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THESIS

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3 1293 10589

This is to certify that the

thesis entitled

Peta-D-Galactosidase of Aberrant Coliform Eacteria

presented by

Antone K . Fontes

has been accepted towards fulfillment
of the requirements for

Ph. a. degree in_!’i_.c_r. o__biology

WWW

Major plofessor

Date Tfay 16’ 1957

 

0-169

 

 

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MSU

LIBRARIES

 

 

RETURNING MATERIALS:
Place in book drop to
remove this checkout from
your record. [Lugg will
be charged if book is
returned after the date
stamped below.

 

 

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A..Dfla Tuq‘fi.-‘u.l. C .JLJLF‘ .13; :3 J .LJLKI‘K

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by

Antone K. Fontes

An ABbTRACT

Submitted to the 3chool for Advaeced Greidate tudies of

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lichigen State University of AIpiCulture and

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Applted Science in certial fulfillment of

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the rquiremeuts for the d gree of

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She presence of j—D-gelpctosidase in several aoerrent
coliform OFTQHLEflS was deno.etreted. These organiens,

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recivered from lakes and rivers in Selective end

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differential media used in roitlne water enelysi
to utilize lactose in the ty-icel dsnner.
Cell-free extracts creger i from one of tneoe atypical
organisms was compared to like extracts from a tyfiicsl
coliforn organism. Sinple kinetic stedies of the enzymes,
in both extracts, indiceted that tkey are very much alike
in res ect to affinity for the synthetic suostrate Chi}.
Studies, on intact cell susyeuelons and cell-free
extracts of tnese aberrant organisms, snowed that the ac-
tivity of the enzyme was much lower than from like pre-

parations of typical ccliform organl;ms.

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A;tone K. Fontes

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dvancod “radiate jtldies of

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Submitted to the School

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Lictigen Stete University of Agriculture ind

Asfilied Science in eartial fulfillment 0:

tie requirements for the deeree of

4

Dejsrtlent of licroolology 3nd idolic health

1957

ASK}. *‘Cil'JLiL'Eltl-Llir. T 3

 

The 84thOP wishes to eXpress sincere thanks and

aoorecistion to Dr. L.

and guidarce.

He wishes to thank

the pOrt they nlsyed in

rloed in this thesis:

ONE} used in this stod]

Dr. 3. A.

in this

. , y» . . he '- . -. fi
origami 3.1; :3) L13= C1
The

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who gave no so mich in

L. hallmann for his sole

Broitmsn for their

author is dee;ly indebted to his

order that this

advice

the following lrdivldisls for

heloing to coniict the work des—

Dr. J. C. Séeck who orepnred the

Lula. Io 7L0 Dghl;?lfi: qfij

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84.1ch

help in isolatini some of the

work.

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Iilltr‘OdJCt10n o o o o o o o o o o o o o o o o o o o 2

EXCGPlHfiSI-Ltal Fina RSSUltS o o o o o o o o o o o o o 16

DiJCdSSj—Ofl 0 O O O O O O O O I O O O O O O O O O O 59

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)leril r1 Py O O . O O O O O O O O O O O O O O O O O O 63

:(‘sefepelrices C O O O O O O O O O O O O C O O O O O O 64

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jBCEJSJ of the ingor ence as in ic tors of polldtion in

‘f " "’ 'P “ . ’ " p '. ' fl ' ' {:1 " Tl 'E' ’ "1‘ . ~ a" ‘ 1 ‘ (‘\t . , fl
tug examination oi water, tool and ignites, the oscteria o:

, , - ‘. — ‘ r' 1x v F\ L‘ ‘ v . _ A - JI- -\ I 1“
the COle-ISPOQC-CJ red: have seen sidely stilloi. in:
< ‘ e A “ 2 n ' .-, -c~ ‘ age-e ~.— .~, . .. 1
stdnusrd' for the Cissslllcstlon oi thee: oibehleh- he s

,s. - «:1 7—. . , m+ ,e
s PC.11PU¢o nor the most pert

‘..~ vs .‘\ . 4." I] " y" ‘ hf‘ “. .‘ ‘,“‘ 3‘ ' 7‘ 4' 1’ "e .' .‘ ,‘.'. "1-" Ir ~ ‘-
tl‘e D>.€C.LLJ_C.tJ-O$Jj lo: 21:} eiih1-3i-.‘/~ t'll.eU--CP 314 Oféfluibm 09101138

to tje coliford groin is Quite adeodste. however, it has

these organises or“ he slow lastose fernsntin; tyge, tie non
. _. ,, -. - .,. ., ,_ _ .3 .y A . . . .. . I t ,_ . 9 H
lactose fermCutin type enl the pagillse forming or ‘niteslle

type.
Stuart et al (1) called these forms of coliform oscteris

I ‘ ~10; ll 4 _-.-,,; 1,-. ~.,._ A -. « ,,.:,...
'sberre t COllfQCm dnl CliSSlfltJ then into the {OllOnlhs

l) llcro—serogenic coliforms

2) fSCuiOmiCPO-36POLBLLC collforls

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(Z) ragill e-for in; coliforns
(4) ALBéPOQdLlC colifo ms

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3) non—loctoeo fernehtlué colliorne

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it in oi extrete interest to MancPS in this Lflaoc o:

3 .. ' ’ ~,h .. r‘ - o r + e - r ." ‘ “q '. ‘ ‘ . ” ‘y‘ '\ h l 1, ~ ' r‘ . .’\ .
Oflélnatel. in lgooraeory dtdiioo on the colliorh eectcfla
4-} A, 7-1", ° < ‘ u 1‘,‘ “ " r ,_;. "1 :3 t‘ww' _. '
LI‘-~J- C l.) LLJLLA. CV;AVLJC\J ..’.A.B OJ
n l! 5'. or wan-r; in n 1;: tr. ’5: n "o. (urn v'>‘~ . :_~ ,-.,,..q, ' r ~ fix
b0 .LiOL .4 'J.blvU‘»/F.L:L ’Alv Luv ybi.l.l3-.1i.oo l[.4ov Ulshlllau- :- 4,14.

nave been attenuated in the source free which they were re-

covered 3rd ssoseouentl; fail to confirm when glsced in VEFlOdS

ted

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If A .' ‘/“' h “ ‘ 4' .‘z. 1'“ -«‘* "s 3“ , i r " a ';v1 1 f In -
LJUVLLLB (C) £3413. Uff‘lt “1’5. oef‘Ceiiu 3:. 3011;135de 1.33.1.

woter supply were soerrdnt COlifOPlS.

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LOebal (4) P393Pt31 that sole coliforio, trained to grow

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sewage o cterie to flu; colilorw or lulzmo w.icn do not
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fUPmcut loctooe hue“ CAlth1tQA under leoorauory co- lttone.
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while othors may bresxdoWn lecuo:e out fail to istsoolize

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-ir ability to f-rment lectoe= cr inhibited their

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Dulaney and Slith (7) sh wed that culture tubes which were
negq ive for coliforh in 4Q hOirs contained coliform o: tne

slow lactose ferteotin; vsriet<.

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reviewed many Still») in welsh aoerrant

‘1‘- , . A ,, V‘ - '. > v 1 -‘ r~ , 4- “ ‘, - v —~, .1 2 ‘ ’-
the a l yoeltlve evidence that Juadrd;bgic collfort

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oacteril are assoc; tel Wit; 3.3tc-03utefic lisodrc ices.

" , ...l V ', ,: .. 'Q .- A, : .-\ .‘ | . - y~ 3 4- ~-l,~ 1. \ . \ - -\ . 2.—
;srr Sud Prelllan er (7) Oc” netrateu ch‘h fecal ssh lee con-

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Ldloney sh} Lic el on (l0) isolste- slow lactope ffifub.tl44

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cs Oi a group oi children with soldemic

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coliforms in the f

diarrhea. At the or esent time nun-e rous investi;ators are

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concerned with tie relationshii Oi infantile diarrhea and
certain aoerrant forms of E. coli.

Massini (ll) was the first to describe a strain of §;*cgli
which existed in two forms. One of which is able to ferment
lactose and the other wnich appears to have lost this cagacity.

The non lac toss fernenter appeared to regair its lactose
ferhenting properties after several succiltures ir the
presence of lacto 5e . Kriebel (12) isolated non lactose ferhen—

ters from human patients and was able to adapt tiem to fernent

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after aging several days in the p

lactose. Uadsworth an; Hitchner (13) found that certair aberrant

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lactose fermenting coliform of the interne‘iate :roup coal; oe,

converted to rapid fermenters after aging in lactose nutrient
broth for four days. Lewis (14) working with the classical

s. coli mutabile, resorted that this strain was actially a
nixtare of two types, a non fermenter and a le ctooe fbfdvut -r,
regardless of t.e growth medium used. He stated that the non
lactose fermenting variant contained about one lactose

ferule ntiii variant in 103 cells. Growth of this v riant in

the gresence of lactose enhances the aspeqrances of the fer-

U)

ment'ng variants end b" subsequent olection daring subculture

become predm inately lactose fermenting. An ineeic-tin

0)

study with aberrant coliforms was r; orted by Leer (15) in

which he founi that the HOfl-fCi$;Atlfl* variant of this strain

vhen dried or t sated with tolue 119 could ierme nt lactose at

the same rate as tne fernentative variant. beers su:;ested
that both types contained the same amount of enzyme but the

lactose was unable to penetrate the cell wall of the non—

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hV ot: esi zed that suocultivation in the presence of lactose
mi ht cause an increased pern.eao oility of the cell, resulting
in fermentation. Unfortunately Deere was unable to supcort
his theory with eXperi maental evidence.

Lederberg (16) was the first to report on he deter-

mination of the "lactose" of i. coli by a meti od other than

 

by growth or res.iratory studies. In this now clas ic work

(.0

he showed that the lactose enzyme was a n—L
which attacks the oeta linaage of the lactose molecule. oy
utilizing a synthetic 3-D galactoside composed of ;aiacto;y-
ranoside linked to O-nitroyhenol, he vas able to determine by
simple rnOLomotric tec nice, the splitting of this substrate
by the enzyme 3—D $9.1? cto iiase

Cohn and honed (17) reported that a lactose hydrolyzing
enzyme could be extracted from E. coli in a cell free state.
In all appearances the enzyme manifested similar grocertios

to Lederberg's 3—D galactosidase. Huey and Lardy (18)

(I)
p.

establish‘ the Kinetics of a :urified 5-D galactosidase

extracted from s. coli K 12.

Rahn (19) stated that the adaption of bacteria in the
utilization of certain nutrients reores nted the de novo
formation of an enzyme as a sgecific response to the presence
of its substrate. This hypothesis has been challen3ed by
Gale (20) and others, who felt that the presence of a

specific substrate was not a :r recu 1 its for t_e formation

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of an adaoti ve enzyme. The studies of Xotnan and o oL
(21) clearly show conclusive evidénce thet under certain
conditions §;_coli does not contain apc recia cle a cunts of
tue precursor of inducio e 3-3alactosida3e. a si ilar inves—
tigation was studied earlier by Lonod and Cohn (22) who
stated that the crocegs of induced enzyme for;m ti'on is
essentially one of a is novo synthesis.

