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LSU Historical Dissertations and Theses
Graduate School
1957
Studies on the Aerobic Carbohydrate Metabolism
of Leuconostoc Mesenteroides.
Mary Knettles Johnson
Louisiana State University and Agricultural & Mechanical College
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Johnson, Mary Knettles, "Studies on the Aerobic Carbohydrate Metabolism of Leuconostoc Mesenteroides." (1957). LSU Historical
Dissertations and Theses. 213.
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’ULIEii ON PILL APdOnIC CiuidOHYD; .Ai’E M •/Tj.ijULI
ON LEU CON OS TOC MESLIfT U-.OIDEd
A Dissertation
Submitted to tue Graduate Faculty of the
Louisiana State University and
Agricultural and Uechanical College
in partial fulfi 1lment of nhe
requirements lor the ae*;ree of
Doctor of Fnilouophy
in
The Department of Botany, Bacteriology,
and Plant Pathology
by
Mary Knettles Johnson
B.S., Louieiana State University, 1954
M.3., Louisiana State University, 1955
August, 1957
ACKiJOWLJiDGLIFNT
T he a u t h o r v/iahea to e x p r e s s h e r a p p r e c i a t i o n
C h a r l e s J. U c C l e a k e y T or h is
preparation
from
Institutes
and ad v ic e
on
r.
the
of this raanuacript.
T h ia w o r k was m a de
lowships
suggestions
to
po ss i bl e,
the N a t i o n a l
J ci e n c e
of H ea l th .
ii
in part,
by p r e - d o c t u r a l
F o u n d a t i o n and
fel­
the N at i o n a l
TiiHL:
acknov.
e e ;j.
'.'-:ei :t
rl31_ii'* v
i.'I*1 o 1
-
11
.........................................
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vii
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ijX 'L
/v
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„oxti-u-Ji prv . n o t i o n ...............................
.iluco.te dissimilation by Leuconos toe meneuteroices .
l uii)X i ^ti.iC1
« • * • * * * * * • *
ilA k'lljil1kki>^ .k-4■.J 'Jj..',k'lA/
ta *
•
•
Cx eriments on d'-xiran u t i l i z a t i o n ................
j'he effect of pH. on .;lucose o x i d a t i o n .............
Growth ex;;eri,iOi.ta................................
feasurement of cyanide acnj il.v.
................
^oLi^cii'ikjotiO* . trains
* • * * * • * • • •
i trux i'!uiUi
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D I G C U C G I O N .............
i ii
PI
r-4
c\
hex Iran utilization ......................................................................
The effect of p;i on jluco u o x i d a t i o n ............................
Growth experiments
................................
Cyanide sensitivity ......................................................................
Gtrain comparisons
......................................................................
The effect of carbon aoarct- in the growth medium
Ena products of aerobic glucose dissimilation
. .
End products of aerobic t juconate dissimilation.
Pentose oxidation .............................................................................
Peroxidase activity ................................
«
•
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f7
il
u
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'/1 i'A
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‘1^
ia die
1.
The et':'ec t of ^>'1 on fluoone OiiuhUui] ut. Leucunoiitoc
teroimjs a train > ............................
f.
Coiajarir.on of toe v, tes of o ? r;e.. a.. «.nhe on ,du>;u30 ovres
cell aaajmii:; ion 3 of Lencorua toe meat- n to 10 ,de n
j,.
Coimar ison of lue rn tea oi‘ otk.,;ei: u :•tnk<- bt/ rev Iin.f
cella of Lenconoij toe meaen teroid 03 strain y>\L 011
,;luuc,.e t fri.ctoae and sucrose after ;ro .th ii, ..led m
containing eac.i of trrse r. i0 -r a ...................
4*
Cotn.,ariaon of tue rates of os, e., u, n.<e by r e ^ f nL
cello of feuconoatoc tnesentero mfea strain 44o on
e;l'icoje, r ctoae and sucrose after , rowt,, in ul .*•<> -e
mm
one i' m u mod ...a *
•
*
*
*
*
*
«
•
•
•
•
>.
Tne effect of lncose on sucrose ox i-.a t Lon by
Leuconoa toe meaen terumiea strain f4 ' ................
o.
Proaucta of aerobic ,m o o s e breakdown b,, Lor m r m too
me sen teroidea 3 train ff t............................
7.
products 01 aerobic
iuconate cream own b\
Leuconostoc meaenteroideo 3 train 5 4 8 ................
8.
Dx; ren intake on vario 10 ru;a m by J-xyl oa o-r;rown
cells of Leaconoatoe meaenteroiden s'.r.in iff •
;}.
i eroxidnso activity anti oxygen consumy tion by
h lucose-.-.rov;n cell suspensions of Leuconostoc
mesenteroidea a train 54 8............................
'
LIoT
u F FIG.uih.J
Figure
i'he eiYec t ol‘ increuaed .:uri':.ce area ana y..&.kin,j on
the .;rov;t:i ol' Leuoonoa tuc :nea«n teroidea a irain . 40
in r’lucosie m e d i u m ............. ... ....................
vi
A Bo JitAC i1
The fermentation ol' gluuoae by .rowing cultures of
■
Leuconoatoc has been reported to result in the formation of car­
bon dioxide, lactate, ethanol and a relatively high percentage
of acetic acid.
..©cent analyses of strictly anaerooic ;;lucoue
dissimilation by resting cells of Leuconoatoc me sen teroidea strain
39 have shown no acetate unonj tue end .rod sets.
This work iiat
also shown that strain 39 is unable to utilise oxygen as an
electron acceptor in the dissimilation of glucose.
In the present studies, it was found tuat although resting
cells of strain 39 did not consume oxygen on glucose, represent­
atives of five colonial types of L. mesenteroides showed consid­
erable oxygen uptake under the same conditions.
I*he uptake of
oxygen on yluooae was completely inhibited by low concentrations
of cyanide.
The oxidation of glucose by jL . mesenteroides wau found to
require approximately one mole of oxygen per mole of substrate
and to result in the production of approximately equimolar quan­
tities of carbon dioxide, lactate and acetate.
Glucose—grown
cell3 oxidized gluconate with the uptake of one—half mole of
oxygen and the production of approximately one mole each of car­
bon dioxide, lactate and acetate per mole of substrate.
Glucose- and fructose—grown cells oxidized both these sugars}
vii
however, no OJcyr;en uptake was observed on sucrose rith cells
(grown on glucose, fructose or sucrose.
Cells t;rown in sucrose
medium showed no oxygen uptake or carbon dioxide production upon
incubation with purified dextran produced by uie aaiue
train.
Glucose—grown cells gave inconsistent results witu respect to
pentose oxidation.
D-xy 1ose-jrowi cello shov.x-o a low ra te of
oxyjen uptake on (glucose, D-xylone and b-r j bose, but no oxy, on .-.as
consumed v/ith i>- or L-arabinose.
destiny cells of L. raeaen teroides showec peroxiviasu activity,
as evidenced by utilization of nydrayen peroxi.e in the presence
of substrate.
Of the numerous compounds tested, only f,lucose,
(.gluconate and fructose 3erved as substrates for peroxidase activ­
ity .
Lj. me sen teroides exhibited ueaavior typical of an organism
which is facultative
ith respect to utilization of oxygen in
that cultures grew more rapidly in glucose medium under conditions
affordinj increased oxy ;en supply.
The results of this investigation show that several strains
of L. mesenteroides are able to utilize oxy yen as a final hyc.ro(gen acceptor in the dissimilation of jlucooc,
thereby producing
acetate as an end product in place of the ethanol which is formed
anaerobically.
These results explain earlier reports of the
formation of both ethanol and acet te curing ,glucose fermentation.
It is probable that in those experiments air was not completely
excluded from the fermentation flask3 and acetate was produced,
instead of ethanol, in proportion to the amount of oxygen avail­
able to the cells.
viii
INTROJVJCVION
Surveys on the fermentative activities of numerous strains
of Leuconoatoe were conducted by le-.eraon (lShfi) and Hueker and
Pederson (1930).
Growing cultures of all of the strains were
found to produce from glucose relative],
high percentages oi' vol­
atile acid in adaition to approximately euiiimolar amounts of
lactic acid, ethanol and do.,.
The procuc tiou of volatile aciu was
<L
so constant that this ciiarac teris tic was su geu teu as one crite­
rion f>-r the differentiation of Leuconon toe from the true streptococci•
h determination of the „nu. products of glucose fermentation
by resting cells of Leuconoa toe mesenteroides strain 19 (ueLloas,
Bard and Gunsalus, 1 »p 1 '• produced results consistent with the
older work with respect to the u entities of lactic acid, utaanol
and CO . formed from glucose.
However, no mention was made of
volatile acid formation.
In the course of experiments performed in tnia laboratory on
another phase of the metabolism of L. me sen teroides
utiliza­
tion of dextran by resting cells), control tests showed that our
strains of this organism exhibited a considerable
glucose.
This finding was in conflict with the +■
uptake on
BeMosa and
Gibbs (1955) for strain 39» and it was considered that a survey
of the aerobic activities of several strains of L. mesenteroidea
1
inijht yield information by which the apparently anomalous result-3
of studios on jlucose dissimilation by this organism could be
explained.
Included in the survey were studies on tue effect of in­
creased oxygen supply on growth of the organism and the effects
of cyanide, pH variation and tue composition of the jrovvth medium
on oxygen utilization by resting cells,
hnd products of aerobic
glucose and gluconate dissimilation and peroxiaase activity were
also investigated.
hEVIKv; OF LI PiuiA'AfkK
Dextran product! op .
In studying the Function of a prucoas
in a cell, it in in general presumed that the ability of a cell
to carry out a particular reaction is, imuor some conditions at
least, of value to the cell.
