrogenlyase,
nitrate reductase, as well as thiosulfate and
tetrathionate reductases. In E. cofi some of
these mutations map near the gal region, prob-
ably between gal and bio, and others map near
the mtl ( mannitol) region. The mutations appear
to affect not the production of the relevant en-
zymes, but their assembly in functional form
into a particulate element, which can be recon-
stituted by in vitro complementation between
extracts of mutant strains. This situation sug-
gests the existence of specific assembling sub-
stances in the cytoplasmic membrane of bac-
terial cells and recalls the apparent role of
structural protein in the functional organization
of mitochondria (5, 15). (A set of rol mutants
from the present work was tested for ability to
reduce nitrates and for gas production from
glucose. All behaved like their parent strains.)

It is tempting to believe that the bacteria1
membrane contains a number of such substances,
which act in the coordination of complex en-
zymatic processes such as those affected by vari-
ous colicins and whose mutations may confer
specific tolerance. The identification of these
substances, their functions, their interactions
among themselves and with enzymes and other
components of the membrane should prove a
difficult but rewarding task.

ACKNOWLEDGMENCS

This investigation was supported by grant
GB-5304X from the National Science Foundation and
by Public Health Service grant AI43038 from the
National Institute of Allergy and Infectious Diseases.

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