Ihe purpose of this study was to determine if the aberrant
coliform bacteria, which are frequently found in rivers and

-‘

lahes, are sitilar to tyrical co ii forms with rescect to their

.

enzy e. An attengt will be made to use

:3

the teanic of Lcderoer: (6) for measurin3 the 5—D 3aiactosidase

in these organisms.

At the present time there is much need for newer research

-'

mettods aimed at linking he various 0; 3an isms founv in water.
The classic methods by wLLich this grou: of oacteria has been

classified are suo oct to great error. It has been shown, in

media and reazents used for selection

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and diff er “ti tion of o-a cteria can be C uite toxic. Thus,

biochemical reactions which are excected to OCCdr in odlture

m dis frequently fail to tags olace. Ens worn oy Lailnann

(1)

and Laroy (é) attest to this.
J.

In recent years, stddies on the ontigenic relationship

of the ‘acteria from rivers .nl lanes fave contributed much

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or an' Other newer research

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tools edch as oacteriopna3e ty in and in ra—red sgectro—
photometry are now being used for siniiar purpo es.

It is t}-

( I)

hope of the author that this study may add to

*4)

the ex;andin3 Knowledge 0 the relationship of the many

bacteria found in water.

 

The aberrant stains of coliforn bacteria used in this
study were isolated fro; river an; Leke water. They were

lactose broth, leuryl

recovered from the following media

1

tryntose brotn, eosin methylene blue 9 er, brilliant green

bile oroth an? Lallmsnn's enteric nedium. ike aberrant

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enes group of bacteria. Any culture wnicn apneared to
be a mutabile tyye when tested on eosin tethylsne clue ass?
was not included in this grouo. Bi ht ty ically aberrant
lactose fermenting organisms were used for this study.

The cultures were maintained on dextrose ajar slsnts

at refrijeration ten erature (403). Subcultures on fresh

‘-I fi'1, "" 1 " ‘.' " f“ ' .’~ 1 1 a“ ‘ ' ' r‘ a V." ‘
a3vr slantr were node hodtnly. erotn Cultires were 3r0wn on

In addition to these aberrant CDllfQPLfi, the followihg

. ’ r1 . “1 " , . .q A ’3 ...., A . T '1’ Y. 1 t ' f fi fi ' . "' o - .- _
0P37Liars were uecu: L. Coli.nn (a dutHOlld utilfl), s. coli nL

 

(a stable lactose ferzenting), &._c;_i 3 7663, and E. cg_i MM

(an anaerogenic colifori). The following typical coliforns

were u.:d: E._coli K 12, g. coli 3 and E. coli 5 766.

 

 

 

Med a:
Tie ells for the ass

KAncitrate-EH O

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Carbohydrate

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ire carbohydrate was

salts and the two were com

f-qw T' D 4.1 r~ y\' r. ‘

ine on oi ens medium wrs 6
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ii; seal; doLl for re

lactose fern-

Laboratories

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ay of J—u gaiactosidase were 3rown

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following formula

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nine: asettically after cooling.

8 - 7.0 after 3 e”

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isolation of tne slow

of Lifco

ucts

11

Growth and Harvest of cells:

Unless otherwise stated the ;rocedur= for 3rowin3 and

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harv:3tinv the cells was as follows: one ml of an 18 hour

glucose grown culture was seeded into 100 ml of lactose
synthetic medium in 500 ml erlenneyer flasns. The flasks

.were azitated in a reciprocal shaLin3 a;paratus at approxi—

' LI

mztely 80 strokes ter minute at 37 C for 18 hours. At the

from the s axer and the sustensions poured into 50 ml cen—
trifu3e tuoes. The cells were sedimented in an an3le head

centrifuge at 3“SO RPM for 15 minutes. The res ltin

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d. Twenty-five ml of sterile physio-

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supernatant was decent

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logical saline were poured into the centrifu3e tub s, washing
down the walls of the tuoes carefully so as not to disturb

the pack;d cells. The saline was then Quicily decanted.

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y.)
H

. A \ .A ' A ~ J- ' y -, r» .— I‘ v.. ,-
liu thh CBLuPlfu351 find

«a - “‘2“ 4- T‘I A ‘ x «’1 q v 1" ~
PCJJS end—d twice. ihe fi oi cell r es was made us Nltn on
I
' ‘ . ' ‘ 1‘ ‘L ‘ ‘ ‘ y ‘ fl V‘ 1“ C. . ‘,' ." 7n)“ “ 4 "‘ 1“ :I "‘~ " r W
approXiiztely eguai VOl“'u o; saline. inese restin3 cells

were either used immediately or were stored in a ti3htly

stopeered t be in the freezing coigsrtnent of the refri3er-

S;

ator for use at a later date. several eXperiment5vdesi3ned
to test the stability of the 5—D galactosiSase under these

conditions of storage were made.

12

’ ‘3
(,3
c+
u')
n
‘ J
9-)
Lu
O
U)
3"“
i...)
('1
5.-
L
H
r
O
U)
.0

:reterst'ons
1‘1

inr013 hout this worn, where intect cell sdspensions

. , . 1.5 . Vr' - . 3 f“, - ~ v ' ‘- a “—2 o“ '- ‘. I 3‘ — I ‘ ‘fl‘ r1 -"‘ 'A
WQPS 4531, n8 susoensions WBPS SuduuuPllch With restoct

resting cells were dildtei with

l’<
*3
m
t?)
J)
W
m
L)
m
L1
H)
O
H
H
0
d
c+
3
(I)

rive 9n opti el density reading of o.c3 at 420 mu

in a V and L Q‘ectronic 20. A nitrogen deterline -tios of e

W
(D
U
'"S
(I)
(D
(.3
A-
C+
,1
c+
H‘
<1
(D
03
L
m
(J
D
OJ
H
0
II)
0
W
O
I)
i"
H
U)
x \
’ 'J
(
L)
'J
u
('.
‘JJ
F“
D
c+
'V‘
H
U)
:3
i ')
E}
b-
(u)
5

was made oy micro- -Kje ldehl method. One ml of re sts: d.rd

cell SJs: ension re

if}

rese :ted 0.78 mg cell nitro.en

A ""irtis tissse hOLOLQHlZEF was used for disruoting
the oecterial cells. A flat bOttOL tuoe, 10 c; in ~ength
and 2.8 CA in diameter was used as the grinding is oer.
onsgoer was glsced into a oesker WLiCh contained

. ,~A ' "'1 2 , ..-.- . . A.” '- M ‘. A
CPJyueu ice. ine ice cololcte y sirrounced tie griniiur

(I)
*5

~ .— 3 - A , 1 at.» J- “A -- 1 1 ' . —' . . .x ,
cneluer in order to gees tne cells at seltin; iCe tel
' ‘ I. ‘5 f‘ 1 .‘ '.' f- . ‘ I." ‘.! ' , z _' . 1'\ ~ ‘ ~ -" " ‘— r“ ‘ q 1 f
tdre. fine cells were COiUincd Hit; superbrite ,* giess

bonds (40/50 :esh) in 9 ratio of C e;

\

0
'3
c+

J
O
(U
r—J
H
C;
C+
C
L t

')
5
d.

filess seeds. The mixture wss claced into the grin ing

chamber and 1omoxenizeF for l5 minutes at aoordximetely

v '- A.

r n

“i“..dfccturrd ’oy 2-;1m'nse.:olis fulfilil; and 2;:ndicotdrin oo :;;.9.nJ .

Follo~irs dizi“te

‘5
(i
F1
0
D
O
i- 0)
ff-
('1)
O
(T)
f -
H
U}
C+
1 ‘5‘
(I)
H.
r'\
C1“
(4
U
(D

\D

O
m

rt

*3

P.
l

was made do to 25 ml with saline, transferred to

fdie tdoe, 94d cl ced in a refrigerator for 30 n-1nutes. The

,- ». a.” e.+ "‘cgfi : town fire 5 ~ 2 ~‘— n , e
.T.lXt\lre M30 Ovuurifxvjva fit L'u‘ux) Sift; 1.0L“ J 1n.Ll-JtG"3 1-; 3.1....
.,. .‘_ 4 F . ' ”- . . A . ‘. w r. -q ‘, . ,- . . ,5 - r V, ,. .-~ 3, ,T - fl 9,.
enéle centriiu e. ihe sdcernatant was decanted and recen—
,—;, , ”.5 q a m . V. , n - A. . .:; n' o m ‘1‘. .. 2 _ 7.
triiu ed to (CMOVC any retaininJ \cDrls. ine PJSJltiL’

,"\f'\

suoe rn atsnt was decanted and centrifi;ed et SC, Oou x

g for

‘A - 4 ~’ '» IN "\ 'I"’\ w -. " ' ‘ I" .h ‘C‘ vr‘ ‘ ‘ . . r ' .
one hour in the hi3; eke:u head oi an Interretional PofPl-

gereted centrifu;e (model :31). The finol eagernetant was

for obtaining the crude enzyme extrect from the cells. The

extract was eit

:fl?J
1;
L0
b4
OJ
4)
V1
(3)
I J
H
U .
L):
,—
.3
L+
(
F
O
W
[’3
c+
L
PX
( ;
L}
3,.)
cf.
.1)
C)

under a drag Of benzene.

 

Assna of the 5-L galactosidase in cell free extrac s:
The aet‘o- for ass y of the ngjlo M'S es eitielly tne

tes ed was made up to 4 ml in greded nitroren concentra—
tions with sodium phosdhate buffer r3 7.4. “he dilitions
were made airectly in sfiectroohotoneter cuvettes. The tdoes
Jere ola ce —d in a co13t9nt temiereture water bath at 37C for
five minutes. Initial readings of ea h tuoe were iade at

420 mu. One ml of O- nitroohenyl 5—D galactoside (CAEE)

I

. a i 7 , 1
at a concentration oi 3 X lO‘/ was add ed to eacn tuoe,

14

mixed by inversion, and incuoated for 15 mindtes st 373.
At the en? of the lfiCJD*tl3u :eriox 1 ml of l percent

sodium carbonate w's added gaickly to each tuoe to stop
the reaction. Ehe tubes were 3 axon and read in a s ectro-

photometer at 423 mu. After correction for initial readings,

the o tical density valies were converted to ix OLE}

(U

hydrolyzed and these values wer- plotted against t;e resdec-
tive n trogen concentration. Only the valdes which snowed a
strict linearity oetween extract nitrogen and am0dnt of GREG

hydrolyzed were used.

FA

. ,2 Lana
Lin-431" UI;U C

.nditions of the test, a change of O.

O

octical dewity unit was eiuivalent to 100 uh ones LJdro-

L

lyzed per minute. The "snecific activity" of each extract

was recorded es mm ogre hydrolyzed /u5 h min.