If the process were of no value to
the cell, it would represent a waste ox' metabolic material and thus
constitute an evolutionary disadvantage.
In the case of dextran production Dy Leuconostoe, information
is not uvailable on tue function which dextran serves in tne over­
all metabolism of the organism; what, if a ny , value the production
of dextran lias for the or^; .nism is unknown.
oince gum formation by bacteria is frequently associated
with encapsulation of the organisms, it might be tnought that the
production of dextran by Leuconos toe provides a mecuaniain of pro­
tection for the cells.
Hovveve. , this seems unlikely in view of
the large quantity of dexIran wnich is founu in the medium, rather
than attached to the cells in tue form of capsular material, and
the fact that the medium, rather than tue cells, has been found
to be the best source of dextranaacrase from this organism (Carlson,
Rosano and 7/hiteside-Carlson, 1953)The production of dextran as a mechanism for the disposal of
unusable waste material seems equally unlikely oince dextran con­
sists entirely of glucose units and glucose is rapidly metabolized
7
4
by the organism.
Other organisms, <>•£• Aspergil jus ap . {'.Vui teuide-Garlson and
Carlson, 1952) and Penicillium a p . (Tsuchiya, deunes, dncr.er and
'Ailham, 1952), have the ability to hydrolyze dextran.
suggested that Leuconostoe might also bo a b l e
30
.’bin i'act
to u t i l i z e d extran
that, under some conditions, it would serve at a reserve Pood
material .
Glucose dissimilation by Leuconostoc meson teroidea .
Gayon and Jubourg analyzed
In 15.-1
the products of glucose l’ermen tn ti>-n
by an organism which wu 3 very probably a species oi Leuconos toe.
I’hia organism was isolated from spoileu wine and prodsceu mannitol
from fructose.
Under varying cultural conditions,
the organism
produced 5 *2 - 1 2 . 6 per cent acetic acid, 25.0 - 4 4 - 5 per c..nt
lactic acid, lc .7 - 29.2 per cent ethanol a n d 1 ' .2 — 2 2 . 8
CC^ from the glucose fermented.
per cent
.mccinie acid (0 . 6 6 g per 1 00 g
glucose) and glycerin (9 * ^ 8 g per 1 0 0 g glucose) were also de­
tected .
Pederson (1429) reported that growing cultures of Leuconoatoe
mesenteroidea fermented glucose to approximately equimolar quanti­
ties of lactic acid, ethanol and CO .
A "relatively high per­
centage" of acetic acid was also formed.
Further study of a
series of Leuconostoc cultures from a variety of sources (Hucker
and Pederson, 1930) showed that all these strains produced ap­
proximately 4 5 per cent lactic acid, 20 per cent CO^ and 25 per
cent volatile products, including both ethanol and acetic acid.
A high degree of volatile acid proauction was described as a
5
criterion for diii'erentiat ion of Leuconoa toe from streptococci.
A study of the fermentation of glucose by L. ueaen teroidea
was unaert: ken much later by jeMosa, bard and Gunaalus tl‘>pl; in
an attempt to determine whether ethanol is produced by bacteria
by means of the same pathway (Kmbden—Ueyerhof) as hac been founu
to be operative in yeast.
Resting cell suspensions of strain 39
were used and glucose was found to be fermented to euuimoiar
Quantities of lactic acid, ethanol and C0„ .
of any volatile acid production.
No mention was i;u.de
Variation in pH <1iu not in­
fluence the ratio of the fermentation products.
Cell-free ex­
tracts were found to contain di duosphopyrid ine nucleotide (ldJN)
linked dehydrogenases for ethanol, lactate,
triosephosphate and
2,3-butjlene glycol.
that tue extracts
No evidence was found
contained any aldolase,
iaomerase or carboxylase activity,
fhese
workers concluded that u departure from the ..mbden—Meyerhof path­
way was indicate., altnough ethanol and lactate seemed to arise
from their usual precursors, acetald'
ie and pyruvate, respective­
lyGlucose fermentation was investigated further (Gunaalus and
Gibbs, 1952) by means of tracer studies.
In the case of cells
fermenting glucose-l-C^, all of the radioactivity was found in
the CCh produced.
When glucose-3,4 - C ^ was used, none of the
labeled carbon appeared in the CO^, half wa3 found in the ethanol
(carbinol carbon) and half in the lactic acid (carboxyl carbon).
It was concluded that the CO^ arose from carbon atom 1 in the
glucose molecule, ethanol from carbon atoms 2 and 3, and lactic
acid from carbon atoms 4» 5
6-
The fact that glucose— 1-C"^
6
was fermented in such a way that ail of the label appeared, in the
CO^ waa considered to be evidence that the hmbaen-hleyerhof path­
way v/as not operative, since this median ism would have reunited
in the formation of methyl-labeled ethunol and lactate, and un­
labeled 00 •
In addition, it waa found that small quantities of acetate
increased the rate of glucose fermentation, presumably by acting
as hydrogen—acceptor.
methyl-labeled ethanol.
Acetate— 2-C^^ waa reduces by tht cells to
It was sugjestea
that acetate jives rise
to the ethanol formed in the fermentation of glucose.
This study revealed also that cells of L. niesenteroiues con­
tain a jlucose-6-ph03phate deh,, drojenace .
fhe enzyme was partial­
ly purified (helloes, Junrsalus and hard, 1 9 3 3 } and found to utilize
either DPH or triphosphopyridine nucleotide ( illi) as coenzyme.
The presence of this enzyme and the data from the reports pre­
viously discussed led these workers to suggest that the fermenta­
tion of glucose proceeds by way of a hexose monophosphate type
pathway, in this case an anaerobic one, involving deiq,drogenation
of glucose—6—phoaphate, followed by oxidation, cecarboxylation
and cleavage reactions to yield
and Cf moieties, precursors of
ethanol and lactate respectively.
The participation of a pentose phosphate in the pathway was
oonsidered unlikely since no compound of this type was found to
accumulate during fermentation or to be consumed by the cell ex­
tracts (DeMoss, 1953) ■
It w aa suggested ohat cleavage of a keto-
6—phosphogluconate mignt occur.
Enzymatic experiments indicated
7
that oxidation of 6-pho3phogluconate precoded cl- ..vage-,
.'he enzyme catalyzing the o> iu^uon of 6— phonphogluconute was
partially purified ( Doliosa , 1994; heI.Io3a an., bibbs, 1959).
A
3tudy of the products of the activity of tni3 enzyme, when coupled
with reduced DP'N oxidizing systems, indicated that CO^t riculose
phosphate and at least two unidentified compounds were present.
It was proponed that the ribulose phosphate and CO,, mrght have
arisen from the chemical decomposition of a heto-pnosphogluconute,
the hypothetical intermediate.
In discussing the results of this series of experiments on
glucose fermentation by L. me 3 enter 0 iJ.es, Jel/.oss ana bibbs (1955)
noted that the growth of this organiam is uninhibited by conditions
of complete anaerobiosis .
They stated that resting cell fermenta­
tion of glucose, either in the presence or absence of 0. , led to
the same end products.
It was conduces
that the electrons re­
moved from glucose-6—phcaphate arm 6-phosphogluconate do not trav­
erse the same pathway as in yeast or Ischeric:;ia coli under aerobic
conditionsj in L. toesenteroidea the electrons rnus l be „rnnsferred
to acetaldehyde and pyruvate, forming ^he end products ethanol
and lactic acid, while in yeast and h . coli the electrons are
transferred to 0^•
A quite different type of pathway for the breakdown of glucose
by L. meaenteroidea has been proposed by Antoniani, Federico and
Fleischmann (1956)*
They suggested that glucose is split into
two molecules of glyceric aldenyde, one of which is changed to
lactic aoid and decarboxylated to ethanol,
the other being oxidized
to pyruvic acid followed by reduction to lactic acid.
8
Peroxidase activity.
bumner and homers vl933) state mint
nearly all plant cells contain peroxidase.
^ever'.l animal per­
oxidases have also been studied, _e.^« salivary peroxlcase, lactoperoxidaae, tryptophan peroxidase and myeloperoxiuaue.
1'hese
enzymes catalyze the oxidation of a number of compounds by hydrogen
peroxide.
Clifton (1957) states taut there is doubt as to the
presence or activity of peroxidases in bacteria.
however,
there
are in the literature numerous reports of peroxidase activity in
various species of bacteria.
Douglas (1947) found that suspensions of Lactobacillus brevis
had no activity upon lactate alone anaerobically, and no activity
upon hydrogen peroxide alone, aerobically or a n m robxcally .
How­
ever, when the cells were incubated anaerobically with lactate plus
peroxiue,
tue lactate was oxidized to acetate and the nydrogen
peroxicie reduced to water.
i’he author concluded that this
rganism
contains an enzymatic mechanism for activating hydrogen peroxide
as an oxidizing agent, and that this enzyme would, by definition,
be called a peroxidase.
He pointed out, however,
tnut the reaction
was not sensitive to cyanide, in contrast to the peroxidase in
plant and animal tissue.
In addition,
the usual tests for per­
oxidase were negative - that is, substrates usually oxidized by
peroxidase were not attacked by these ceils.
In spite of those
differences, Douglas stated that it seemed probable that in the
normal metabolism of Lactobacillus brevis, the peroxide which
theoretically should be formed as the first reduction product of
O^, is activated enzymatically aa a hydrogen acceptor and reduced
to water as rapidly as it is formed.
hydrogen peroxide did not
9
accumulate upon aerooic oxidation of glucose and lactate.
