Reprinted fron

Medical Rese.arch

GOVERNISG BOARD

IRVINE H. PACE, Chairnzan; A. C. Ivy., COLIX 1.1. P~IXCLEOD,
CARL F. SCHMIDT, EUGESE A. STEAD, Dx\m L. THONSOS

Volume 3

RALPH W. GERARD, Editor-in-Chief

GENETICS OF MICRO-ORGASISMS, s. E. LZl,-ia, EditO,.
ASSAY OF NEUROHUMORS, J. H. Gaddum, Edito,,

SELECTED PSYCHOhIO-lOK hIk:ASL'RL-:JIEST ;\lEl-HODS,
      li'alter K. Miles, Editor

hlE-l`HOI)S FOR STUDY OF PEPTIDE STRUCTURE, Clzoh Hno Li, EciitOt

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Isolation and Characterization of Biochemical
       Mutants of Bacteria

J. LEDERBERG, University of kVi.xorzsin

ALTHOUGH nrosr mutations ma!' be ultimatelJV referable to
biochemical changes, the term "biochemical mutant" generally
refers to a mutant detected by its effects on the nutrition or on a
specifically recognizable enzymatic process of the organism (1).

The use of mutagens to increase the proportion of mutants is
discussed by Witkin {pp. 23 ff.). The methods presented here
aim at separating mutant cells from the nonmutants T\-hich usually
outnumber them. The techniques have been applied especially to
Escherichia coli, among bacteria, but hal-e also been used with
other organisms.

Media.-The recognition of a nutritional mutant clcpends on
a comparison of its growth on "minimal" and "complete" medium.
These terms are relative to the purposes of the investigation. A
Itlininlal medium consists only of components essential for the

T.-\BLE 1 .--;\fEI)I \ 1`01: Zl.~c//ericlrin coli*

     .I. ~IISIAIAL MEDIIJN?               B. COMPLETE MEDIUN
        (B. D. Davis)          C:iseill digest (N Z Cascj      10
Gl IICOSC
KZH1'O,       :        Yeast esti-act              5
                        &HPO,                 3
KH,l'O,                2       KH,PO,
S+ citrntcGH20          0.5       Glllcose        ;
1IgSO,-iH,O             0.1
JSH,),SO,                1

C. RIrr;rara~ E!WB MEDILQC ("ENS") (20)      D. COXPLETE EMB MEDIUX
Sugar                 10       sup                10
SodirlnI sL[ccillnte         5       Cnsein digest (S 7, Cnqe)     8
SZICl                  1         I'emt extract             1
MgSO,                 1         sac1                  5
K,HPO,               2       K;,HPO,                   r)
(TH,j,SO:              5         Eosin \          0.4
F.osi II I'                0.4      lfettiylcne blue          O.OG
~Ieth~lcnc blue           0.063

*Agar is always used at a final concentration of 1.5% if required. All concentrations are
g/I of distilled water. The media may he prepared by adding the materials and autoclaving
together. However, minimal medium A is better prepared by sterilizing the sugar (and agar)
in a separate aliquot and mixin, v with the Sterilized solution of the other constituents just
before pouring agar plates.

*Trace elements have been dispensed \vith, being p-esent in adequate amoun:s in other
chemicals of ordinary chemical purity.
                          5


6        GENETICS OF MICRO-ORGANISMS
growth of tl le wild-type strain; a complete medium contains a
variety of supplements covering the range of interest of growth
factors for which mutants are to be sought. Yeast extract, pep-
tones and similar organic preparations are commonly used as
complex supplements lvhich are espected to contain adequate
amounts of such <growth factors. 1\%en specific mutants are being
looked for, however, it is important to verify that the "complete"
medium is sufficiently enriched in the particular factor and that
it does not contain intagonists Tvhich might interfere with the
expected responses (23).

Media suitable for E. coli are tabulated in Table 1. The!, are
certainly not the only formulae that will pro1.e satisfactory; for
specific purposes it lvill alwals ,I be necessary to formulate the
appropriate substitutes.

RrWDOXI ISOL.4TIOS OF SUTRITIOS.4L hICTASTS (2, 8)

PROCEDURE

Cells grown from an irradiated inoculum in a complete medium
are diluted to IOO-500/sample and poured into or spread on the
surface of complete agar plates. .kfter 24 hr of incubation, colo-
nies are picked at random with a fine platinum needle. It is con-
venient to cool the needle in a small tube containing 1 ml of
synthetic medium? touch the colony lightly, immerse it in the 1 ml
tube, and then spot the needle on a complete agar plate. One can
also use a 2d tube containing complete medium for maintaining
the isolate. After 24 hr the tubes or spots on complete agar
corresponding to clear minimal tubes (no growth) are trans-
ferred to slants for later verification as mutants. 1Vhere a spot
shows no growth, there may not have been successful inoculation
into the minimal medium, and the test is discarded.

EunEuation.-Tatum (35) recovered about 1 y0 characterizable
mutants from E. coli treated with x-rays or nitrogen mustard.
Roepke and Mercer (29) obtained yields of I-2oJ0 from cells plated
imn~ediately after s-ray treatment. They noted that storage of
treated cells in electrolyte-containing medium resulted in a
markecll~~ louver yield. In Rn~ill~s sftbtilis, Burkholder and Giles
(4) obtained 3--Gy0 yields of mutants, a high yield which may be
related to the resistance of their material to lethal effects of radia-
tion. Substantially the same procedure has been used for
Neurospora, where Beadle and Tatum (2) report the isolation of
380 mutants from 68,000 tested ascospores, or about l$$yO. How-
Ct\`Ct-, the genetic procedure used in Neurospora is only 50%


      BIOCHEMICAL MUTANTS OF BACTERIA       7
efficient because the ascospores are the progeny of a cross of treated
with untreated nuclei.

    DELAYED ESRICHJ[EST (LAYER PLATISG) (21)
Much of the Tvork needed to obtain a collection of mutants is
spent on separating them from their nonmutant neighbors, which
often predominate 100: 1. The most direct but least efficient pro-
cedure is random isolation followed by individual test on minimal
medium. By delayed enrichment of minimal agar plates, se\*eral
hundred colonies can be screened at once.

PROCEDURE

Plates are prepared with 15-20 ml of minimal agar medium and
allowed to solidify (bottom layer). The bacterial suspension is
diluted to contain ZOO-400 cells/sample; each sample is incor-
porated in a few ml of minimal agar and poured over a plate
(seeded layer) . 1Vhen this has hardened, 5-10 ml of minimal
agar is poured on top (cover layer). Thus all the colonies T\.ill
develop in the agar and will not be disturbed by subsequent manip-
ulation. The seeded plate is incubated for 24 hr or longer to
permit the development of prototroph cells (i.e., cells as nutri-
tionally competent as the wild type) into uniform large colonies.
The mutant cells will have developed very slightly, if at all, and
\tyill be invisible to the naked eye. They are brought to view b!,
pouring a few ml of complete agar over the layer plates (supple-
mcnt layer). The growth factors diffuse down through the agar
and cause growth of the mutants, which are picked up as smaI1
or as new colonies after 6-12 hr of further incubation. `iVith some
practice and use of a fine needle, the tiny mutant colonies can
bc picked to complete medium for verification.

!\lodificatio?zs.-Other methods of delayed enrichment suggest
themselves. The supplement need not be poured on top, but
may be injected below the bottom layer with a hypodermic needle
through a penicillin cup, or placed in a penicillin cup itself, or
may be poured in a trench cut out of the minimal agar either with
a sterile spatula or by pulling up a strip of cloth sterilized r\*ith
the plates. These and other modifications, e.g., transfer of the
entire agar layer to a plate of complete agar, may be particularly
useful in dealing with aerophilic bacteria or molds which l\.ill
not develop readily as deep colonies.

      MARGIS.4L OR LI.\IITISG ESRICH,\IENT (6)
X marginal or limiting concentration of growth factor can
usually be found at which a mutant will form colonies character-


8         GENETICS OF MICRO-ORG.ASISMS
istically smaller than prototrophs in a supplemented minimal
agar. This principle is especially useful in counting a particular
mutant (whose quantitative responses al-c already known) in the
presence of a preponderance of prototrophs or of other mutants
(31, 6).
The greatest drawback of this procedure is the uncertainty of
the proper concentrations of supplements to use. One can use a
graded series of concentrations in the hope of finding the correct
level empirically, though uneconomically.

In principle, a more rational pre-estimate can be made by
determining the content of a given factor in hydrolysates of wild-
type bacteria grown on minimal medium. On the assumption
that the wild-type content is not very different from the needs
of the organism, the amount of supplement which xvi11 limit
growth of a mutant can be calculated from this content. This
assumption also implies that, in general, a level of bacterial
hydrolysates which suffices for any 1 growth factor requirement
will also be suitably rich in other factors. Conversely, a concen-
tration of hydrolysate that is marginal for 1 growth factor should
be approximately marginal for other substances, known and
unknown. