8
MUTANTS OF ESCHERICHIA COLI AFFECTED IN”INDIJCER EXCLUSION”
Eugene B. Ackerman
INTRODUCTION
The uptake of carbohydrates by Escherichia coli is affected by two
processes: active transport, in which the substrate appears inside the cell
chemic1ly unchanged; and by the PEP dependent phosphotransferase system, in
which the carbohydrate is phosphophorylated as it enters the cell. Although
the mechanisms of active transport are entirely different from those of the
PT—system, it is known that mutants in the PT—system may show reduced rates of
growth on sugars taken up by active transport. Furthermore, it is known that
glucose and non—catabolizable analogs of glucose may inhibit the induction of
sugars which are taken into the cell by active transport; this phenomenon is
known as “inducer exclusion”.
The present work attempts to analyze inducer exclusion on a genetic level
by directly generating mutants in inducer exclusion and then locating the
locus of the mutation on the chromosome by phage transduction.
METHODS
E.coli K12 strain HK743 was incubated overnight at 41°C on the.foiIowing
medium:
--
0.2 ml 10 mM glucose -and 10 ml Basal Medium containing 50 mM Tris, pH
O, half strength
2
, 0.1 mM FeSO,•7H
•311
4
P0
0
H 2
2
7.4,190 rnM..NHk Cl, 0.33mM K
) and 0.1 ml tHALT.
H
2
CaC1
0
2
M
and
0.02
M
KC1,
,
0.02
O
2
MgSO7H
ASW, (0.4 M
day on a 5 mM lactose
next
the
plating
for
680
OD
This medium permitted a high
a well of (0.1 M) 3—
then
the
plate
tHalt plate. The bacteria were •spread on
a zone of inhibi—
next
day
The
deoxy-3-fluoroglucose was made in the center.
of inhibi
zone
the
tion occurred nd the following day mutants arose within
grow on
to
tion. These mutants were screened for their continued ability
1
methyl-a-D— glucoside. The
’C]
C] glucose and 11
4
glucose and to take up [‘
picked mutants were grown overnighton the glucose medium previously
described. The next day 2.0 ml of cells were spun down for 5 win at 10,000
rpm then were resuspended in a solution containing 10 ml basal medium, 0.15 ml
tHALT, 0.1 ml 0.5 M glucose, 0.1 ml 0.25 M lactose, and 50 p1 [C]-lactose
(6tci/ml). Growth curves and [lLCJlactose uptake was noted for both the
parent and the mutant by shaking the cell suspension at 30° then filtering 20
p1 of cell suspension for radioactive assay. This uptake study was repeated
using 0.1 ml of 0.5 ml N-acetyl glucosamine.
Examination of the lac operon was conducted when glucose grown cells were
further grown in media containing 2.5 mM lactose and either 5 niM glucose of 5
mM N-acetyl glucosamine..
Growth of phage:
1)
.
2
3.5 ml of soft (.75%) nutrient agar.at 42°C. Add 0.125 ml 50mM CaC1
Add 0.1 ml of mutant culture. Pore over nutrient agar plate and set.
2)
Put 0.1 ml of phage pius 0.3 ml nutrient broth on plate.
but control area.
Spread over all
.
.
9
Incubate 4—6 hrs at 37°C.
3)
1)
5)-
-
with 2.5 ml 0.9% NAC1 and
Scrape soft agar off into sterile tube and wash
add to soft aqar then centrifuge.
s of chloroform, leave at
Decant supernatant into sterile tube, add 2 drop
room temperature for 15 mm.
Transduction
1)
ent.,
Subculture H1C761 (1 10) for 60—75 nun in nutri
2)
2l at 37°C for 30 mm
Incubate 0.1 ml phage nd 0.1 ml 50 mM CaC
chloroform.
3)
Add HK761 (4 ml).
4)
Incubate for 20 mm
5)
plates.
Plate 0.05, O2, and 0.2mi onto appropriate
s.
plate
of FK761 and phage on separate
to remove
at 37°C.
Control is 0.2 ml
RESULTS AND DISCUSSION
mutants in inducer
The first objective was to directly generate and screening out ptsG
nt
pare
exclusion. By using. a ptsM strain as the
ose we obtained mutants in inducer
mutants which could rio longer take up gluc
Figures 1 and 2 indicates that
exclusion or some other regulatory property. the parents functioned while the
for
it is inducer exclusion that is affected,
.
sion
exclu
cer
indu
in
tion
mutant did not func
operon while the parent strain
Our mutants readily induced the lactose
mutant also lost “catabolite
failed to do so. Figure 3 shows that the
etyl glucosarnine was used in prefer
inhibition” since neither glucose nor N—ac
dependent phosphotransferase system
ence to other sugars taken up via the PEP—
such as fructose.
CONCLUSIONS
tion which affects the
These results show that we have a novel muta uptake and utilization of
the
g
rolin
regulatory role of the PT-system in cont
or not.
sugars, whether taken up by the PT-system
o.
10
15O
CU
mutant
0)
Cu
Cl)
0
E
C
Parent
0
mg/mi
Figure 1.
Mutant and parenL CHIC 743) grown on glucose
overnight, then suspended in 5 inN glucose
‘I
11
b
e150
°
10
G)
Cl)
mutant
0
;50
0
E
Pa rent
:0
0
0.2
• mg/mi
Figure 2.
Mutant and parent (K 743) grown on glucose overnight
then suspended in 5 mM N—acetyl glucosautine
—4
.
12
•
E
‘1
800
fructose
401
glucose
Cl)
0
E
mg/mi
Figure 3.
Mutant of ilK 743 shows “catabolite inhibition
for glucose does not supress the uptake of
other PT—sugars