An adaptive peroxidation by dtreptococcus faecalia waa re­
ported by .jeeley and Vandemark i,2 9 19 *
Although no disappearance
of ad. ed hydrogen peroxide could be detected upon incubation of
cells in the absence of substrate
there waa no cataiaae
activity), peroxide waa rapidly decomposed by cell suspensions in
the presence of glucose or glycerol.
Lactate aluo servec as sub­
strate, but the i'ute of peroxidation waa decreases.
ihe peroxida­
tion waa "adaptive" in that aerobical ly-grov/n cells snowed much
higher peroxidase activity
than cells grown under condition.-, of
limited oxygen 3upply.
Another study of the peroxiuase activity of
. faecalia
(Seeley and del Kio-bstradu, 1951) indicated tuat riboflavin
plays a role in the format j.on and disposal of nyCro. en peroxide
by this organism.
Cells grown aerobically were found to contain
over twice as much riboflavin as anaerobically-grown ceils.
grown aerobically and transferred
Cultures
to fresh aerocic medium did not
form free hydrogen peroxide in the medium regardless of the ribo­
flavin content.
On the other hand, cultures grown anaerobically
and transferred to fresh aerobic medium formed considerable amounts
of free hydrogen peroxide in direct relationship to the amount of
riboflavin supplied.
conditions,
After a short time of exposure to aerobic
the hydrogen peroxide initially formed was disposed
of, provided sufficient oxidizable substrate was available to sup­
ply reduced carrier.
These workers stated that the evidence indi­
cated that in cultures having the peroxidative ability, the disposal
10
of free hydrogen p-roxide
takes place by reaction Aitu a reduced
flavin compound.
Bolin il9Sj) discussed the findings of various workera that
cell suapenaiona of certain strains of streptococci c.rry out
peroxidase-like reactions in the presence of some organic sub­
strates.
He suggested
that this might explain
the fact that the
terminal respiration of streptococci does not invariable ^ieia
peroxide although it passes through flavin carriers ana these
organisms contain no catalaae or oy tochrorne components.
extracts of o_. faecal is were prepared and to m d
potent reduced DPN (DPNH^) oxidizing system.
were no demonstrable cytochrome components,
Jell—free
to contain a
Alt:ioue_.i i, a n t
cytoeurome c acted
as an electron acceptor and iA its presence, DPlfd^ waa oxidized
about four times as rapidly aa in air.
iiieie was no cytochrome c
oxidase or cytochrome c peroxidase activi y.
Jhe oxidation of
BPKK^ in the absence of cytocurome c occurred
vita tue uptake of
-i- mole 0
per mole of DPNh
<1
utilized, althouga tue extracts were
C.
completely devoid of catalase activity and no peroxide accumulated.
The oxidation waa shown to involve an intermediate peroxidation
as follows:
DPNH
+
DFNH2 + H20 2
*
DFN
+
H^02
*
DPN
+ 2 H20
The enzyme responsible for the peroxidation was completely separated
from the oxidase by ammonium sulfate fractionation so that aerobical­
ly there was no reaction in the absence of added hydrogen peroxide .
A departure in the nature of this peroxidase from that of the
classical peroxidase was indicated by the fact tnat there was no
inhibition by 0.01 Id cyanide.
The properties of this enz. me wert investigated m r t n o r by
Dolin (1934)*
i'he enzyme did not utilize reduced TfN;
substrates for heraatin peroxidase,
c, were not attacked.
typical
including reduced cytochrome
The purified enzyme contained flavin
adenine dinucleotide and small amounts of lipoic acid.
However,
there was no conclusive evidence for the function of thi-se com­
pounds in the reaction.
The enzyme was not inhibited by Versene
or 8-hydroxyquinoline or by exposure to strong ultraviolet light.
In a search for other peroxidation systems, it was found that
cytochrome c catalyzed the peroxidation of DPNII^} the latter reac­
tion had the same pH optimum as the ji. faecal is enzyme,
but it
was heat stable whereas the bacterial enzyme was extremely heat
labile .
In a discussion of the role of the peroxidase enzyme in the
metabolism of J_. faecalia, Dolin (1955) stated that the enzyme
offers these organisms a competitive advantage over strict an­
aerobes.
It enables the organisms to utilize oxygen aa an alter­
native hydrogen acceptor without accumulating peroxide, even though
the terminal oxiuases are flavoproteins.
There is no loss of ef­
ficiency compared to the lactic acid fermentation, since neither
the oxidation of DPNH^ by pyruvate nor its peroxidation is ener­
getically coupled.
Dolin stated further ohat it is still a
question for conjecture whether conditions can be found under
which the oxidation and peroxidation pathway can be coupled to
phosphorylation.
In any event, pyruvate is removed from its role
12
as the obligate hydrogen acceptor, and it is tiien capaole of being
utilized oxidatively in a reaction which yields one aubutrade­
linked phosphorylation via acetyl coenzyme A.
A 4-0 per cent in­
crease in cell yield under conditions of aerobic growth was said
to illustrate the importance of the peroxidase to the organism.
Peroxidase activity has also been reported in dtreptococcus
roitiu (Cla per, Meade and Haiby, 1954)*
Aerobically-grown cell3
had much higher peroxidaue activity than anaerobically-grown cello,
while boiled cells would not catalyze the reaction and catalase
activity was shown to be absent by the fact
required for peroxide utilization.
that suosurate was
o u l f a t h i a a o l e - r e s t a n t strains
produced a much more active peroxidase than the sensitive parent
strain.
In a discussion of the mechanisms of electron and uyui-ogen
transport, Kaplan (LcElroy and Glass, 19i4) stated than an extract
from Pseudomonas fluorescens had peroxiuase activity in the presence
of reduced dyes or reuuced DFN an., hydroyen peroxide.
Cells ,;rov;r.
under good aerouic conditions had very little peroxidase activity,
whereas cells grown at low oxygen tension showed extremely hitlh
peroxidase activity.
No information is available regarding the formation or disposal
of hydrogen peroxide by Leuconostoc mesenteroides, although it
might be expected that this organism would
3I10W
peroxidase activity
in view of its close relationship to the lactic acid bacteria pre­
viously discussed.
u a t e h i a l ^ a ;id m e t h o d h
The following strains of Leuconoatoe mesenteroidea were em­
ployed in tnese experiments:
1 5 4 * 730, 719* 5 4 8 , 8u0 and 2 4 8
(McCleskey, Favilie and Barnett, lp47) •
strains 154
73^ ure
repi'esentative3 of colonial tt pe A and strains 719* 54-8 <md 860
represent types B, 0 and F, respectively,
intermediate strain.
strain 2 4 6 is a u-F
A culture of strain 35 vvaa secured from Or.
f a1ph LeMoas.
The basal taedium used in all the experiments contained 0*5
per cent tryptone, 0.25 Per cent yeast extract and 0.3 per cent
KjHPO^, at a pH of approximately 7*
glue ose was used for maintenance of
This medium with 0.5 por cent
3
took cultures.
Uas measurements weru made with the Aarburg constant volume
respirometer using the "direct” methods of 0^ and CO^ determina­
tion as described by Umbreit, Burris and Stauffer (1951)*
Air
constituted the gasaous phase, and tue temperature was 31 0.
Ma­
terials in the side arms of the 7,'arburg vessels were tipped into
the main oompartment after a 5 minute equilibration period.
Experiments on dextran utilization.
produced in the following manner.
Purified dextran was
One liter of basal medium con­
taining 10 per cent sucrose was inoculated with a stock culture
of strain 730 and incubated for 6 day^. at room temperature under
stationary conditions.
The medium was then diluted 1:1 with
13
14
distilled water and paa.'.ed through a Jlmrple 3 centrifuge to remove
the cells.
Dextran waa precipitated by addition 01' an equal vol­
ume ol' cold 9? per cent ethanol, followed by centri fuga t-ion to
collect the precipitate.
The dextran was then iaken up in water,
reprecipitated with ethanol and again collected by centrifugatian .
The precipitated dextran was aried for 2 dayu at 95 0, pulverized
and suspended in water at a concentration of 1 per cent.
Cell suspensions of the same strain were prepared by inocula­
tion of 300 ml of the basal meaium plus 0.1 per cent sucrose, fol­
lowed by incubation at room temperature under stationary conditions
for TO hours.
The cells were collected by centrifugation in an
International refrigerated centrifuge at 4 C and 2,Su0 rpm and
suspended in 20 ml of Allison's solution, w ich contains 0.7 &
K^HrO
. 3 H^O, 0.3 g KH^PO^, 0.2 g DaCl, 0.2 g ligOO^ . 7 H^O and
0.1 g CaSO^ • 2 H^O per liter of distilled water.
The above— listed
temperature, speed and model of centrifuge were used for all cell
suspension preparation metnods.
Cultures used in this and all of
the following experiments were checked Tor purity by smear and/or
streaking on raw sugar medium.
Dextran utilization was teatou by measuring 0^ uptake and CO^
production by cell suspensions incubated with thi3 material.
Each
Warburg flask contained 1 ml 0.063 M phosphate buffer and 1 ml
oell suspension in the main compartment, 0.1 ml 10 per oent KQH
in the center well (except for CO^ measurement when the center
well contained 0.1 ml distilled water) and
in the side arm.
ml dextran solution
Control flask 3 ido arms contained ^ ml distilled
water or 0.1 11 glucose instead of the dextran solution.
15
The effect of pH on glucose oxidation.
Cell suspensions
were prepared by inoculation of 12b ml medium (with 1 per cent
glucose) from a stock culture of strain 730 .
The i hi ik was in­
cubated for 2? hours at room temperature, after which the cells
were collected by centrifugation, washed twice with phosphate-free
A1li3on•s solution and suspended in 10 ml of the same solution.