Assay o” the 5-D galactogidase of intact cells:

 

Ihe D-u alactosidase activity of intact cells was

determined in a manner :imilar to the assay of the cell

free extracts with the following changes: a standard cell
susrension of the Lesired culture was ;repared as greviodsly
descrioed. ine cell adagensions were diluted to suitcole
nitrogen values with sodiim phosphate cuffer. Four nl of

,AL v. 4— A A. ...,;3 3 . - 4— a .,
irto Cuvect~s exxi.racuoatea in

*JJ

each dilution wer- +l<cec
the Witef Seth for five mindtes et 370. Initial re dings

—-

were made after cerefully s shin: the :Ja;:h5ioh.

Q3
0
34
ct
L.
(J
(D

e_.

7"!)

O

15

Che ml of 5 X lO'b Cir} was a ded to each tdoe, mixed by
inversion and incibated for 15 mindtes. ho sodium CTPQO-
n“te was edded to the reaction tubes. Instead the tJOBS

ed into an ice water oath and the readin;s were
taken immediately. Lederberg (16) stated that sodium
carbonate causes a marked decrease in the absorbency of the
cells. The activity of the enzyme was defined as EN OLPG

hydrolyzed per ml cell sus ension after 15 Rinutes.

"U

16

.J
!
LU

\J
L:
S...
H
(‘f
(1]
co

 

 

Determination of extract nitro en
In tke study which will oe descriLed in this pager, the
enzyme activity of the cell—free extracts will oe referred

to in units of "so.ecific activity" ex; mi of edbstrate

v

~

changed per u~ extract nitrogen per minute under the scecifieu

('_)

re tie odahtity of cell

conditions of the azsay. Theref
nitrogen of the variods cell free extracts must of necessity
be known. Lester and Bonntrr (2 ;) re; rted tlst the cell

cts stowed a ir-ct and constant

in

nitrogen of cell-free extr
relationshi: with the values ootaine i by oiuret color ot the

trichloracetic acid :recioitaole oroteins of the extracts.

 

there cell free extracts have s high nitrogen concentration,

the biiret rr tein- nitrofen r lationsr i: is linear and holds

true; however, with extracts of low nitroren content tLiS

 

relationshio is not reliable. Lowry it. a_. (24) showed
that minute BnOJHES Of soluole an: insoldble protein in
serum could be ec04rately deteriined colorinetrically with
the p;en o reagent of Folin-Jiocalteu. These adthors siégested
that there was a possibility of using this metho; for estim-
ating the nitrogen of cell-free enzyme extracts.

Preliminary RLteiftS, qualitative in nature, iniicote

that the technic of Lowry COdl) be used with cell free ex—

tracts and promoted tie develo;ment of a method for its use.

A.

l7

Lstoblishment of the standard curve:

Cultures of four strains of coliform bacteria §;_goli
K12, E. coli 8, ;;_co li ML and g;§31_; were grown in lactose
synthetic medidn as previously described. Each culture was
seeded into a series of five flasxs. At the en} of 2, 4,

8, l2, and 24 hOurs, one flash from each strain was r; oved

harvested. then the

m
*3
(I)

from the incubator and the cells

(D

cells from the 24 hour flasx had b en harvested, five pools

were made of the four strains with resgect to incubated tine.
Each of the five pools was disrum. ed and the cell-free
extracts were made as described earlier. The pool of the
coliform strains used was a representative seiple of the

tyoe of organishs which were used in the course of this

VIOP ..

Re—wents: Solution A — (1) sodium carbonate 2 percent dis-

 

solved in l nercent sodium h droxide solution. (2) 0.5
percent coooer sulphate dissolved in l percen solution of
sodium tartrate. Combine 50 parts of (l) with l gart of (2).
Solution 3 - Folin - Ciocalteu ohenol res3ent adjusted l to
l with l N hydrochloric acid.

Cne ml of extract was placed into each of two cuvettes.
5.5 ml of distilled water were added to one of t1e tuoes .
Five ml of solution A were added to the second tube and

the contents mixed raoidly. After stendin 10 minutes,

0 PTICAL ENS I11 (-60 Inn/u—

F
0

9
In

18

 

 

l L

:3 5b i3 :60 :25

l

)u. cl NITRObEM/ml tum?

-Fig,-l Relationship between extract
nitrogen and protein. Nitrogen de-

termined by micro—Kjeldahl method.

{o

 

19

0.5 ml of solution was ad3ed a1 d mixed rapidly. A r arent

blank was nade by using distilled water in since of the

O
U

extract. At the end of EC minutes of if “ti n at room

D

."\
V. r;

(V

retire, the tubes were read in the s;ectrophotometer at
6 oO nu. The blue color was stable as long as two hours.

The obiical density readings of the test extract were cal-
culated oy subtraction of the optical density, if any, of

he extract control. The corrected ootica l densit of the

(<1

test extract minus the optical ensity of he reagent blank
was taken as the true color due to cell protein.

A determination of the nitrogen content 0: eacn extract
pool was made by the nicro-ngldahl technic. Five a1 gortions

1"”

of each pool were taken for analysis. rne extract nitrogen

was calcula t d as ng extract nitrogen per ml of extract.
1h 4 values were taken as the abscissa of a plot against

the 0,? sticel density tahen as txe ordinate. The relationship
of the cell protein and the extract nitrogen is in agreement
as s own by the fit of the line to the points. In the
studies described in this ca er, tie extracts were reacted
with the Lowry reagents and the values of extract nitrogen
was extrapolated from the standard curve. Fig. 1
To test the accuracy of this metnou for the deternin-

ation of small amounts of extract nitrogen, the fc llO‘iLS

:eriment Ias set u:; 18 hour old cultures of gL_Qoli K 12,

d in the usual

( D

E. coli 3 and ALF f 1 were 3rown and harvest

 

 

 

fll. extract
samnles

Calculated

 

 

 

 

60

 

 

21

t1

manner. cell free extracts were made from L?CG strain.

Three 1 mi :ortiozs of €?C; extract were aneljzed ior cell

D
*-
r+
'3
D
(D
:3
(T
‘<‘.
6+
(1:
1.4.
k:
*3
O
I
w'i
(.4
(0
FJ
£11
~‘O
L
C’
0
Ci:
*1
t N
(I
m
<
(P
"3
:J
(V
O
P?
c—+.
CT
(D

d
I \J
'3

eo secera e determinations was taken as the value for
txe flitPOJQL of the extrects. Three 1 ml portions of tLe
same extracts were reected with the Lowry r-e:erts and the

xtrecte were celCJlated. The

(T)

Optical density of the

extract was extrapoletei from the

73.4

nitrogen content of ego
standard curve. The Tesults Table 1 show trot the nitrogen
ree—

values, obtained from the standard curve were in gooi a

ment with the values obtained by the micro—ngljahl methoi.

elis:

O

Keckenicel disregtioi of

Many methods have been descrioed for the mechanical

DJ

isrittion of bacterial cells for the purpose of Iregerine

active enzyme extracts. The majority of technics have

p;

yielded excellent preparations but in most cases reruire

elaborate ani exzensive epoqratas or soecielly deoignod

l
)-

equifment. Kelnitsky e . al. (25) described a metho* for

(

grinding e glass-cell—paste mixture in which he forced the

mixture be ween 9 pair of concentric glass cones. Tne
fragility of this specialized eooeretus limit“ its ise to

small quantities of cells. The Boot: Sreen mill on the other
hand can handle larie ciertities of cells but it is large

and quite cumbersome. Docxsteder and Helvorson (26) using

.e'm tissue hOuogehizer regorted

_1
b

a modified Eotter—nlveh

(1‘)

that with alumina abrasives this method is V ry ejficient.

The method however, must still be comrared to hand grinding

i\

. wdsre 3tandardization from one extract

(U

in a mortar and p stl

(

to another is practically iupossiole. Lamanna and fiallotte

{L
F.)
U
"S
{L
r r W
(+-
(0
Li.

(27) reported that bacterial cells could be easilt

’5

by the use of linute glass oeads in either a Laring blende
or a motor driven tissue homogenizer. The authors did not
elaborate on their technics, nor did they “resent quanti-

tative data of their study. Tnis technic Su

i
O
(D
H
FJ
5‘
H
c+
a
CD

’ J
7
Q)
'3
to
d
L
U)
:"
I""
O
:3‘

promising methOi for disrupti;
are readily available in most laboratories. The following
exnerinental results are described to show the efficacy of

the technic.

One ml of a culture of ;. coli_K 12 was seeded into

 

100 ml of 0.1 rercent lactose synthetic medium. The cells
were incubated for 18 hours. The cell suspension was
harvested usin; sterile procedures throu4hout the washinj
and centrifuging. The resulting cell paste was suspended in
an equal volume of sterile saline. Cne tenth ml of the cell
sustensiou was diluted serially in salire ard elated on

for 24 hours and

C)

lactose synthetic agar; incubated at 37

the colonies counted. The remaining cell suspension was
divided into three equal parts. Three aorasives Lere
selected for study: (a) finely ground acid washed sand,

N
‘1

(b) powdered alumina, (c) "Superbrite" “lass beads. mach

abrasive was mixed in a ratio two parts cells to five parts

aora ive. This ratio was selected after a series of pre-

(0

lili cry trials were made with different mixtures. The

as: of handling, recovery of ex-

0)

l ction was based on

U)
(D
(D

t ts and efficiency of the honogenlzer. The cell-abrasive

*3

(3

,0
J4

mixtures were houogenized for 5, 10, and 15 minutes. At the

end of each griniin oeriod he respective honorenates were

A
J - v

diluted up to lO :1 with sterile saline. The abrasiv was

(D

allowed to settle to the bottom of the tuoe. One ml of the

~-« .- - - ‘- . .. J-AA ' ‘ . .. ., a. a. 1,.
suoernutant was taxen an: slated in tne saie taller as tne

of

(i)

original cell suspension. Table 2 shows the percents

.-
‘k
9

cells destroyed with the various aorasives at different

’)

l

\J‘I

(D

intervals of tine. These data indicate that aft

0.)

b asives

"5

minutes of grinding by this technic all three

0

‘5 oercent of the cells. However, at shorter

.o.

disru ted over

\

grinding ti es up to ten minutes, only the glass beads were
effective in destroying most of the cells.

A second experiment to test the efficiency of this

([i

method of disruiting cells was made by ccdparing th

activity of the crude extracts. The cells were 5rown

(T)

enzym
and harvested as described above. After homogenizing for
15 minutes, each homoge.°te was diluted to 10 ml, trans-
ferred to a centrifuge tube and spun for Live minutes at

3000 REM. One ml was taken from each supernatant and

24

lists II

I

 

 

 

 

TJE EFFECT 0? TI L 0A THL biSffiUCTlCh CF CELLS
3X GRllDI\3 WITH EJLEAL AJIASlUES.

Percent of cells disrupted
Abrasive 5 min. 10 min. 15 min.

Alumina 44.0 50.0

\0

\0
KC) \0
\O 4} U]

Glass beads 96.0 99.2

 

 

The number of viable cells was determined by

plate coants on 0.1 percent

H

actose synthetic

mediumi-l.5 percent a sr.

