"The Significance of Adaptive Enzymatic Patterns
jin the Study of Terminal Respiration."

R. Y.-Stanier

University of California, Berkeley

The analysis of adaptive enzymatic patterns, “simultaneous adaptation",
can be used to advantage in unravelling microbial metabolic reaction chains.
This represents an extension and refinement of the kinetic approach to problems
of intermediary metabolism made possible by the substrate-activated nature of
many microbial enzymes. The principles of simultaneous adaptation can be steted
as follows:

le If the dissimilation of a given substance A proceeds through a series
of intermediates B, C, D, BE, F, Ge «+.» + and, if the individual’ steps in
this chain are under adaptive enzymatic control, adaptation to A will
produce cells that are simultaneously adapted to B, C, D, #, F, Geogesee

2. If adaptation to A fails to adapt the cells to a postulated intermediate
X, then X cannot be a member of the reaction chain leading from Ae

Although the general validity of these two postulates are self-evident,
certain problems arise when they are applied to a given set of experimental data,
In the case of postulate 1, simultaneous adaptation to the actual intermediates
is a necessary consequence but does not exclude the possibility that the primary
substrate may also activate adaptive enzymes not involved in the dissimilation
in question.' Too little is known about the specificity of activation of adaptive
enzymes to assess this possibility accurately, but it should be kept in mind when
interpreting cases of simultaneous adaptation. For exanple, we have recently
shown that a bacterium capable of attacking both isomers of tryptophan
adaptively shows only partiel specificity in its adaptive responses to the isomers»
Specific activation of the l-tryptophan-oxidizing system occurs as a result of
exposure to the l-isomer, but both enzymatic systems are activated after exposure
to the d-isomers Rigid interpretation according to postulate l, might imply that
the l-isomer is an intermediate in the dissimilation of the d~isomer. However,
there is other evidence to indicate that the most probable pathway for the
oxidation is:

l-tryptophan d-tryptophan
l-kynurenin d-kynurenin
kynurenic acid
and hence the adaptation to both isomers after exposure to the d-~iromer alone
appears to represent a case of non-specific activation. In cases where non-
specific activation occurs it is likely to be evident from a consideration of

over-all adaptive patterns,

In the case of postulate 2 it has been suggested by several workers that
permeability may constitute a major problem. According to this argument non-

adaptation to a postulated ‘intermediate might mean that the intermediate when

externally supplied fails to penetrate across the cell membrane. The force of
this objection is. greatly diminished if it can be shown that adaptation to the
substance in question brings about its immediate rapid oxidation,

If this is so the argument for permeability barrier can only be maintained
by assuming that adaptation per se represents a change in permeability, and there
is already substantial evidence against this beliefs A decisive refutation of
the permeability argument can be made by the use of dried cell preparations.

Our work with such preparations has already shown that pre-establishment of a
specific adaptive pattern is a factor of crucial importance in obtaining in vitro
activity against presumptive intermediates. An illustration is provided by.
studies on the oxidation of aromatic compounds by Pseudomonas fluorescens»

Recent work has shown that polyhydroxy aromatic compounds are involved in. the
dissimilation of aromatic acids. Cells grown on mandelic acid or benzoic acid
(both of which belong in a common reaction chain) show complete simultaneous
adaptation to catechol but not to protocatechuic acid, while cells adapted to
parahydroxy benzoic acid show the exact reversal of this pattern, Dried cells
grown on mandelate or benzoate show enzymatic activity against catechol but

none against protocatechuic acid, while parahydroxy benzoic grown cells are active
against protocatechuic but not against catechol. The biochemical implications

of this work will also be discussede
“Bacterial Enzymes. as Biochemical “Tools.”

Fritz Lipmann

Massachusetts General Hospital, Boston

The contribution due to microbial systems in the elaboration of
the present metabolic picture will be assessed from the earliest work
on fermentation through the, development of the vitamin-coenzyme
relationship to the present approach to understanding of synthetic
mechanisms «

In some detail the steps will be retraced leading from the finding

ft
of acetyl phosphate in Lactobacillus delbruckii to the presumed general

 

role of carboxy] activation in biosynthesis. In particular, present
results and outstanding problems will be discussed regarding the two-

carbon intermediacy in fat and carbohydrate metabolism,
"Enzymes Involved in Bacterial Metabolism of
Disaccharides and Polysaccharides."