Oxygen uptake on glucose was measured at pH 5.0 and pH 7.0.
Growth experiments.
The basal medium plus 1 per cent glucose
waa used to measure t:ie effect of shaking and increased surface
area on tue growth of strain 246.
triplicate
.rlenmoyer flasks
of 1,000 ml and 250 ml capacity containing 200 ml of medium were
inoculated with 2 ml portions of an Id hour culture waich had been
grown on a rotary 3 haker .
The larger finals were incubated on
the shaker at room temperature anu the smaller flasks under sta­
tionary conditions at the amae temperature .
the amount of growth
was measured periodically on duplicate aliquots by determination
of the optical density at 5^0 n^£ on the B & L dpectronic 20
spectrophotometer.
Measurement of cyanide sonsitivity.
Cell suspensions of
strain 246 were prepared by inoculation from stock culture of 5 0
ml of basal medium plus 1 per cent glucose in a 300 ml flask.
The flask was shaken for 20 hours and 5 m l of this culture used
to inoculate another flask of the same medium.
on the shaker for 18 hours,
After incubation
the medium was centrifuged and the
cells washed twice with Allison's solution.
I*he resulting suspen­
sion had an optical density of 0.68 measured at 4 2 0 ngj after 1 1 10
16
dilation witu dis tilled water.
(i'kis wavelength waa uueu f >r all
meadureinonts of tiie optical density of cell suspensions -)
Cyanide
3ensitivity of oxygen uptake on glucose was determined by measur­
ing the rates of 0
10 ^ M KCN.
uptake in aie presence and absence of 4 *6 x
This concentration waa maintained by tue method de­
scribed by hobbie (1948) which involved addition of u.l ml of 4*35
M KCN to the center well, the main compartment uaving a KC.. con­
centration of 4 •<-' x 10 4 LI.
One ml of cell suspension and 1 ml
of phosphate buffer, pH 7*0, were incubuted with _ ml 0 ,02 il
glucose•
Comparison of strains.
fifty ml amounts of tne basal medium
with 1 per cent glucose in 300 ml flasks were inoculated from stock
cultures of strains 39, 154, 246, 54o, 719 and 8 o 0 .
fhe cultures
were incubated at room temperature for 24 hours on a rotary shaker
and then added to 390 ml of fresh medium in a l,GuG ml flask.
After 20 hours incubation on the snfiker, the ceils were collected
by centrifugation, washed once ..ith .-til is on 'a solution an 1 final­
ly suspended in Allison's solution.
-ne suspensions were adjusted
to give a reading of optical density (J.75 upon 1:20 oil ution .
A
comparison was made of the rates of 0^ uptake on glucose by the
six strains.
The above described method of preparation of cell suspensions
was used for all other respirometer experiments with the exception
of the ones for which methods have already been described.
The
sugar supplied in the growth media, which war glucose unless other­
wise noted, was added at a concentration of 1 per cent.
17
Chemi cal analyaes .
frior to c h e m i c a l a n a l y s i s ,
-rnrbur,-; i'laak
contents were centrifuged at 2 ,8 0 u rpm and 4 0 to remove tiie cells.
The supernatant fluid waa frozen unlesa tue analysis c o u l d be per­
formed immediately.
dilutions of this m a t e r i a l w e r e made wi th
twice distilled water.
Glucose was determined aa reducing sugar by tue method of
Somogyi (1941 ?) using the technique of color development described
by Nelson (1944)-
The copper reagent consis Led of 28 g anhydrous
Na-IIPO , 100 ml 1 N NaOh, 40 g Nochellu 3 &lt, 8 g crystal 1 ine
2
4
CuSG
. 5 H^O and 18 g anhydrous
per liter of water.
Nelson's
chromogenic reagent was prepared by dissolving 25 g ammonium
molyrbdate in 4p0 ml water, addin
3 g Na^HAsO^ . 7
21 ml concen ir ted 11 30. and then
<- 4
dissolved in 25 ml water.
The test was per­
formed by a..ding 2 ml of the copper r-.agent to 2 ml of a sample
containing between 10 and 200 ,ug glucose in a te3 t tube.
The tubes
were covered with loose-fitting aluminum caps, immersed in a boil­
ing water bath and heated for 10 minutes.
,-fter cooling to room
temperature, 2 ml of Nelson's reagent were added and the solution
diluted to 10 ml.
The intensity of the color developed waa measured
at 5 ^ 0 rqju and compared v,ith Uiat of une standards containing known
amounts of glucose•
oucroae gave a negative teat with this method without prior
hydrolysis.
Hydrolysis was accomplisheu by the addition of 0.2
ml Difco invertaae to 1 0 ml of the diluted sample and incubation
at room tempera lure for 20 minutes, after which reducing sugar
was measured by the method described above.
18
Lactate was determined colorimetricully by tne metnod of
jJurker and hummerson (l94l)*
A aarnjile containing from 30 to C>G
Mg of lactic aciu was placed in a 15 ml graauaieu eti: tr ifugo tube
and made up to a volume of 9*5 ml .
CuSO^ . 5
One-naif ml of 20 per edit
and approximately G.y g CatOIi)^ were then adued and
the precipitate diaperaed by snaking.
The tubea were allowed to
stand for 30 minutes at room tempera turn with frecuent shaking to
disperse the precipitate.
After cen trif ugation for 5 minutes at
1 ,5U0 rpm, 1 ml of the aupernatant waa carefully transferred to a
large tent tube.
The tubes were then immersed in an ice bath,
and to each o ml of concentrated
a 50 ml burette.
were adued dropwise from
The contents of the tubes were mixed thoroughly
by constant ahnf.ing during the addition of m e
acid.
:'he tubes
were then heated in a boiling water bath for 0 minutes and cooled
to below 30 C, after which 1 drop ^acii of b [ er cent CuSO^ • 5 H^C*
and of para-hyriroxydiphen; 1 reagent 5 I .p g para-iiydroxydiphenyl
in 100 ml of 0*5 per cent NaOH) were added .
.’he precipitate wuich
f rmed wus immediately dispersed by shaking and the tubes incubated
at room temperature for 30 minutes .-its occasional shaking to redisperse
the reagent.
After heating in a boiling water bath for
9 0 seconds and cooling to room temperature,
the samples were
transferred to colorimeter tubes and the optical density read at
5 6 5 nyi.
Comparisons were made with standard solutions of lithium
lactate .
An attempt was made to determine volatile acid production
by the method of Conway (1950) •
At best, only 66 per cent of
known amounts of acetic acid could be recovered, regardless of
19
incubation time or sealing u/:fcnt used .
L’he iefore, tiie volatile
acid was sopara ifd by u team d is ti 1 1at Lon ( the aiatil 1ution : luuk
con tainin.r
, 2 ml 10 0 ILSi-.
4 c
t' ••
J a 10 ml sample
j- J
t; 4 and ^
^'.gS0.
4 • 7' H,,0
2 ’'
and titrated with 0.02 N ha' H from a micro-burette, using phenolphthulein as indicator.
Twice distil .eil water was ured for di­
lutions, steam generation, blanks and for rinsing ol' all
*he
equipment .
The volatile acid was identifier a
acetic by mea..ureinent of
its ether-water partition constant according to the method of
Pel czar, Hansen and Konetzka (l
'- V . ) .
a ..ample of 12 mi of aia-
tillate was placed in a ot ml separatory funnel and to it was
added 20 ml of etner.
After shaking for 1 minute, the mixture was
allowed to stand foi 2 minutes.
A 1... ml sample of the aqueous
layer was removed and titrated.
The partition constant was calcu­
lated by dividing the ml of alkali consumed in titration after
ether extraction by the ml consumed in titration of tne same quan­
tity of distillate before extr o t:on .
Peroxidase testa.
Cell suspensions ».en
ly described except that after wasuin^,
prepared aa previous­
die cells were suspended in
a solution containing 1 volume of Allison's solution and 1 volume
of 0.25 & phosphate buffer, pH 7*0.
Cells were tested for peroxidase activity as follows.
One
ml of cell suspension was placed in a test tube v/ith 1 ml water.
One-half ml of substrate (50 uM) and 2-^ ml H^O^ (75 us) were added,
and the tubes were allowed to stand at room temjierature for 60
minutes with frequent snaking to keep the cells in suspension.
20
At the end of trie incubation period,
trr. cells were removed oyr
centrifugation at 2,50o rpm and 4 0 for ru minutes, anu 1 ml of
the supernatant fluid was analyzes for residual
spontaneous peroxidation, each suostrate was
of cells.
The
tion with H„0o
£_ i-
•
To detect
tested in the absence
cells were tested for catulaae activity
by incuba­
in the absence of substrate.
I.ydrogen peroxide was determined uy the me tnou of iiain and
Shinn (193 ') using a potato extract prepared as follows.
One
hundred and twenty" g potato were ground with 6b ml of U.12S M
phosphate buffer, pH 7-0, in a 'baring blend or •
The suspension
was poured into a bCG ml rrlemneyer flask, mixed sith op ml glycerol
and covered with a layer of mineral oil.
alter refrigeration over­
night, the supernatant was decanted and centrifuged for 20 minutes
at 2 , 8 0 0 rpm and 4 0 to remove the debris.
ine supernatant was
again decanted and tnen stores in tue refrigerator.
f .r detection
of H^O^, from 0.1 to 0.3 ml of tnis extract (depending on the
length of time since its preparation) was adued to 1 ml of sample,
containing approximately 15 US ^2^2* an^ ^
w a ^e r *
i,wo drops
of ortho-tolidine reagent (1C per cent solution in glacial acetic
acid) wer.
added, and trie color which developed after a few minutes
compared v/ith that of a standard
containing a known amount of
^2^2 (PrePare<* from 30 per cent licrck Reagent Grade) and a blank
containing no
In the presence of
tlie fotato e*tract
catalyzes the peroxidation of ortho-tolidine to a blue-green
colored compound.
reaction.