UH

 

25

 

C)
'1"

 

 

Abrasive

 

Cotical density/l cc extract
extract 1 sxtract 2

 

 

 

 

 

Sand .4: .45
Alumina .50 .5

Glass beads .80 .84

Ratio of cells to abrasive is 235. Coxosrison was
based on the ootical density due to kydrolyeis of

OKPG/cc of extract in 15 minutes

diluted with E ml of sodium phosphate buffer pH 7.4 in a

i
(I)
*3
(l)
r(1’
l...)
in
O
m
p;
H.
H
y.)
c+
:‘s‘
(D

spectrophotometer cuvette. The tubes
water bath at 37 3 for five minutes. a reading was tagen
and then 1 ml of 5 x lO'EM OHPG was added, the contents
were mixed and incubated for 15 minutes. The c anre in
Optical density was read an? corrected for the initial
reading. Table 3 shows that the extracts obtained by

with glass beads had the gr;atest B—galactosidase

arindln

v

i H

activity. These data augment those f the previous experi-
ment. Not only do the glass beads disrupt the cells more
efficiently with time, but tne release of the intra-

,ter. 0n the basis of these

(1‘)

cellular enzyme is much gre

slass beads for extracting d-galactosidase

'7‘
9

studies the use of

from the bacteria was adopted.

Stability of 3—D galactosi'ase under storaje coniitio 3:

Due to the considerable length of time required to
prepare the bacterial cells for assaying the enzyme activity,
it was not always possible to perform tne assays on the same
day that the extracts or standard cell suspensions were
brerared. Therefore, the resting cells or the cell free
extracts were usually stored in the frozen state until they
were used. Lederberg (16) reported that extracted
3~D galactosiiase is unaffected by storage under refriger-

ator temperatures for ssve.al months. There has been no

27

report on the stability of the ens me when intact cells are

stored by freezin: grior to extraction. *he following

I)

'n
L

(
(H

stidies were cerrisd out to coniirm the results of Lederb.

O
Q‘

‘ n the frozen state.

(U
1

and to justify stora;e of intact c 11

V

Caltures of E. coli K 1?, E. coli IL and AL§_1 and

(D
o
H
(J
K
h
0
(l
{N
d
H
(D
6*
C)
(I
(U
(D

b:
b

I

§;;_ggli B were grown in 200 ml of
‘tiztic medium for 24 hoars. The cells were harvested in the
Lisual manner. The final cell mass was suspended in 4 ml of
ssterile distilled water. Two m1 of each sdsoension were
jplaced into a glass vial, tightly ealed and placed in a
;freezer chest (-200) for a period of seven do s. The remain—

'ng portion of cells were disrupted and cell free extracts

(H
m
b...
ii";
O
(+-
0
01
f _ 1
L I.
go
0’)
(E)
(l)
O
C+
F"
<
F).
c+

‘<.‘
O
H:
(1}
i0
0
t3
(u
(+-
*3
,0
F‘
p...
'.

«obtained. The 5-0
‘wat deterainsd islediate y.

At the end of seven d ya, the fro en cells were removed
:from the freezer. The vials were placed under running water
‘to thaw the cells. The cells were disrdpted, cell free

' V

extracts prepared and assayed for 3-D galactosidase activity.

U]
O
C"
J
(‘f‘
C+
H4
,J
(D
L-
I
L
B
i"
J
O
C+
O
L
f.
p,
in
( u
b
O
rt

’The resalts (Table 4)

(I;
H.
:3
6"
(I )
h '3
*3
C)
[\J
U
:3
07
(‘7'
l)
d
(D

'4‘
v

:intact cells is unaffected by storar'
*Et least under he conjitions stated herein.

A second ex :riment was designed to study the effects

J

Of‘storage of intact cells un;er ordinary refri erator

(l)

P

”temperature (4 G). Since there appeared to be no d'fferenc

QLn ensyie activity of the several coliforn strains after

28

TABLE IV

VAEARISFL CF Th; 3-D GALLCICL'LAS; ACIIVIEX C?
s..

(1""r 1-5 ‘f 7‘ gm an (1' 7 "M"; V "_\ ' ‘. f1 “ v ‘ “3f“: '» '3: '2" .’
ULLL $53.21.; LxxiRnUEJ .L/L.L.L¢LU ERL‘JA wags...‘ .Jl‘v-.iL.J In

TEL FRCbih STA;; F93 DlViA DRE" hiffi SImlLAR LR—

H
r; J
L)

 

s specific
1 :f extrects
3 Frozen cells

 

 

 

E. co___i_ K 12 108 me

E. coli ML 29 30

 

h. on i B 175 180

ALF to. l 85 SO

 

 

 

Cultdres harvested after 24 hours in 0.1 perceht

lactose synthetic medium.

*Average of 3 readings

 

TA

29

, -

jhE V

A. a ”1."? -—-r'~ t “ ‘ ""N 1" J r" *1 ' “I
JHLAAakJi‘w Jig“ 4...; in .ULJ—fii‘ul 0.1141414.)
f\ f" r,"—"‘ " a“ W -.

ti 4 C Fa}; V1-1.L'v'i 3 IL‘LLVL) C3

 

Eeriod of storage
in refrigerator

Units sgecific activity

of 3—3 galactosidase

 

1 week
2 weeks
3 weeks

months

KM

Fresh cells

111

108

\C)

7.5

 

 

 

stora e in the frozen state, only one organism (i. coli K 12)
was tested in this exeeriment.

f"

One ml of a caltdre of E. coli K 14 was seed d into

(1

e>ch of five 250 nl Lrlenneyer flesxs containin; 103 m1 of

'n hetic acaidm. Ihe caltdres were

(I)

0.1 percent lactose

. ' , , , ,‘ 1 _ .. A .. *- 0 . ,_ A, '1 r. - . "11,, ,- ., . i e
inedoeted and fl.(cht:1 in tne usiai manner. ins Pebuloih

U)
5..
I

O
(U
Fx)
f4.
C )
D

3
U
u
f—
F
El,
H
C
H
U
f‘
FJ.
(t
(D
Q
L
n)
H
O
"5
d‘
0)
V i
C)
5s
“”5
O
H:
(+
I

( {1

(.1.
(T ,
L 11
L.)
"1
»
*2)

periods of tile. The remaining portion was disruk

cell free extracts were made. The 3-D :alectOsilase activity

('

of the extract was assayed immediatel". At elven intervals

erator. ihe cells were broken dg, extracted and
assayed iiiediatelv. All the conditions of his excerimert
were the same with the exception of storage ti e. lhe resdlts
of this exveriment are given in Baal: 3. Ihese data show

that there is no significant difference in the extracted
<ensyme from cells wLic? have oeen stared n the r‘frirerator

'for Varying

‘
~./

2:

,--: —‘,r» v a " r', ‘ 'L' 7- ‘. J- ‘ "f 3r- “; ‘ \.- . , ~,- , , - . h ‘ , . y . ,'
oil. :LOh a diffeient L.ictdre i; not gho.n. a col? rison

31

finrr.r'1-'-nj-"f.A f. {1 v1' .
J,-....i".ui.., 1.LU:~.JL IL. Vii—Ila E

 

baits s;eciflc activity
of 5-D galactosiiqse

 

h)

 

 

 

 

()3
(\3

bed some effect on the concentration of enzyme w ioh was
suese’oegtly extracted.

'—

Sane msny of the cell-free extracts used in til; stqoy

2‘
(D
*3
(U
u)
c+
O
*3
(1)
LL
u)
0’]

long as tree weeks, so ex;eriiert to etqdy

C+
;., l
(‘2

stsoility of 5-D oiectoeiies: in cell free extracts

was set up es follows: one m1 of a CaltJFS of i- coli A 12

‘3
l

WELS S ““

(
(

iei into loo m1 of 0.1 porceit lactose synthetic seiiwt.

C‘?‘
p.

C,

After 24 hoers incaoetion the cells were harvested, disroo

3‘ y' ' r‘, f 1 P ‘. r . L .- «“4. A - - r\ -" A 7“ .-\ ,-‘ n . 9A . x , "1 ‘ .
ehi tn: cell iree extract' ygetsrea in L: Jodil manner. ‘xe

U

_ _ ‘ “ - ,1. .- _ A q A . , . “- (a A. "17.- .—~ ,- fl, . ‘ r~-.
extract was oiviiel luuu foir eolsi QJPUSO lures exits were

glscei into SL911 visls snfl seal 3 tirhtlg. Ike Visls were

"l

e refrigerator st 4 o for veriOJs periods of

k. JO
3
(‘+
Q

then out

time. in: remaining portion of extract was s ssyei imie—

di"tely. At tie eni of l, 2, ani 3 weexs, the storei extracts
were removed from the refrigerator ani the Q-s 5sLectosiisse
activity determined. Ere fists (isble 6) show thfit

3—D gsloctosiiese activity of cell-free extracts is not

affected oy storage at 4 C u; to e perio; of three we;Xs.

c+

m a“ -w . - -, . . «- $‘ »-, 7..-,9. -‘ ‘ -' .1 : _ , ' was
LL3QC restate, (3.1-; lat/1:3 Jile..bf' Linc CQiiiiClQl“; “goCI‘LLJCyL are

in swreetegt with those of Lederoerg (16).

35

Part II

yorwel lacto;e fertezter

,_\

Kinetics of g—L galactoeiiaee of

‘

.A‘ 'r" A . -D A x ‘Y K ‘-
aha an aoerraht lacto:e ierieiter:

 

Tie kinetics of the 5-D galactosiiase systea in g. coli

L

rate for this

(D
U]
('9‘
so
0
H
P.
L .
o
p;
c
, 3
(l
(‘
U
r.)
r.-
(1.
Fl
d
‘<;
O
H.
O
f
H1
L, J
>0
(I)
d
y .
(T)
(P
i
(J
(.0
C’-

(

enzyme. Lederoerg's resalts show that the depehience of

3-D galactosiiase activity ipon on}; conceitration agrees

1)

SD

sim 1- 'nayme—sobstrate coaplex. lbs

with the theory for

'0
<

7

tichaells constant (it) for OLE} was foam: to oe 1.3 x 10'4h.

(I)

The high affihity of the enzyue for CLE3 males possiole th
assay of ehzyme activities in very saall concentratio;e.

Lederoer: etajiei tLe j—L IalECtOBidQSS in craoe extracts
s e

of ehzyme-suostrate will oe foani in more refines enzyme
preparations. A portion of this work will oe ievotei to the
iiuetics of the enzyme siestrate reaction with oertially

A

”it e zyme extracts. Since in the main part, this

LL

(-5
in
"S
F.
*‘fi

work is concerned with the lactose spliting enzyme of
aberrant coliforh organiste, it was necessary to establien
the fact tiat this enzyie was iaehticsl to the 3-3

of a. coli K 12, a normal lactoae feriehtihg coliforl. Ihere

have oeen no re orts up to this time on the e—t galactosiias

. (l)

in the acerraht coliforms. Cf pEPBuOQHt itportagce in this

consideration is the COmJEPiSOfl of the Km and Vaax of ooth a

34

normal lactose ferleuter and an aberrant lactos fersehter.
The following phase of study will be devoted to estaoiishih:
the "unity" of the 3—D ralactosiiase in the aoerraht lactooe
fermentihx colifors and in §;_92Ll K 12.

‘2

n i -»- ..* v .14.! . n r r“. .- .--. ,-_
oooceherat oh aha goriiicsoioo o: 5-1 _alactosi see:

 

The early kinetic st11ie

(,0
O
M
,_
c,-
(D
L».
i
C
be.
1.0
i, J
.J
0
Cr
C)
b
.—
L J
(
(
( l
O
to

i. coli were for the QOfit 31rt eerformed on crude extracts,

Lederberg (16) 3L1 hohoj SE 81. (28). Ruby -nd Lardy (18)

 

extracted 7h; fractionateo 5—D gslactosiias; from ;. coli K 12

in a highly p1rified state for he p1rcose of choracterisin

V
5‘
m
u
5:.)
O .'
H
(I)

the cur ysicsl prooerties of the eisyme. Lester (29

Q

*3

to 0‘

‘-

eoare a partially purifizfl enzyme extract by the simple
technic of acetone fractionation followei by eutohium s1lphate

pre cipitatioim Rita several mo1ificatiohe,the metho: used

ealactosidasd or 11.22L2

( \

in this work for treolrio the j—
K 12 so: gggfi l was tLat iescriOed oy Lester.

Five liters of lactooe synthetic seianwerejiviied into
500 nl portions in 1:00 11 Erle dhzye" flasgs. Eive ml of an

18 lo1r oli c1lt1re of th

U)

~ r‘ I ‘rv '\ f‘ r r‘ I“: ~ ‘. .‘ ‘L ' "K . H "
OPiflAiSm were seeoeo ihto CiCL

.)
(I)
k)
{3
-\
;
rd
H
o—
‘
:x
0
ya
#4
.—
5.
(I
“J
F
D
3)

flask. his flas as were pla cei in t:

(T

ihC1Oate1 for 13 ho1rs. ii; cells were harvested by cehtri-

fugation. The resiltin: cell

11,...2 4.- ,1 I 1
I". ‘V ‘V 1 ">—- 'V 2
AQte honour Wr918LLM/J {JV-LCD lVit J»

"0'

,0

130 ml portions Oi physiological saline tieh res1sgehde1 in

7'“

an eqml volume of’ saline. the cell

U1

' ('1

re vionly descrioed. :1; fra3'uehte1 cells were tkeh iil1ted
to 50 ml with salihe 941 centrifa e1 at 2539 33H for 15

, ' .- . + - _ ,x‘ a ~ - «G», I ~—— n v o "N I, '~. ’ " ~ 1
11h1oe3 to e1ove l?r3e cell fra31euts ani 211s) oeaas. r.e

kl}

s1t-.h taut was decart;1 ani centrif1re1 at 23,000 x: for 01'
the s1fer 0,9ht were carefully Pi¢3v€i with

a Cipette -11 glace1 into a 50 11 cegtrifu3e tube. ILree 11

O
$1)
0
(D
O
1’

f:
(I

h)
(u
L0
1 A
Ll:
(D
(‘23
(+
0
cf
DA
(1

C"
C.
U
(D

the cohtehts were 1ixe1 by

4 C

{a
p...
H
5.
O
{3
C+
0)
Ct
$J
(J
(.j
U)
'7.)
H
i ‘2
O
(U
$.24
P-
D—J
6*
0
d
’ ..
(D
*3
SD
(-9-

refrigerate

.| . .9 ‘ -’ ‘_ ~17” Y, ‘ . . v a . .3 (9 ~ \ - I ,A. r. a ., .
for lu mihates. l.e 1ixt1re was cehtr1113e1 at 4,000 1E1
“ . , #- 1,_ . 34.1-, .,. .1. .- .- . a! 1,: n _. '; ‘1 4, .,.
for 13- 111111183, the 8.1;..Cp’iqbetilt glgttteu Of]: , 0141;;‘3 Ere-
- 4. '- .- ..4, .11 ,4.“ 3"". . - .4 . . . 1 , ‘ ' ,1 ' . . : ,1
cioitaoe oiscarieu. ins Supernatant was co1c11eo with ah

eq1ai \ .tity of acetone in a do 11 cohtrifuge tuoe 611 the
cohtents shaken ahi refrdugu¢tte1 as above. f1; t1oes Wc”e

centrif1ge1 at 4,000 REI for 13 minutes, after which the
s1eerh ta at was :01re1 off. Ihe precipitate was jissolve1
in a 31a11 a1o1ht of I/lS sodiur phosohs e o1ffer eh 7.4,
co1bihe1 wit? an equal volume oi sat1rate1 a mohium s1lghate

for 4 hours. Ere

(I)

and allowed to sit at roou temeerat1r

Hi :ture was cehtrifuge1 at 4,000 REM for 3 1111tes an; the

+

suoerha-aot discar e1. Ihe re' lti:: erecieitate was oiesolved

C

C'"
P
H
r...‘
0‘
{)1
5;:
(

c

d
(D
w
3
$41
* )
r4
L)
O
G)
Lb
Pa
{5
$0
(3
G

’1-
H
O
F. I
F)
:13
p-

’1

(D
(

HI
(I

( I
<3
(D
>0
“f
p.

.1
1
x.)

*4.

in a

d
p
F
L‘—
c+
F).
H
’1...
(i .1
$.
N
(n
o.

j131312.31 azaiss

~.J

1 water for 24 hours. The iialy

C‘"
"5
r;.
L.

extract was can e1 at 20,000 xg for one Loar. Ine

"pcPOthnt w ich r " lte1 fro1 this fractiohatiou constit1tei

(T)
(I
L.

the oertislly purifi=1 3—D alactosiaass which was 1331 for

 

 

 

Extract + 33% acetone
4 C for 10 minutes

Centrifuge 4000 REE 15 minutes

ppt (discard)

Supernatant

50$ acetone

4 C for 10 minutes

Centrifu3e 4009 RPM 15 minutes
r .
Supernatant (discard)

ppt - dissolve in M/13 Sodium
phosghate buffer pH 7.4

Dissolved pot-+ 50% saturated ammonium sulfate

is

(W

>m temp. for 4 hours

Centrifu3e34000 RPM 15 minutes

FF—
Supernatant (discard)

ppt — dissolve in ”istilled water

dialyze 24 hours against
distilled water

Centrifu3e 20,000 xg for
one hour 1
I

pet (discard)
Final supernatant

37

the studies of the kinetics of this enzyme systen. 1 tea
on he activity 01 tniq gartially gurlfiei e1zy1e manifested

'r the cr13e extracts.

m
0
<
(1.)

acoroximetelj a 200 foli increas»

is shown in

n>
PU
H
O
1:.
(

()
c+
0
ct-
' v4
‘ \
W
’3
LL)
0
c+
F
L)
I

\I)
(+-
r].
C)
A
L)
O
(H
L

L.

*5
(l)

EL 12 and 5.31722;

'11
i...
1‘
[U
0
ix)
I
t
I
D
F.)
,1)
O
C?-
U
a
p.
lb)
\
H
P—b
n
C)
O
(J
ct
L“
Iv
O
0
FJ
f.

 

3
LJ
1*)
'3
(J
O
(D
‘41
H.
D
d
H.
L
7,6
T3
r;

T
r3

 

4- r J- ' TV _‘ 'x
Deter inotior oi 11 on) .1 :
~11,- ’ 1 ‘ \ ’- ‘r‘ " .q 9 — A ‘\ ‘ ' ‘ v q a ‘ w’:
i.is .ha o o: the -t11y was 1631:HC' to shew the 3833“-
, C- '5 ”‘1 1., 1, ‘ . .. . 0 ' . , 1, . ', , r "
dance 0. the 3-D 3alactosidase actiVity on the concentration

('1‘
J

of OIPG. The partially eurifie1 enzyme e; ract was ad;1ste1

(D
N
c f'
" S
a:
0
Ct
5
L)
(h
' N
3..
F
(I
(J
t...)
(U
c+

with distilled water so that one 11 of

(‘9'
O
0
PW
,—
(u
C
(J 1
:3
Ho
r—r
')
U
(u
k.—
O
P\
t
(D
f—l
O
r T)
“
(1‘
Lu
P.
H
L
(1"
(n
x. J
(I)
‘7‘:
(+-
"5
'x')
O
c+
.1“
(J
U1
r( ’
H
\0
O
p,

'. "‘ fl 1” ’1 1' ’ "‘ "‘ ' ‘1‘. «~ -‘~ ‘ ' ' ‘~ r "fi ‘. "‘ V "x
into each 01 tro SyCCBPQPLOtUheteT cavette‘ in 1 series oi *.

3h ee 1l of M/SO 501111 ghosthate buffer at tn 7.4
to eacr cuvette. The cuvettes were glaced in a constant
temperature water oath at 35 3 for several minutes until
temrerature equilibriJn was reached. One 11 of CEP} at the
following concentrations was a11ed to each series of 2 C1vettes;

. *7 -.vT.. ~, '- . ~A ’- 1' by! s-“ A --.,.— . v, y H‘ ,~. --9 -
41114.“- , 211111 , 1111.31 , O o Diifl'l , 3? 111 \J‘ o U4111‘A PC ED 6 Ct! l. \J 3 1-} o If} 1'; C A ‘I v t t e 3

' a“ a _ 7 .'~ A ~ ‘ I a a ' .'\ .g .: r ‘ -' I '\ ‘v r" 2‘ ‘- —‘ - V. TI 1 -'
«re sraxeh 921 neuoatei for 13 11n1tes. at the en; 0. the

:E.‘
(l)

‘. ' Ir 0“ . "‘ “\"- ' I: 'v‘.‘ p 1' 'J‘ "\ A. r‘ ”-1 .' 1 fi's r“ ‘ . r
inCioation ,erioo, 1 1i oi a l sorcent soai-u c rbon te

(J)

I

ol*tion was adiei to each cuvette to sto; he reaction. The

l

J. n
(at ‘ :3 ‘97

ere 111ediate ; read in the s ectronhotometer at

(l)

0

3.1V

(1

ensity re~din s were converte1 to

(a.

420 mu. The optical

[#] OPTICAL DENSITY uns‘rs

 

58

2.40
Lao
le I0"‘M

 

l ‘ 4

HT] sz ONPG:

Fig, 3 Activity of ONPGase as a function of substrate
concentration. Partially purified enzyme ex-

tract rrom _If._.coli K12

39

 

 

 

..oL
: 3.H)

:3 = L40
3 : 2.2x10‘4M
s.
t
3&5
U
D
J
<
2
E
o
4?

04‘-

l l 1 l i
2 3 44

I

[t] mM ONPG
Fig. 4 Activity of ONPGase as a function of substrate
concentration. Partially purified enzyme ex-

tract from.ALF l

40

2K Cir} hydrolyzei / m3 nitroge' as previously iescrioec.

fhe suostr

\O

.te concentration and corresconiing relative velo-

city were plott;d fly the seigle reoigrooal transformetion of

Lireweaver and ourk (:0). Figures 3 an: 4 Show th“t the clot
of tie recigrzoal suoctrate conCertratioi results in a fit

of points to a straight line, which is in a'reeneit with the

theory of a simjle inS coi:lex. Ice ordinate intercept of

C"
1.1
t)

('2:

straignt line r gresenta the l‘leJE velocity (Vnax

the activity of the ennyie saturatci wit} suostrate.

m
w

Pd
:34
U
P
¥
0
J
t. _J
O
C
+—
‘ D
H
(
L):
H:
r
(
(4'
(a)
U)
H
O
(U
P.
H
, ,
(—+.
(n
*3
O
(:f-
C+
")
‘D
C‘
l-
U
y.
z:
\
q
‘

"S
(0
v(‘
'3
(T)
U;
(T)
’\
’ J
C+
0.
6""
L",
(D
O
L)
(D
.)
*1
d
'3
\D
(.5
f.
C)
p
5-
O
f...)
U]
L.
(J
(1)
cf
*3
3
(+
(L
5
E
0
t
(4’
r
a
l
O
C"
t.
(
Ho
(‘1‘
L;
,—
U)

H

he f maximal. The L; for 3-D galactoeijaoe from E. coli K 12

elsewhere Lejerberg (16) a,o Lester (29). Ire extrcgolatei

aLFfl was 2.2xlo L. 1115 value is nearly iuenticil to the
valae ootaincd with E. coli K l2. The Vmax for gLFil'was

714 units. Thi: ie lower than tne value ootaineu with L. co i

" Lu~. ;..--..,,.-.- 4. .. .-,.,. ‘, .1: .t, ,
1’: 1.9. I'.O'.‘4":3ch’, 811109 ULLC} MdLCL‘niliiT-‘uuldiri wait, 11.3~Jv Vaioi.

4 ’ , .. ‘ . . o , i , A : I —\ +1 ,
UTFti" L1,; curli l - i $1-2“; .n: ‘_.,f.:~:°-'3.bLOll'-. --- i. ierc. 35‘ iii on:
5 e. V o x ‘ 1 r - )
Vmox oi thJ th is iore sic rent tr“; r ”l. Fi*. 4.
'fl*fl _ ‘ _, “r r\ e: I. A " F *3 .1 '
Li.» .L “VB". f-JDOVlu-J cViu v1.06 t ' c the “—14 :1“ , Ct 381. " 1v

1.. ‘ pmna- - 4. a i. - a 4. .. .,1'. .

wrjxx. re,ioe~ iii Jot: c . 'aoerrc.m;ilact"~...fer;cntiir;<3oiiforn
"I “ 1 1 p "\. ~~ -‘ A .

sari tie normal ectooe ierreiinni, colifor sec tne ease in

41

:tect to its affinity for CLP}. Eirtn;r ore the extractei

'.‘, .i L —. - t- ". i

in se ‘ o‘tont crit zi« ior co ycrin; urity Oi ehryic”
3 ,.~0 , L 4. .. ‘ - -

VJ \: L LLA-LCClJ' 1:: fiA; 3 Vina t‘c LJ t 00 til 3? ¢Jy1 » j ‘ r v t -AC 4 '3 v C

r , t" J. A ‘ VA. "‘ - -

-u reason:c o- the 3—“ a” cot: loose to LE:

 

()

Lu

P"
('1‘
(I

It is "enerall; Kn; n tgtt ionization at the fictiv~

of the enzyze may aifect it “ telytic activity. ;;e

U]
C

staoility of enzymes is elxo greatly a fect j oy th. tyurogen
ion co.certration of tLe sxterior environient of the cells.

.

m...,\_.,« I.“ , ”d «‘,_‘L “,
L ‘C.‘)U 8f.€ct5 95C; CJlA‘Lyhi'J

or a particular enzyme, ncnce a

U)

comparison of these criteric is e; ertial in con;arin; enzynes
from different sources. The o:tim=n pH for the ass-y of

3-D galactosiiase of E. coli K l2 has already been e;taoii3hed
by Leflerberg (16). Since this study 1; CQHCEPLEd with the

3—D Galactoaiiase fron an aoerrant l cto;e fermenting coliform

it was necessary to -eteriiie whether tne r~e‘ * to t? in

('2
V
bv-u

i
U
*7.
L
(U
U

these strains were similar to i. coli K 12.
Cre ml of the partially purifies enzyie sage up to a
staggeriized nitrogen coltent as previously jescrioeo was
0 o I- ‘. I

placei into each of 2 cuvettes in a series oi o. ihree ml

of K/SO soiium phosphate Differ at varying pH were a Jii to

 

‘ ,\ 0 ‘.~ [I 1 _\ : .'_ 1—: __ .. .1 .. -—
each ScPicg as LQlLOW : series 1, pn4, s fi.b 2, on),

‘ 0 fl "I v~/ ' o r‘ «:r' ’ ’ ‘.o 4— L: 1 . 1 /‘ —vx-‘
Soriws L, 31110, S’ur‘l‘;3 4, piiz, SSf'J...3 D, 574.8, filii -o”iL‘5 \, p119

 

 

 

420 m}

OPTION. DENSIT Y

 

Fig. 5

42

 

l l

PH

Effect of pH on enzyme activity from cell-free
extracts of ALF 1

_ 420m”.

OPTICAL. onusny

45

 

 

 

a»-
q-

PH

Fig. 6 Effect of pH on enzyme activity from cell- free

extracts of §. coli K12

44

The cuvettes were storpered and ke;t at room tetperature

w

or one hour after which they were removed and placed in a
constant water bath at i7 C to reach equilibrium. One ml
of GREG 5X10-3M was rapidly added to each tuoe, the contents
mixed by inversion and then incubated for 15 ninutes. One
ml of 1 percent sodium carbonate was added to each cuvette
to stop the eaction. The Optical density resulting from the
hydrolysis of GNP} was read in the spectroghotoneter. It
was not necessary to c0nvert the OD readings to Specific
activity of this study.

The effect of pH with the enzyme extracts from norna
and aberrant lactose fermenting coliform was siown to be
similar. A typical bell shaped pH res onse curve for enzyme—
substrate reaction was established for 5-D galactosidase
from both strains Figs. 5 & s. The Optimum pH for car} was
found to oe EH 7.1 — 7.2. This value agrees quite closely

to that reported by other workers Lederberg (l6), Lest“r (29).

l)

Th= re

U)

(

ults from this study are further evidence of the

identical nature of the ensyne from the two strains.

J

Pan

Effect of glucose on 8-D galoctoe dase from normal and

aberrant lactose fermentinfi coliform:

 

Lederbers (16) reported that cells grown on ii 0036

showed less B—D aalactosidase activity than cells grown on

v

(0

lactose. Es sujgested thot

(H

lucose might have a degressing

33

 

45

on the elaboration of tLe enzyue. Lester (29) resorted that
cells grown on glucose produced detectable amounts of

5-D galactosida e. It was coserved during the course of this worj

0)

that cells grown on glucose showed little 3—D galactosidase:

lactose. Ereiiiinary studies during this worn iidicated that
lucose night ha‘e a depressing effect on 3-D galactcsiduse

of 2. co 1 K 12. Stechenson and Yuduin (31) found the glucose
had an inhibitory effect on the form tion of calactzymase by

v

yeast. The den essin: effect of glucose on the elaoor ation

F)

alicto 311a s: by a. coli A 12 and n: 1_ 1 was studied.

of 5-D

 

E. coli K 12 and g;§1_1 were grown in 1 ye rcent 51 cose
synthetic medium and the cells harvested as described under
material and methods. The resting cells were brought us
to 20 ml with sterile water. This sus;3er sion was divided
into four 5 ml portions. The cells of Portion 1 were imme-
diatelv disru:t ed ani the cell—free extract obtained. The
extract was placed into a tightly sealed bottle and refri-

eratsd until used. :ortion II was see ed in 20 ml synthetic

(N

(D

d in containing K/BO lactose. fortion III was seeded in

b
, ;
*JI

.-

20 ml of synthetic medium containirgl /50 "ctcsc+-n/;u

glucose. iortion IV was see d ed in 20 ml h 50 glucos' syn-

S,

thitic medium. Bhe tiree ilashs were incuo wit; agitation

; 1.

te ,

at 37C for one hour. The cells were then harvested, disru

p,

i ase

U)

and the cell-free extracts prepared. The 5-; ralecto

46

TAdL: VII

 

EFFICJ C? GLUCC5L Ch 3-D EALACISLILABL IanC-

11”” av *A“~csw
s. -‘. ‘ ... 11.17;.) s...‘ Ji—J o

 

Portion Inducing substrate Units of sgecific
activity
E.coli n12 ALF l

 

I Control-0.12 o 0
glucose

II M/BO lactose 2O 15

III M/SO lactoset 15 4
K/BO glucose

IV K/SO glucose ‘ O O

 

 

 

 

 

Resting cells were grown initially on 0.1 percent
glucose synthetic medium for 18 hours. The cells

were harvested and wished, then incubated for 1

hour at 37C in the oresence of the inducing substrates.
Specific activity of the enzyme was determined from

cell free extracts of each portion.

47

ll

gn

ssayer in the manner pre—

L

activity of t .e four extracts was

0)

viously descrio'; d. In“ results of this experiment are given

in the unaiapted cells of both E. coli K 12 and gLFf 1 is

clearly seen (fortion IV). The extracts pre:ared from the

.4

cells of the initial incubation with glucose tagen in ne
logarithmic phase of growth were also void of ens Lyme activity
Eortior I). The eating ce is which were incubated for two

hours in the presence of h/SO lactose alone manifested a

‘idase activity. The two fold difference

0
1...:
{D
O
C'"
O
U‘

tybical 3—D
in the activity of the normal lactose fermenter, 20 units to
13 units is interesting to note. Ir e inoculum size was of the
same magnitude in both flasks, suggesting that the mechanism
of in;uction by lactose differs in the two strains. A more
striking difference in behavior of this enzy m; system is shown
by the data from (Portion III). The aidition of h/5O Iiucose
to the lactose resulted in a decrease in the enzyme of both
organisms. In the case of L. col' K 12, ther was a decrease
from 20 units to 15 units, whereas in the cas< of AL Ff_ the

decrease was from 15 units to 4 units. ihese data show that

glucose definitely has a decree sin: effect on the induction

of 5-D galactosidass by lectos . lhsre also appears to be a
areater depression in tr:e aberrant la tose fermenter than in

fart 1;;

 

 

V‘fl‘
(b
C I
I

t

A CQJ'”°lJOu of t:' galactouioase activity of lactose

fe .ezti.“ variant; of the colif‘orzn grout:

UJ

The results of the first phase of this work denonstrztes

unequivocably that the 3-D 3alsctosidass of tue normal 13

O
C"

3'38

1i

0
C)

fermenting 3. coli K 12 and aberrant lactose ferientin3
ALFfil is the same. Lietr 'er or not this is true of all lactose
em: entin3 variants of the coliform group is not KhbWh. A

c roarison of the activity, which is a meas ure of the concen-

{TO

tratior, of the 3—D 331 ctosiiase of various 13ctos e ferien—

4:... 1 q

tin tyges Ci colifcrm bacteria has iot been Hade Many

studies concernin3 tne di fferer ices in the lactose solitting
ability f various coliform have been made by coagarison of
reactions in carbohydrate hedia; however, there nave been no
reports in the coaearison of these or3anisms at the intra—
cellular level or more cio01-ely at the eniyno level

This phase of the thesis will be concerned with an analysis
of the 3—D 3alacto3idase activity of norial lactose feraentih3

and aberrant 13ctose fermentin3 coliforu bacteria.