Michael Doudoroff

University of California, Berkeley

Some microorganisms can utilize certain disaccharides more rapidly or in
a different manner than they can use the constituent monosaccharides, In a few
cases it has been possible to demonstrate the existence of special enzymes
which appear to be responsible for the observed differences in mono~ and
disaccharide utilizatione .

Some enzymes catalyze the formation of polysaccharides from disaccharides,
such as the production of dextran from sucrose, levan from sucrose and raffinose,
and of starch-like polysaccharides from sucrose and maltose respectively. .
All of the above enzymes may be considered to be transglycosidases since they
are capable of exchanging glycosidic linkages. In the case of a mutant of
E. coli which is capable of fermenting maltose more rapidly t han glucose, it
has been shown that this ability may et least in part be explained by the
presence of both amylomaltase and phosphorylasee The following reactions
have been found to take place with dry cell preparations:

Maltose <——mpolysaccharide + glucose
|
V HzPQ),
glucose - 1 = phosphate
glucose - 6 - phosphate
fructose « 6 = phosphate.

Another type of attack on disaccharides is the direct phosphorolytic
cleavage, as found in the phosphorolysis of sucrose by Pseudomonas saccharo~
phila and Pseudomonas putrefaciens. The "sucrose phosphorylase” of Pseudomonas
Baceharophiia has been shown to be also 4 "“transglycosidase", which can use 4
number of "glucose acceptors" other than phosphate and fructose.

 

In many cases, the preferential utilization of disaccharides cannot be
explained on the basis of knowledge of the enzymes involved»

Thus, the known enzyme mechanisms fail to explain why the entire molecule
of maltose or sucrose is utilized rapidly and complotely by intact cells of
E, coli and of P. saccharophila respectively, while glucose and fructose are
Hot attacked. Other disaccharides, s uch as melibiose and crehalose, are used
rapidly by Pe saccharophila, although no phosphorolysis can be detected
in vitro or in vivoe Other anomalous cases of rapid disaccharide utilization
are found in the metabolism of lactose by certain yeasts and by Lactobacilius
bulgaricus.

Several hypothes@s for the difference in rates of disaccharide and mono~
saccharide utilization by intact cells will be disoussede
le
Ce
Je

he
De

6.

“Genetic Studies on Lactose Fermentation
in Escherichia coli."

Joshua Lederberg

University of Wisconsin, Madison

Methods of obtaining mutants.

Assay and properties of b-galactosidase, using o-nitrophenyl galactoside.
Adaptive propertiess (Adaptation to lactose, galactosides). Also,
comment on the evocation of the enzyme by lactobionote, which does not ©
seem to react with ite , ° ‘
The genetic types of lactose-negative mutants found. Some are specific,
others affect maltose, glucose, gluconate-fermenting systems,

The effects of the genotype in modifying the adaptive response:

-a) Temperature sensitive allelgof Lacz- which has a different threshold
for the formation of"malto-zymase"~and galactosidase. The point that
temperature-sensitivity is in the enzyme-forming mechanism, not in the
enzyme producede ,

“b) The failure of Lac,=- to respond to lactose, elthough it does respond
to butyl -galactoside, ond these cells. can ferment lactosee Implica-
tions of this separation of the specificity of adaptation and the
enzyme produced for mechanism of adaptation, and for Stanier's
techniques

Patterns of genetic alternative mutations (suppressors) and the uncovering
of interesting biochemical problems thereby (eeg. Mal#Glu- which Doudoroff
will discuss )..
ROUND TABLE DISCUSSION ON

ENZYMOLOGY
naan,
Society of American Bacteriologists, Cincinnati, Ohio
9:00 aem. Tuesday, May 17, 1949

ST aparece

The objective of this symposium is, first, to review the
status of several pertinent metabolic problems and the enzymatic
methods employed in their study, and, secondly, to stimulate dis-
cussion of the data, their interpretation, and implication to
microbiology and to metabolic patterns in generale:

The discussion will be initiated by the workers indicated,-
assisted by Drs» Kaplan, Spiegelman, Van Niel and Wood;’ in

addition contributions from the floor are, solicigteds-

of SL.