Cells of L. mesenteroides do not catalyze this
RESULTS
Observations made in the course of experiments on auxtran
utilization,
the results of which are described below, suggested
that a survev oi‘ the aerobic carbohydrate metabolism of several
strains of Leuconoatoc meaenteroidea might yield information
whicu would explain the conflicting reports in the literatui’e con­
cerning glucose dissimilation by this organism.
fhi3 survey in­
cluded studies on oxygen utilization oy ,*row ing and resting cells
and investigations of peroxidase activity
and ti.e nature of end
products formed under aerobic conditions.
Eextran utilization.
destiny cell aujj-ensions showed no 0^
uptake or GO^ evolution upon incubation with Jextran for as Ion#
as 15 G minutes•
However,
in the control flasks where glucose was
supplied as substrate there was an 0^ uptake of 55
Per hour ac­
companied by an approximately equal rate of CO^ evolution.
Effect of pH on glucose oxidation.
Measurements of the rates
of C>2 consumption on glucose in phosphate buffers of pH 5 *0 and
7.0 showed (table l) tuat the reaction was favored by the latter
level of hydrogen ion concentration.
All of the following res­
piration
experiments were, therefore, run at pll 7*0.The drop in
pH which
occurred during the oxidation of this small amount of
substrate (10 ,11m) indicated the need for a stronger buffer in
21
22
fable 1
ihe eii'ect of pH on glucose oxidation by
Leuconostoc me sen teroides str in 73 0
Initial pli
0^ uptake
in
30 minutes
wl
5.0
75
7.0
108
Final pH
5 -9
Main compartment*
1.0 ml 0.03 1.1 phosphate buffer, pH 5*0
or 7 *0; 1.0 ml cell suspension; U.5 ml 0.02 M glucose.
Center well* 0.1 ml 10 per cent K0K.
23
further experiments.
frov.-1 ix experimen t3 .
Jince t.iu use of an nt-rc; i.e pathway
should enable the organism to grow more rapidly under conJiiioin;
affording; an increased oxygen supply, b'row th ratet, in glucose
medium were compared for mationar; and shaken cult inu.
results of thin experiment are Known in figure 1.
Die
It in appar­
ent that tiiC increased surface area and .-...akiny of tue larger
flask promoted much more rapid ^rowtu t .an that under 3 tationary
conditions.
The leveling off of growtu at If hours in the shaken
culture was probably due to the large amount 01 acid prodsceu,
since the pH uad dropped to 4
Cyanide sensitivity.
at that time.
fota.oium cyanide at a concentration
as low aa .\*o x 10 -4 0 was found to inhibit completely the uptake
of oxygen on glucose by suspensions of resting' cells.
Jtrain comparisons.
a comparison of the rates of 0 ^ uptake
on glucose by 3ix strains of L. meaenteroides is auown in table 2.
There is considerable variation in trie rates, strain 39 showing
no oxygen uptake whatsoever,
since number p.io displayed the high­
est rate, this strain was used in all the following experiments,
although at times other strains were included for comparative pur­
poses .
The effect of carbon source of growth medium upon subsequent
oxidative ability of the cells.
The rates of 0^ consumption on
glucose, fructose and sucrose are compared in table 3 for cells
24
0.6
SHAKEN
OS
0.4
0.1
so
36
HOURS
Figure 1_. The effect of increased, surface area and shaking
on the growth of Leuconoatoc meaen teroidea s train 246 in glucose
medium. The shaken cultures consisted of 2uC ml medium in a
1,000 ml fla3k ana the stationary cultures of 200 ml medium in
a 2 5 O ml flask, both with 1 per cent inoculum.
25
Table 2
Comparison of the rates of oxygen u p u k e on glucose
by resting cell suspensions of Leuconostoc inosenteroides
0 train
(.r up take
ul/hour
'>4.0-lJ
062
7 1 S-B
•■‘
.45
240
37:
154-A
32 7
8 f0-F
l-',
39
0
Main compartment:
1 ml cell susi-er mio nj 2SO
phos­
phate buffer, pH 7*0. oide arm:
50
glucose . Center
well:
0.1 ml 10 per cent KOI!. Final volume: 2.5 ml*
26
’able 3
Coin], a r ia on of ine cater, ol- oxj.-ron
r.y recti nr cel la
o f L e u c o n o a t o c me sen t e r o i d e a a t r a i n rfb on .yluco.-.e , l i M o t o s e
a nd a u c r o c e a f t e r .;row th in m eu L.i con laini;n- e,.oii i,f tueje
0
,. uptake
J u 0 3 trate
01 ucoue—(prov/n
0 1u c0 ae
Fructose
'iucroue
1'ruc
uu.iC- ;i■lvvn
^j 1
Ml
r,C
■.1
u croc e— t;rown
^ul
u
0
500
'\y
0
Uain ccmpurtnient 1 1 ul cell aua^enuion of optical density 1.0 on
It 10 dilution; 2 5 0 ^*' phosphate buffer, pi; ',’.0 . ,.,ide arm:
5 0 m IA
substrate.
Cen ter .-.ell: 0.1 ml 10 per cent nOJI. Final volume:
2.5 nil* Time of incubation:
105 minutea .
27
ejrown in ;.;edia containing each of tnese ju,;ars .
It can be seen
that the rates of 0 ^ uptnKe on f;lucoae ano fructose were inde­
pendent of which of these two sugars was used in t.ie
row t:. med i u m .
No oxyjen up take waa measurable on aucroae by e;lucoau- or fruc losegrown cells, or on any of these aurui'o by sucrose—t;rown cells.
Measurement of residual aucroae, however,
indicated
ina l a con­
siderable amount had been utilizeu by tne ceils.
ladle 4 O'^veo the results of a similar experiment with s - r a m
?.4 6 .
/v,*ain, there was no 0 ^ cons amp t ion on sucrose by eituer
(jlucoae- or sucrose—,-rown cells, and
hie rates of ylucose oxidation
exceeded that of fructose ox i -.at ion .
do. eve:-, sucrose—,jrown cell.;
of this strain did show a small amount of 0
uptake on ,;lucoae and
f ructose.
The possibility
t<*a t
utilization of sucrose was
lu cose ...i
tne aerobic
teste... b,» t.easurinj 0 ... up take on sucrose
in tne presence of a small amount of
5 show
t " irp^.er"
.lucose.
fhe results in table
Lu& t tne amount of oxy yen consumed by cells in the presence
of sucrose plus (glucose was ei.tal to tiiat consumed on jiucoit as one .
Ilnd produc ta of aeroo ;c ,^1 ncone dissimi 1a t ion .
fne i-esults
of analyses of tne products of aerouic glucose t.issimilution by
3 .rain 5 4 8 are listed in table 6 .
these results represent the
average of several tietenninations in which
tne substrate waa ad^.ed
at various concentrations and the suspensions incubated until com­
pletion of oxyjen uptake.
The oxidation of one mole of glucose
is shown to require approximately one mole of 0 ^ and to result in
the production of approximately one mole each of dC>2 » lactate and
acetate .
28
Table 4
Comparison oi' the rates of 0^ uptake by resting cells
of Leuconos toe mesenteroidea strain , 4 8 on , lucooe, fructose
and sucrose after growth in jlucoae ar.ii sucrose media
°2
uptake
Cuba trate
Glucose -tyr own
Glucose
fructose
ducrone
duorose—prown
ij 1
4il
314
->3
3 .*
22
0
0
Main compartment: 1 ml cell 3110; ension of optical density on
lllO dilution of 1.0 i'or <L/1 ucoae-jrown and C . 4 5 f°r sucro.je­
t;rown cells; ?50
phosphate buffer-, p ll 7*0.
dido arm:
jxtl substrate.
Center well:
0.1 ml lo per cent h O h . Pinal
volume:
2.5 nil.
lime of incur), lion t 60 m i n u t e s .
Table 7
iiie effect of ..lucose on sucrose oxidation
by Le iconoatoc mesenteroices strain bh8
0^, uptake
Ou bstra te
lluco.-ie-,rr own
ul/hour
b0 ul! sucrose
.juci'oso-jrown
Lil/hour
0
C
50 uM sucrose +
5 uM plucoae
II 3
O
5
111
0
jlucoae
Main compartment:
1 ml cell suspension of optical density
1.0
on 1:10 dilution; 2^0
phosphate buffer, pH 7*0.
hide
arm:
substrate as indicated.
Center well:
u.l ml 20 per
cent K O H . Pinal volume 2.S ml.
30
Table 6
Proeucta of aerobic glucose breakdown by
Leuconoc tcc meser. t.vrniden a train 54^
Average*
uli
Glucose used
Iian^e
11M
100
Oxygen uptake
i'4
90-103
Carbon dioxide
99
95-106
J.ac la te
0 ;i
Bo-112
/icc t* te
las
102-110
Main coinpartment: 1 ml cell juj;.enaion of O.A ical
density 1.0 on 1:10 dilution; 2 5 0 uii phosph te buffer,
pH 7-0. *jide armi
50 or 60 ui.1 ^lucooe . Center welli
0.2 ml 10 per cent K0 K . Final volume 1 2.5 ml*
♦These figures represent avera&-e3 of several deterininatione calculated on the basis of 100 ^uM substrate used.
31
End products of aerobic gluconate disaim!lation.
.able 7
shows the results of similar unaljaej of t;i ueoiia t.e d i. s iiai 1a t ion
products.
Hall' as much 0^ was required for ;a« ox lea Iaon o; tins
{L
substrate as was required for tar oxidation o;‘ jluco -»e .
.'he
products, however, were the same with bot.i substrates, _i*£*» ap­
proximately equiraolar amounts of' C>... , lactate and uce-t-. te .
ientoae oxida lion.
Ho consistent results wits res.ect to
rates ol' pentose oxidation could bo secured ./its _ Lucose-. rown
cells.
ihi3 result was in agreement with the experience reported
by Aitormatt, Hluckwood and h'eisli
I: ./ •) concerning jen^oae fer­
mentation by ,
■;1ucose—grown cells of L. mesen teroiues .
on D-xyloge, however, 3nowed consistent rates of 0
on glucose ant pentoses (taule 8).
Cells grown
consumption
Cells grown in a xylose me.' ium
to whicn a small amount of glucose had been auaed showed & much
higher oxygen uptake on glucose (jdC ul), but showed tne same low
rates wuen pentoses were supplied as ua t>s tru .ea .
hue to the low
rate of aerobic fjentose breakdown, no attempt \va3 made to identixy
the end products.
Peroxidase acti vi ty .
tiliuation of K^O,, by cell suspensions
in the presence of substrate was considered a demonstration of per­
oxidase activity since controls (cells plus
showed that catalase activity was absent.
with no substrate)
CellH boiled for 10 min­
utes previous to testing showed no peroxidase activity.
fable 9
lists the compounds which were tested for their ability to serve
as substrates for peroxidase activity,
data concerning 0
con­
sumption in the presence of these substrates are also included.
32
Table 7
Products of aerobic gluconate breakdown by
Lcucoftoatoe mesentero ides strain 548
Average*
uM
Oluconate used
..an-;e
uM
100
Oxygen uptake
45
44-4o
Carbon dioxide
97
op-oB
Lac ta te
102
101-103
Acetate
10?
102-113
Main compartment*
1 ml cell suspension of optical
density 0.5 on 1*20 dilution; 250 .uM pnoaphate buffer,
pH 7.0. bide arm*
50 JtM jluconate (supplied as 25 uM
calcium gluconate). Center well:
0.1 ml 2u per cent
KOH. Final volume*
2.5 ml.
•These figures represent averages of several determina­
tions calculated on the basis of 100 mM substrate used.
33
Oaule 8
O x y g e n u p t a k e on ’ra r i o u u s u g a r s by D-xj 'iooe-jrov.ii c e l l s
L e u c o n o a t o c m e a e n t a r o l d e ^ at.r .in 34 ?'
0 uos tr« te
u
oi‘
uptake
cl
lone
01ncoae
c
*
•
f^
O-xyloae
, .3
u— r i uoae
j•
0—arabinose
L arabinoae
Llain compartment*
1.0 ml cell suspension ol' optical density
0*44 on 1*10 dilution; 12^ ^uLi phosphate buiTer, pH 7*0. Hide
arm:
50
3ubstrate.
Center well*
0.1 ml 20 per ce:u KGii.
Final volume*
2.5 ml.
Incubation time:
150 minutes.
34
.'itbit- '
’aroxidaae activity and cxyyen cons-mpt ioi. bc. ;1 \cu.:o--■un wn a-] i;
of Leuconoatoc inest-n tero ides a train S.;3
Jubatrate
n
i'erox niaflt*
i-.c t i v i t ..
ii
typen
d Uii- iiii
t :
n
a one
afi’inooe
Jucrooe
Fructose
Jlucose
1 otaa.ji’.Li ,;1tco.-,e~l-ph.;ai.iiri te
Bariu1;. -1 ;co3e-6-phoapha te
CJttlciuM ,;i ;co:i;iL:
llannitol
L-ribose
j-xylo
+
+
+
e
Jodium c 11r.'tte
l otar-.i .in suc-.-inn te
Jo-1 i at
:ir..ru te
Jodi .m mnlate
Jodi urn oxnlaco tn te
Jodiuiii
:"Jv;.Le
j IVCOJ’i!.
L act ic o d d
Jodi art a ce ta te
*
‘
-
A:
+ denotes reauction in
content ufter incubation ol' 1 ini
cell suspension (optical density of 0.62 on l:lo dilutionj,
75 >ig H_0^t and 5^
substrate for bo minutes; - denotes no
reduc tion.
B:
+ denotes oxygen consumed burin,: Id minutes incubation of 1
ml coll suspension (optical density of 1.0 on 1:10 dilution),
125
phosphate buffer, pH 7*0, and 50
substrate; - denotes
no oxygen co..sumption.
*1
spontaneous peroxidation occurrea in the absence of cells.
5■"
1' f
rill
'i e
S O I'VOli
t!S
o' i mi t . i >n
on
. i 1: i u I ; ■=1 ' e o
.-! II Ll.i t I" :
u,
ho
!>—r i l i o. - , e
variai/J a .
in
the
:•C
x
: on
■ ■;, ■
v:e a
..a.;
. A■r■
o',
f
I
oo.no
niin.i t e r
iii.: i . a
n.
11c 1 ■i
. ■ ;• '
i i' .
as
.
1
1
i,
on
.
a ’ ;‘■
wui
fii . o
* ' r u c t ";•.«> u i . o
o
.
< >
. - o r ; o , "j i j .-.
i
.
roc , i re..
, -1 u c o : . ; - t.o .
’i n I !i
seemed [n -a in', o 1 nt the
0 1* j i ri o n . : i ■ To #
by
Cell l
I
....a
}. U 1
om ioaion
. o ’.1, e V e
.j 11 1 1 '. e o
of
p e r o x i d - . t i on
o;
'c
• r. , • • :
on. 1 e i ui..
i ,i , ; i
,
t, irm
!n
t ;i a t
t 1 1e
pe ro
from
i[i1= . t... t J.
±
.. c l .
t.n*
c o n n t o wa- -
ne ]
me i i i : i
i i'i
.
i.i
.■i
-in ]
of
■■-i i c 11 .
ca u.e i bt 'Soon
.i o .n i ; i . i i d f . i i n
. 1y
:•
i
on , l t
as
no,
in-ie .
,ci.
vi . na
l n o r e a . n •;
ia.ec ;eroj b.at .<>n ..an
(
; hef-p.»a t o
,1
m
t*
<• u .
. i no>
: i a - a * ; ■1 t !i ' ■
. : ■ ■.■.<-
jaoj! Iii:tt-1, Aonaitil npor, i i.» addition ti> the oe :i
per
. a ■* ■
*■ ■n ........ ;
e»- j I .
conn-a:.;
•
V I
'■
r«- v i • ■. a i .• .
i ' i ■o
tii i a
.-■;j »: i i
7 ‘. ill, „ o ■'
i 'i o 1' ri i o
: t']*'■' i '1 a . :<■
: .
-.-w' w n o
I•
only
ol'
o;’
m i one , : .
A r me d t i ■-i u m
' —>.y 1 o. o
rate
f r
.
iieo
t i on
.f < ir I ■<■ t '
ot?! !.s.
;n >;
.'he
tUOHo 0 1' i)!h
i. h y ,
U? o t*"'i , o n 1 y
. ■• P
i m p a ii'tid
con u ition o,
t . ' i t t m' i
t
ol’
,*1 ’l e o n e .
■t r a i n
as
sobat.ru t o .
in cubation
the
on
t9
-lucose
the
war
b' o
tent'" i
u t i 1 i r.a t i o n o f
p eriod .
medium
baker.
:'f>r j . t i r o x i . ai H-
d ro p on
ir.
wl i L o h
i /
n c c o rre>:
p erocm e
a train
act iv .
y<
c<vi i d
h ai
,
■i . i
ir. n . ; a
not
been
be
1 icoue
1 hour
de tec ted
,;rov/n
i ' oi *
lb
in
hours
noted
n i,;l io aru; i.lcdleai.ey >.1
an t .ioi.it:cu 1 tilres of
he'icon<m.too me sen tox’o ulei! in s <c;•o.u- mod in
v
ty
.-os tti•
J
a ! U 11
I . io rr
a mr i m .rid
t.iu •,thecells
i itreri
:n.i 'h*.nnv<-
roar oi •
:n, races
i.it- or. ;- n isms wer- unabl o to iit j I . a«r d e
in ine
trie
rfnvt.h tied vnni
nt
liex tran ’
.van motjLf ica
manner
two
i
on J v . a i d e r
if. 1.. ai.s
c u n a i. t io.-.s
3in
;ii.
i *, . . no
. , u : .;
i ■t. r ; .11
exists
O11
I ini
ol
r--.: e.: a
■’
i.
in
not
an-
,..;:t tne
sac.*
a
n t. i . a .: t. t i n
.<>.!.S i . !*• v;i■ I n r .....m 11
ol’ s larva
.—
cells nine ai ■
unribjln,
i f i e ■■; y.n
o*- iIn -<i it- to j. >• i;a
pmrifiec material.
ri• n
r> s t *:i
iovevfi
u :*in t.r •
in..
.-.om'ce. dr* r e s u l t s
.riven ti a .ion i n o c r
an] e to a t i i i:te ,ox'.ran.
decrease iti
t■,? oe> ii'rii; no., over,
Xt r n n
fit* : «>j•• on r non
an
diovvan
be ■lti1 i 7r
,.I 1■: j <'>"i;1o ill Hi
*A■
.l'I'"Li..)'t*I1
rned iurn.
,'i.e u p . h e
was
of o
'< ii iri l.s
nu:';. ].i-*<: as n ins mai i- wm
De.'ioan and
( s t rai n
d t baa
i
,‘))
'1
b ]■'
was u n a b l e
ton t the ir
to stilir,
is s u f;. ested
p at hw ay
aidered
to be " facu i tat ive" v, i th
(Breed,
M u r r a y and
of ro te s
finsr.
■
..an
. iuco.
, ,n v it .. , f m u
n. t r a j . i i
of
...
ur t
Ir.
nt/aeu t o r o i d e s
a.-.j t’fi as an elect; >m
however,
re
0
a
Coe. tor
tne existence of an
bp tne iac I tnu t Lfuconugloc iu Cwli-
aerobic
hi tenant?,
r'C'l
. ir . r i i i.
in the d i s s i m i l a t i o n of , 1s c ou r .
... c o m p a r i s o n
roil
rvs.me
t to ox; eti util ixa t ic-n
1 '4f i .
of ox p. ,en spi-iko on ,,1acoi.ti lip ..t-vcra i
37
other a tr^.in3 o/ t..e or.^uniaui showed tan I the phenomenon ,.au by
no means ] icii Led to tne i'irs t u train te;-. too .
Altuouph tne rates
varied cona iuerably , represen ta 1 ives oi' each colonial t„. pe showed
some oxy. an up take on , lucoae .
.train j1', uowever, .iu not use
oxygen unde:
inis iack oi' an ueraaic . .tuv,.^
these conditions.
of jjlucose dissimilation constitutes a major 1 iffere nee amon.;
theue cultures which represent strains :>f tja same
ec.c-j.
?ne results oi' tae .;rowtu experiment d e m o n s ?rr ie..
.t
.ra in
2 4 b jjrows much more rap. i:ily under cona Ltion.. affor^ in,; an increased
sup.ly of oxygen.
inis benav ior i-: topical of or,p n isms w..ich are
i'acul tauve w ita respect to ojeyreu utilisation.
The sen ilivity of aerobic glucose breakdown to low concentra­
tions of cyanide indicates tne involvenon t of
conla.ninrt* e;i7, tne in tn is process .
Ir. contrast,
u-.avy me talit was reported
by oeeley and Vandemark (lt'bl ) tha . in tae case of v.tt‘e t tococcua
iaecalis, a closely related or, ;aiiinr.it ;Lucose oxidation waa not
inhibited by the ad--ition of sodium cynniue to a final concentra­
tion of 1 x 10 ^ M.
Ihe reason for lack of o>yt;e:: uptake on sucrose remains
obscure«
due urbanism contains sex-ransucrase (f.enre, 1-41) and
if free fructose were 1 ibex uted in tne process of dextran formation,
it should be oxidized in the usual manner.
In addition, L.
mesen teroidea has been shown to contain sucj'Hae and sucrose phosphorylase (weimberfe- and Doudoroff, 19.r'4)» bota of whies would be
expected to yield utilizable sujars for oxidation.
Al tnou^h these
enz. meg mic:ht not be present in jlueoxe- or .ructose-jrown cells
in sufficient quantity lor activity in t.iis respect, it would
38
certainly be expec ted that they would be present in tae c*. uc
f
aucroae—j'rown c e l l o unlea.- th ie» a e r o b i c me tiloa ol’ ; ro-i ine the
cello prevents
the formation of t n o s o enzymes .
it should be mentioned
that under
In
thi.
connec t i o n ,
these c o n d : lions of c u l t i v a t i o n ,
the ortjanism ,;row much leas profusely in sucrose medium t h a n in
media containing (jlucoae or fructose.
table 5 d i s c o u n t
The results listed in
the possibility that the metacolism of O luco.;e
provides to the cell some substance necessary for aero.ic utilisa­
tion of sucrose.
These results also show that sucrose does not
inhibit jluco.;e <>■.-i,ia t ion, since the oxygen uptake on clut-oue
was
the s a m e
in the presence and absence o f
ihe d iuacchur ulo .
'The results in table 6 show that this strain of L • nea- n teroiicc
utilises approximately one mole of oxygen per mole of ,;luco.:e in
the jii enence of a i r ,
and produces approx i mn te iy ecu imolar ■plan —
titiea of carbon dioxiue, lactate and acetate.
n cnemical
balance shows that a molecule of water is also produced.
The over­
all reaction is as follows:
CrH,^
o i y
+
o
0,
f
C0_
*
+
CK.COoH
i
+
Oil CHOubOOH
3
+
H„0
f
The oxidation of ,;luconate ( ttble 7) requires a; proximatel,, onehalf as much oxygen ana results in the formation of the same end
products.
C6H 1 o 0 7
Ia l„
4
£ 02 — *
— d-i . . . iUOdO r,P ^ Uti
^XUiio 1 Uy:
CC2
4
CK^COOH
•,
4
-*
f.
u *i
.a
ct
u 1 j.
.j
4
over—all reaction in each case.
other minor reactions ire
4
CH^CHOHCOOH
i \i 1
*1 t:.4
1:1.J, » *tub
However,
4
H^O
1 ^
^
w.
W ^VUiAiiail
V
the possibility
that
taking place canrot be excluded because
of the divergence between the results of tables 6 and 7 and the
theoretical values for the reactions.
35
j\ compar ison ol' tae products of f;luooa<_ fe rne a ta 11 on by L .
mesen tero idea (Del.'ou.- , fard aim Gunaalua , 1951 J and
m e prouuc ta
of aerol ic ,;1 ncoae dissimilu t ion ahov/o taat uerobicai i„. , act* ta to
is formed in tae jdact; of etuanol, with tae utilisation uf one
mole of oxyyen per mole of substrate.
This fact indie, ted that
in the aerobic proceaa oxygen nerves a;i the final acceptor . 0 1
pairs
01’
two
hydrogen atoms, wherea3 in tae .anaerobic process the
hydrogen is accented by acetate, or ..ome compound at tne same
oxiantion state as acetate, resulting in the formation of ethanol.
Suspensions of strain 39 are able to reduce acetate to ethanol
(Gunsalus and Gibbs, 195«?)*
As mentioned previously, earlier investigators of yiucose
fermentation by L. mesenteroidea (itdercon, 1 9 2 9 ? sucker and
Pederson, 1930} had found acetate as well as ethanol am one; the
fermentation products.
Altnouj.i tne metnouu used in these siuui^s
were not yiven in detail, it seems likely that no cure was taken
to insure the exclusion
d‘ air from the fe-muniation flasks.
Thus acetate was produced, instead of ethanol, in proportion
to
the amount of oxy, ;en available to the cells under these semianaerobic conditions.
The anaerobic breakdown of ;jluconate by L. mesen teroidea
(strain 39) was reported by lllackwood and Blakley (1 9 5 7 ) to result
in the production of equimolar amounts of carbon dioxide, lactate,
and ethanol plus acetate.
when these results are compared with
those in table 7 for aerobic jluconate dissimilation, it appears
that whereas in the aerouic process one pair oi’ hydrogen atoms
is accepted by oxygen, anaerobic disposal of substrate hydrogen
40
proceeds uy lau reduction ox' one— h lf of
nee fate to e thi nol,
re.jul -..n,-; in the ;rucuc lion of ouf-nalf mol e = .rn ol' ace la to and
e tiu.nol from one mole of gluconate.
The fact tna t only hail1 an tuucu oxygen in requires
i'or
aerobic gluoona l,e breakdown an for rlucoje indicates tnat tiie
oxidation oi ..iucoiu; to gluconate
m
one of the s teps at which
substrate hydiogen i^ finally accepted oy oxy,.on.
iiie ratio and nature -.«f
■ : -i . rod icts of glucose and
gluconate breakdown suggest tnat the 3 a aubatrates may be metab­
olized under aerobic conditions by .at name hcxoae mono, hos.."ua te
type path.vay w.iicli nay been postulated x'or anaerobic glucose
breakuown by tlii. organism (Defoes , Junoal u.. an*: ik-ru , 1 f5 J ) •
Howevf.: , 1..ere appear to be jiffer«nces in
ue aerobic and
tne an­
aerobic pathways '.vita respect to the effect;; of pnojj.aate and of
p.i variation.
Aerobic plucote d insimtint ion in shown in table 1
to proceed more rap id 1 .. a t :d 7 tiian at
.'I
.
fust tae opposite
relation is true for tne ferment ition ox' p f u x u c
dunsalus, 19S1) .
The rate of form* n tat Lou of ,rlucoae , as measured
by carbon dioxide evolution, was resor ted by
.uhoaa (1 ■>[>)) to show
7 7 per cent inhibition in tne p r e s e n c e of 9 x lu
However,
vbe ..oua , bard and
-4 M phosphate.
the aerobic utilization of glucose proceeds rapidly in
0.1 If. phosphate buffer.
Tht utilization of hydrogen peroxide in the presence of sub­
strate by L. mesente 1 0 ides demonstrates that this organism possesea peroxidase activit,
as tne term has been employed by other
investigators working with bacterial peroxidases.
.’he possibility
that catalaso activit.. was responsible for the observed results is
41
excluded by the f-nct that this or,;anism due., not ; os. -usn cnlalnse.
The further possibility
a metal
lust
the cells are tnere 1,, uoti tr ibu t
Ion neeuuu Tor spoil taneouM peroxiu;. t i o n ,
t.it-r m a n
exerting enzytna tic perox Ldaae activi^. , is; also ruled out by the
observation
that boiled cells do not catalyze
the reaction.
L . mesenteroidea is similar to otin.r lactic acid uacteri:-. in
that cell suspensions show peroxidase a c t i v i t y on a limited number
of substrates and do not attack conventional ,oroxicase su in-tra tea
(e . g . t
ortiio-tol id inc*) .
7-ie function of the p e r o x i d a s e enzyme
system in the norm,, i tnetaboli31.1 of the organism remains to be
established, ul tiiour;f! it seouis iihcly th..t it carve* as a mechanism
to protect the ceils i'rotii the action oi' n.vsrojen peroxide formed
in disposal of substrate h.-dro^en.
The absence .a’ pe.roxic.aae
activit,, In ..train y ,• is undoubtedly conr.ec ted v;Ltn tne lack of an
aerobic patnwav in this strain.
o .JLIMAhY
Tne reaul ts of preliminary experiments witn reatin(: colls
of Leuconoatoc mesenteroides aust'fij i-ej luat a survey of lae a«robi
carbonydra te metabolism of u.ii. or, an ism .nigh t yield informs. tion
which woulu explain tne conf 1 ^ctin.; reports in the literature
concerning glucose dissimilation by this organ ism*
nesting cells of _b* mesen teroldea slioweu no oxygen uptake or
caruon aioxi..e evolution upon incubation witu purifieu dextran
from the same strain.
Oxygen uptake on glucose was demonstrated with resting cell
suspensions of representatives of five colonial types of L.
meBent^roideat strain 39 showed no oxygen con.'un, tion under the
same conditions .
Aerobic breakdown of one mole of glucose required approximate
ly one mole of oxygen and resulted in tne promotion of approxi­
mately one mole each of carbon diox lie, lactate anu acetate.
Glucose oxidation waa founo. to be cyanide— sensitive and to proceed
faster at pH 7 than at pH [3*
Increasing the surface area and shaking of tne culture re­
sulted in more rapid growth of the organism than it displayed
under stationary conditiona.
Gluconate waa oxidized by cell suspensions with the utiliza­
tion of approximately one-half mole of oxygen and the production
of approximately equimolar quantities of carbon dioxide, lactate
43
and acetate per mole od suoatrate.
Jlucoae— and iru c tone —(;rov;n cc-.i io ox id iced bo t.i
,;u :ara;
however, cell;; jrovn on t.;luoo v , j’ruclose or cue rose showed no
oij',;ea up take on sucrose .
i’ue rate o:’ .unlo;ie 0 : 1 .a l ion '.vas
variable v/itii olucoce-^roAn cells and 1 ov, with u— xv'looc-6'ro».n
cello .
L . me sen te no idea *H3 found to ex;ii. i
fructose, glucose and gluconate.
:;f;'OA ijnne activity on
►jKLhCTED .j 1BLKJGiht*'hi
Altermatt, II. A., Blackwood , a. C. and. j'eiaa, A. d. 1935 ■.*’he
Anaerobic d is., imi 1a tion of ./-xylose-1— C , */-xylo:,e-2-(J
and
J-xylo3e-5-C
by Leuconoatoe meaenteroidea. Janauitm ^ .
'iocbetii. and ihysiol., 3J^ 622— o2 6 .
Antoniani, C ., ?'ei:erico, A. and kleisciuuuun, L . 1 -bo •ducoce
dogmol&se and beta-lactic acid.
Boll, soc • ii.nl. uiol . jper.,
32 1 811-813 . (Cin.r.1. AbsLracta, ^1, no. 67744, 1957).
darker, C. B. ana Bummerson, .■>. ... 1741
f2ho colorimetric deter­
mination of lactic acid in biolo,n.Lcal material . 2. .iol . di.eni.,
1 3 2 , ;• . - 3 3 4 .
Blackwood, A . C. and Blakley , m . A * 1757 J..etubol i:jiii cl1 ( l,ico!ia.o
and 2-ketopluconate by Leuconogtoc tneaentc roidc-s . .;acajlul.
rroc ., 121-122.
Breed , l\ . o *, ./.array, r * j. b . and a 1tc.iens , a . i
arKf* y 1^48
Lor tjey 1a manua L of ae te .rdnative Due r jolo^y , nth ed . ine
Williams ax) 1 ..ilkino do., dal timor* .
Carlson, darner » ., Aorc.no, da m e n A. and .-si »ea "de -Carlson,
Virginia 173b ...tudies on the dex .rtiuuc ot of Leuc^nostoc
dcxtranicum. d. Bacterid., t/j, 13---14G.
Clapper, William A., Liead e , Grace il. and fail>» aubriel
ly'54
Peroxide production and peroxidase actlvi
of sulfa tuiazole
sensitive and resistant a trains of ..troy 1 .
xus ;.ii .is . i'roc.
.Joe . hx..tl . Biol . Aed ., 6 7 » <•00—602 .
Clifton, d. A. 135 7 1n trod uc tion to uact r al paygiolo&y, p. 13C.
McGraw-Hill Book Co., Inc., New York.
Conway, lidward d . 195^ A. orodifi'usion anaiy.. in ana volumetric
error. Crosby Lockwood be Con, Ltd., London.
DeKoss, k&lph D. 1953 Houtes of ethanol formation in bacteria.
J. Cellular Comp. Physiol., 4 1 , 207-224*
DeLoss, ralph P. 1954 Oxidation of o-phogpho^luconate by
Leuconostoc meaenteroides . Bactoriol. Proc ., 109 .
DeAosa, Aalph P., Bard, R. C. and Cunsalus, I. C. 1951
Ike
mechanism of the heterolactic fermentation:
a new route of
ethanol formation. J. Ractedol., 62, 499-511*
44
45
NeMos.., l.alph D . mid Cibos, Margin 1 35 c-I'ho^pnc. ;lucoriu te dehy­
drogenase iron: Leu conor. toe me sen t«-roidea .
. .ae r .ci ., 'j\ ,
730-73/1 .
Jtii.iOa.i , I:. Ijili j ., jUnnali.j, I. C . a
n.ru , a . 0 • l.'.o
• iucose—
<>—phospha te dehyd o,;enaee in Leucouo.-;. toe muaen tero ides . J.
‘fciC^GI i01 ., O0 , 1^ 1O •
Bolin, i... I. 1953 i’iie oxida tion and peroxidation oi m Nii
extracts oi’ b ire p tococcuo faec;, 1is . arcu. Biocneiri. nn 5
phys., £6, 4^3-'1o5 .
l)ol in , M .I . 1;J•'.•••
Proc., J_31 POO.
t.yme tic :« ro.-; i in t ion of Li Mil, .
in
10 -
Led era uion
Dolin,
M .I. l3Lli:'he DPNH-oxid if.in.
;z-me.,
of trap toooccua
faecal in . II. Tnc enzyme:: util iz inc' or y.yen , ey toci.ronie c,
peroxide an-: . ,o—dichl orop.ienol-incio(,:o'i.oi or ierricyinidi; an
oxidants . nr cn * .’iocht,m* .:. 1 . r :jh..
^
. .11 .—.._,).
Jouplne, h. C. 1^4, h droyen i-.-rn ide in tue meluoolism oi'
Lac tobaci 1 lun brevis . J . -ao te r iu 1 ., :,4, .72.
Jayon, U . and jubourt;, ii. 1V 1 iouvei i>v; r> chercues our le
l’ermen t mannitique . Ann . Ir..<t. .a. tear, 1 , .■<>'}o9 .
Civile, D . fc. and i.lcClesruy. , J . .J. 1 •
Put formen La t lor. of
yucrose by Lencono toe moyen l-roidey » • • .m c u rioi
65, 7L-7--•
C tumulus , I. C. ana Oi ubs, Martin 1 J'7J int- ue terolact lc fermon tation.
11 . Poa ition of C * in ::ie ;rod ,ieto of glucose aiuaimilation by Leuconos toe mescntoro Lies ■ 7 . ,iol. Cnem., 194, 871—873•
Hehre,
v. J. 1941Produc tion from /ucrose of
a aenolof; 1call y
reactive polysaccharide by a sterile bacterial extrac .. science,
23, 7 37-238.
nucker, C. J. arm Pederson, Carl
1
studies on trie coccaceae •
XVI.
the , enua Leuconoa toe . ,. . i . ^tate A, r . Ex. t. ,jua . i’ech.
Bull. Mo. l<->7 .
Main, K. H. and Chinn, L. E. 1935
peroxide in bacterial cultures.
7'iie determination of hjdrofe-en
3. Biol. Chem., 126, 417-423-
i.IcCleakey, C. b., Paville, L. '.7. ana .arnett,
u. 1947 Char­
acteristics of Leuoonoatoe m e senteroidea from cane Juice . J .
Baoteriol, 5 4 , 697-703.
MoElroy, W . I), and Class,
chanism of enzyme
action, p. 456. Johns Hopkins rreas, Baltimore.
Nelson, Norton 1944 A photometric adaptation of the bomojyi
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46
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A.
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Enzymatic
VITA
:,lary Knot ties aounaon was born on ^o^teinb&r ^ , 1 (v2y in
Detroit, iiicbi^an.
Che jraa.iato^ l'roiii Lanuitii; Central
ooiiool in Luiilov/vil'ie, Hew York, in 11■.'6 .
iir.,a.
Cue received uer
de(;ree in Bacteriology in June, 1C:,4 and nei'
... .
decree in
Bacteriol o^\. in Jane 1955> both I'rom Louisiana C lute Univ-.-rai ty .
Che is now a candidate i'or tne ueji'ee 01' Doc .or of i'hilouopny in
Bacteriology at this university.
47
EXAMINATION AND THESIS REPORT
Candidate:
»iarv knettles John aon
Major Field:
Dac teriology
Title of Thesis:
Jtudies on the aerobic carbohydrate metabolism
of Leuconostoc raesenteroides
Approved:
M ajor P rofessor and Crfairman
Ml
G raduate SehAol
D ear
EXAMINING COMMITTEE:
yL/
Date of Examination:
J u 1j l .25j_ 1 2 5 7 _
/
.if