(1)
In

The or3anisms eel cte to be assayed were :roun in 3lucose
synthetic medium for 24 hours. Ihe culture was centrifu3ed and
washed twice. The final pellet wai suSpehu ed in 5 ml of

sterile distilled water. Extreme care was taken a
cortanination. One ml of this susgensioh “as see ed in 130 31

of 0.1 percent lactose synthetic medium in 500 ii Erlenmeyer

m M ON PG Hvouowzeo

 

 

49

[.4 q N \‘I’ROGEN

Typical assay of standard cell—free enzyme
extract from g. coli K12. Cells grown on
0.1 percent L.S.m. Optical density units

at 420 mu are converted to mM’ONPG hydrolyzed.

an ONPG HYDROLYZED

tn
8

50

 

 

"‘4.

L6 ' 6A

,u C] N ITROGEN
Fig. 8 Typical assay of standard cell-free enzyme

extract from ALF 1. Cells grown on 0.].
percent L.S.M. Optical density units at

420 mu are converted to m: ONPG hydrolyzed.

51

anisgs were incubated, harvested, and the cell-

B-D galactosidaee Iron both a normal and an aberrant lactose
fermenter. A linear relationahip of enzyme activity versus

cell nitrogen is clearly indicated. ILrOighoat saoseoaent

D;

assays which are escrioed in thin work only those values

which showed a strict linearity were taken. The 3-D galactcsid

C)
V

go

s
activity of the various extracts were converted to AK ORE}
hydrolyzed per ug nitro;en at 37 C for 15 iinates or "inics of

scecific activity." The ssecific activity of all the extracts

I

are re?orted as the averar: of thr

‘4

(D

e

(r

‘eparate assays.

The results of the assay of the various organisms chosen
for this stddy are shown in Table 8. fhe specific activity of
the extracts prepared from the normal lactose fermenting coli
(1, 2, and 3 in the table) range from 135 to 120 units. These
data are within limits of eXperimental error in tLEL a single
extract may differ as much as 5 urits in either direction.
This is the unfortunate circumstance which resilts fro; the

use of angurifie* enzyme pretarationa. ju3se dent work showed

L

‘

however, that this characteristic did not influence the

validity of the tata for co parative purgoses.

D

The rates for the aoerrant lactose fernenting coliform
(4 to ll in the table) shows activity ranging from 20 units

to 82 units. flitn the exceftion of ggfifii and E. coli LL,

 

52

 

CCL;ARI30E CF The 3-D GALA "” "5 '

:fiTI/‘r‘ - ‘,
J; u.z.ai./X.Ji.‘.a g-

()

nLn UKLL
_ ‘r. v: " (1- '3 '\ {\“‘ "‘1" '3 '- x‘ 3 A ' r A ('1 "ruff __ “ 3— ‘_ n ~.
M .1. h-{A- J E J F.‘ V‘.- ‘3; V; .‘LL A L\ Va “Io—LUALJ ”Lid .1: \l N)“ E L- KLiAL-JAK L LL\ J
,1 x- -v ‘. ". — . ,I .r T 1 , . . f. . -. _ -" ’ ~.‘,- fl 3 .. — . o A “.1, I? .I; T. --- . .7— ‘

a uni - L in. c-1194. .L. Jr; .) AMI; Azania \iL‘.J.'\I‘ Ltd. v I - a... I." rut-nil I‘-

In} CREE‘m I 5313 .

 

culture used Units specific activity

 

 

E. 0311 K12 llO

 

 

1p
t4
*1}
:R‘
\N
[U
O\

>
IT‘
'11
:ui
U I
CD
L?

'p
t4
2h--
(:0
\

 

 

 

 

53

f r-

(D
(T)

the 8-D galactosidag; activity of the tserrant lactos

.
\J

)

menters is significantly lo'er th an the norms l lactose fer-

menters. Tre3e data su33est that the potential for lactose
breakdown in the aoerrant lactose ferisiters rasts in concen-

tration of 3-D *"lactosi ase. Si3r nificantly however, i: the
fact that all tie strains which were analyzed SFOA d s'me

3-D 3aiactosi ase activity, in most cases at low levels. fhis
is antithetical to the theory that slow lactose fernenters

may be deficient in 3-D galactooidase ym'en grown in poor
media, since the medium use d in this study was a minimal

synt etic median.

3-D galactosidase of coliform mutants:
The studies sh wing thst enzyme synthesis can ce inhibited
or retarded by mutagenic asehts are now classic. flitro3en

A

mustard, X rays, ultra violet rays, and bacteriopha3e infection
have been u3e wi h success. There has been no report in the
literature of the effect of ultra violet ligk t irra iiation on
the 3-D galactosidase of the coliform bacteria. The preliminary
study reported here su vests an area for more extensive research.

of the study were E. coli

(D

The cultures used in this has

'0

B756M and §;_coli KM which were irradiated by the method
described by within 32), a. col; KL a typical s;ontaneous

mutabile coliforn an? E._go i KL‘ the lactose cositive

t‘;

progeny of E. cgli_h

 

 

54

 

’T A f .1- “1’-

i 1 JLL Li

“'1'?“ . '\ ”4 *r ‘1 1‘ m ’1 v‘ ”A - .- h. ‘1 “I?“ ""‘j‘ [I T {"7315 ‘5‘ fir", ‘3 h " " Vv
in; 3-D Jinaaioo_bnoa W: onvntan L TAnT o-iain)
A r—~ -~ f" ,1?

U? L o ‘J «L: o

 

Culture used

Units Specific activity

 

 

(U

H H K»!
R) U1

 

 

 

55

The cultures were grown, harvested, and extracted in

the usual manner. The 3-D alactosiisse of the extracts were

assayed as before. The data shown in Taole 9 manifest a

‘

striking difference in enzyme activity. The srecific activity

P...-

of E. coli 3766M was 33 units. inis is considerably less

 

9

(D

activity than was shown in the unirradiated cultur Tabl-

E. coli KR had 15 units as compared to 82 units from the

 

sin a significant decrease

'4.
v

unirradiated source L. coli KM. as

 

was manifested.

B-Dggalactosidas: activity of intact aberrant lactose ferientin.

gglifor

H

 

The results of the preceedin; studies have shown that the
3-D galactosidaoe activity of extracts fron aberrant lactose
fermenting colifora differ significantly from the extracts of
normal lactose fermenting coliform. This is evidence that the
lactose solitting potential of the organisms in the coliforn
is not constant within the groufi. Uhnther these differences
are due to activation of the enzyme as suggested by Deere (15)
or to cell wall impermeability to the substrate retains to be

detersined.

The following study will be concerned with the

to

5-D 3.lactosi”ase activity measured on the washed intact cells

1

of the aberrant lactose fermentin“ coliform. Inasmuch as the

,A“
v

activity of the aberrant lactose ferienters was made on a

56

-fi-‘b~-

 

eletive activity
1 extracts (9.coli
K12 taxen as 109)

 

:. coli K12 500 100
1

26.6

K)!
K)!
O

19
18

;>

t—1

sz

:n

\rJ R)

' \O \O
O 0‘)

KM W -\1 \N

10

p>
t4
*1]
=1: :ti’
J:-
U]
R)

ALF # 5 96.7 19-4
ALF # 8 69 14

E. coli KL 0 O

‘. ca112~LL* 96. 19
E. coli 33

R)
\_\'
o

E_.‘.__coli 1-11.; 2"

I:

F“
\JJW-Qx')

CO

ALF}? 6

\y!
\.
O

ALF 1.; 7 21 4

ALF‘? 8 41.6 8

 

 

 

 

 

«\- .Irw;.l , ‘13....{5‘3 ll! aflcrh I

J ,
IA

 

57

 

 

,_,‘ ‘ j‘v'fl: f "w‘ ‘t.- »‘ 3a .. A ‘. ,1... .A 1. x . .‘ “1", ‘- 7"}
Cq.‘-.L'71-LJ.~/OI\I y': 1-1.1.4 D-JJ J.‘1i_.:‘.qi .. JliJIZJL .1uLLVlii

o .F‘ 5 " ’— "'. ’1. N " T
J £401“ l IL'\ LAA dI‘ U ALL’LJ
r‘ .“ T v~ .3 - T- 'v - w I A R ‘ " ~ 3 A “'1 T .3 fl 7‘ "\ V "‘ f" ‘_"‘ “\ ‘ ~' . .
J via-L: - -kii C: XJ— I‘“-.L J-". -) .‘l‘b‘.‘1u h DL-L.%1L\l LJ'dAlJJ, v J; DAL.JL‘-:‘;‘.I‘-

IN} CZLIEC11- Cl3%;13h3.

 

erahi"m Jnits absolfic a tiV"ty shits relative ac-
1ron cell free e;tracts tivity intact cells

 

E. coli K12 110 100
ALF # l 63 26.6

R)
K 9 '
O
H

0

\r'
m
0\
H
(n

U:
("n
O
H
v
4:

ALF

a)
r4
w _
m- §t =% m» a:
4:.
\N
\O
H
C)

 

 

 

58

com arative basis in terns of the

ferznenter, measure ehts of specific

e perinent. Instead, the a

in this

quantity of ORE} hydrolyzed per nl

Inspection of the data (fable

ficantly lower 3-D galactosidase ac

O

V

lactos fert

activity of

enters as conbarcd with

a normal 1? ct se

activity was not necessar

“C

ctivity was based on the

Q

of a standard cell sus ension.

go

10) again sho signi-
tivity in 11

the

n' ‘\ v~ A "t J— M " "w p
fermenters. Ab LOobd in assays 01 the CLll- f'ree extracts
n‘ ‘ f‘ 1 "1 I " p ’ "‘ ‘ . r: :' P I’- 1' ' C‘ -‘ 3' V V“ r
EPOM tn; ZDBTPEJC idthuu LCT1ewaPQ 3 Wldo Fin;€ Oi Chque
J-‘-'. - . air-‘ 1 ,. ,1 «‘11, an 1 ,1 P~.‘
activ1ty res11ted with tn- ‘1 ay of intact ceiia. 1hio 1 ain

‘\‘ 1‘ 1» Lia

‘. . 4-1. -
V , '3, _3
1.. -' --3UL..LI

1 -. z - . 'N »r\ -. VA ‘ — r" 5 .~,— -‘
[111.101 liffef‘siitly tin-.11 ILvLfiJIaJ.

I)

:01? cto i 1.3 induction by lactose.

s} coli.hL. UnLer the

i

0)

3-3 galactosiucse acti vity 03311 no

11

U)

C3

Table XI shows a comparison

activities 0' tie aberrant lactose

in both extracts and in act cells.

4.
L1

aberwwuit lac

of the 5—D

.. A . 4.
tOLJs CP.L.L::ILJ

rmenters to s-L

P

A fart cr i1terest n

(1

co :1iitions of th

06’

(D
(I)

1110.

L);

J_
U

galfctosidase

ferienter to ' col; K 12

v1
Jud.

' ‘r‘ 1:)
‘iv

activities iven a

are 5

the

the ratio in

x 12 as 100.

lactose

3‘": n
-JV‘

11.1th

ter.

0 " ~‘_
me chanisn for

terms

fermenter”

OI

'7‘ ‘1 'fi

line d-u

;ercehta5es

5alactosi1ass

akin; activity of s.

activity of the aoerrant

are not as hi h a- the11orrvel lactose ferien-
this difference in enzyme concentration is the sole

aberrant

lactose breakdown

r‘f\

to 3ete rr;n1ei.

(NC
VV

0
1.1Vr‘rzl‘.’ '
rv...'_— .2---)

59

 

D

A comoarison between the 8-D galactosidas= o1 tycical
col1forn bacteria and aberrant coliform bacteria has been
hresented. An enzyme, which afoeared to bis identical to the
3-D galactosidase of §;_ggli, was SLOWH to be present in
the aberrant coliforms.

'he first phase of this study was devoted to exocrineits
designed to standardi7 ze tie technics by which cell-free
extracts of the bacteria could be studied for enzyme activity.
The method for disrupting the bacterial cells using a "Virtis"

1

issue homo ehizer was studied in order to Justify its use.

C”

Many preliminary tests were made to arrive at the optimum
mixture of cells and abrasives. The results inficate that a
ratio of 2 parts cells to 5 parts glass beads was very effec-
tive for dierurtin: the cells. It was considered very in or-
tant tr at this technique be standardized in order that the
enzyme activities of the extracts would not be influenced by
variations in breraratio . It was found, under tie cond it 13113
used, that the 5-D “elactosiias is unaffected by this method
of extraction

A method used for measuring small amounts of soluble and
insoluble.trotein in serum was moiified for use in measuring
the :rotein nitrogen of the cell extracts. It was found hit

this method gave reproducible determinations. Since all the

60

assays performed on cell free extracts were oased or the
activity of the enzyme in terms of mg of nitrogen it was
necessary that the method used be rapid and accurate.

I The results from a study 0; he Kinetics of partially
purified enzyme preparations obta ned from a typical coliform
and an aberrant coliiorm show that ChEG was specific for the
enzyme of both organisms. ihe hictaelis constant for ORE}
and 3-D galactosidase from §;_goli_Kl2 was 2. 2le'4M as
conpared to l.7xlO-4M for the aberrant coliforn. Likewise,
the effect of pH on tie rate of enzyme-substrate reaction
was shown to be very similar. Tim se criteria suffest that
there is no difference in the gala ctosidase elao rated by
these two organisms, at least, when the enzyme is extracted

"in vitro" with a synthetic sub-

fron the cell and assayed
Since the aberrant coliforx have been shown to possess

3- D gs lacto i dase, it is possible that the‘ are ineffective

in utilizing lactose, because of insufficient Quantity of

enzyme or inability of lactose to re so 1 the enzyme. fhe

hypothesis that cell wall permeability may account for

differences in induction of "lactase" in aberrant coliforns

is worth con-,iderinz. We might visualize aberrant coli f rn

as he MVi a temporary or partial barrier toward lactose allow-

ing only minute amounts of the sugar to enter the cell.

Although this theory is without significant.eXperimon al

61

evidence, the results ootained in this study sux

this may be one of the possible mechan sms in these organisms.
It h“s been s own that there is much less enzyte present

in the aoerrant coliform t;an there is in the typical coliform.

This was found to be true in cell-free extracts and intact

resting to note however, that

(T)

cell suspensions. It is int
assays on the intact cell suspensions of the aberrant
organisms showed an extremely low level of ensyme activity.

sions and not with the extracts

t—‘

i

(1)

why this occurs with the sus;
is not known. It could be that the cell wall of the aberratt
organisms are more impermeable to ORE}. Thus the full
enzyme activity is not measured.

The fact that some of the aberrant coliform organisms
recovered from water possess the enzyme for utilitzing lactose

is very significant. Perhaps many Lore of the bacteria

which we recover from rivers and lakes have the capacity to

'U

roiuce this enzyme and subseiuehtly utilize lactose if they
were not placed into selective media. Despite the fact th't
an organism w1en placed into a medium such as brilliant green
bile broth loses its capacity for utilizing lactose does not
mean that the "lactase" has been destroyed. It is gaite
possible that some other unknown mechanism has been altered.
Often some of these altered organisms regain their lactose-
splitting capacity when placed in a non-toxic medium, others,

however, a;p:ar to remain permanently altered.

62

It is quite possible that our methods for deteriihing
coliform populations f a body of water are not adequate.
Eerhaps many trae coliform organiias are missed by our
cultural techniques. we can rot be sure that certain
aberrant forms of coliforms which are frequently isolated are

not closely related to the typical colifarm until new methods

for studying then are developed.

jJVVAéY
o n-n-«. a
__

.u_—_—_

f"

mhe 3—D :alactosidase from aberrant coliform organisms
was investigated. Kinetic studies on partially purified
enzyme extracts from an aberrant coliforn and a typical
coliform showed that the enzyme activity was similar.

Kethods for disruption of bacterial cells and prepara-
tion of active enzyme extracts were studied and develo;ed.

A comparison of enzyme concentration assayed from intact
cells and cell-free extracts from both aberrant and normal

coliform was made. Lower levels of enzyi; w re foun‘ in the

aberrant coliform.

10.

ll.

12.

13.

 

Stuart, C. A., Mickie, F. L. and Bormsn, 3. Suggested
Grouping of Blow Lactose Fermenting Coliform Organisms,
Amer. Jour. Publ. Lealth, 1940, 30:499.

Levine, N. Determination and Characterization of Coliform
Bacteria from Chlorinated Water. Amer. Jour. Publ. Fealth,
1941, 31:331.

Revis, 0., Note on the Artificial Production of a Permanently
Typical 2.0011. Centbl. Bakt. Abt. 11, 1911, 31:1

Lommel, C., Modifications des PrOprietes Biochemique due
Colibacille souls 1'influence de Hilieux Additionnes de
Colorantes. Compt. Bend. Soc. Biol.. 1926,95:7l4.

Darby, C. W. and hallmann, W. L., Studies on Media for
Coliform Organisms. Jour. Am. Eater Works Assoc., 1939, 31:689

Mallmann, W. L. and Derby, C. W., Use of a Lauryl Sulfate
Tryptose Broth for the Detection of Coliform Organisms.
Amer. Jour. Publ. Health., 1941, 31:127.

Dulaney, A. D. and smith, E. F., Slow Lactose Fermenters in
Water Analysis. Amer. Jour. Publ. Health, 1939, 39:3.

Parr, L. 3., Coliform.Bacteria. Bact. Rev., 1939, 3:1.

Parr, L. W. and Friedlander, 3. Studies on Aberrant Coliform
Bacteria. Amer. Jour. Publ. Health, 1942, 32:4

Dulaney,.A. D. and Michelson, I. D., A Study of B. coli
mutabile from an Outbreak of Diarrhea in the flow Born.
Amer. Jour. Publ. Health, 1935, 25:1241.

 

Massin1,R., Ueber Einen in Biologischev Beziehung
Interessanten.Kolistamm(Bacterium coli mutabile). Archiv.
fur Hygiene, 1907, 61:260.

Kriebel, J. A., A Comparative Bacteriological Study of a
Group of Non-Lactose Fermenting Bacteria Isolated from
Healthy Food Handlers. Jour. Bact., 1934, 27:357.

Wadsworth, C. K. and Hitchner, E. R., The Nature of Change
from Slow to Rapid Lactose Utilization by a Member of the
Colon Intermediate Group. Jour. Bact., 1936, 31:22.

65

14. Lewis, I. M., Bacterial_Variatiun with Special Reference
to Behavior of Some Nutabile Strains of Colon Bacteria
in Synthetic Media. Jour. Bact., 1934, 28:619.

15. Deere, C. J., On the Inactivation of the Lactase of §.coli
mutabile. Jour. Bact., 1939, 37:37.

16. Lederberg, J., The Beta-D-Galactosidase of §.coli strain K12.
Jour. Bact., 1950, 60:381.

17. Cohn, K. and Homod, J., Purification and Pr0perties of Lao-
tase of §.coli. Biochem. et. BiOphys. Acta.,1951,7:153.

18. Kuby, S. A. and Lardy, H. A., Purification and Kinetics of
B-Galactosidase from §.coli K 12. Jour. Am. Chem soc.,
1953, 75:890.

19. Bahn, 0., 0n the nature of Adaptive Enzymes. Growth,l938,2:36

20. Gale, F. E., Factors Influencing the Enzymic Activities of
Bacteria. hact. Rev., 1943, 7:139.

21. Batman, B. and Spiegelman, 5., On the Origin of the Carbon
in the Induced Synthesis of B-Galactosidase in 3,0011.
Jour. Bact., 1954, 68:419.

22. ronod, J.and Cohn, h., Induced Biosynthesis of Enzymes.
hdvances Enzymol., 1952, 13:67.

23. Lester, G. and Bonner, D., The Occurrence of B-Galactosidase
in 3.0011. Jour. Bact., 1952, 63:759.

24.Lowry, O. B., Bosebrough, I. J., Farr, A. L. and Randall,
R. J.,Protein Leasurement with fiolin-Phenol Reagent.
Jour. Biol. Chem., 1951, 193:265.

25. Lalnitshy, 0., Utter, I. 5., and Xerkmen, C. 8., Active
Enzyme Preparations From Bacteria. Jour. Bact., 1945, 49:595.

26. Dockstader, n. B. and Ealvorson, I. 0., A Study in Grinding
Technics for Bacterial cells. Science, 1950, 112:618.

27. Lamanna, O. and Nallette, K. 3., Use of Glass Beads for the
beehanical Rupture of hicro-organisms in Concentrated
suspensions. Jour. Bact., 1954, 67:503.

01

28,

66

honod, J., Pappenheimer Jr., A. N. and Lohen-Bazire, G.,
La Cinetique de la Biosynthese de la B-Galactosidase chez
§.coli Consideree COme Fonction de la Croissance.
Biochim. et BiOphys.Acta., 1952, 9:648.

Lester, 0., The B-Galactosidase of Lactose Yutants cf
§,coli K 12. Arch. Biochem. and 1i0phys., 1952, 40:390.

Lineweaver, E. and Burk, D., The Determination of ?nzyme
Dissociation Constants. Jour. Am. Chem. 300., 1934, 56:658.

Stevenson, h. and Yudkin, J., Galactozymase Considered as
an Adaptive Enzyme. Biochem. J., 1936 30:658.

Witkin, E. M. Genetics of Lesistance to radiation in
§,coli. Genetics, 1947, 32:551.

u I P I: .R a! VII-Ilse:

 

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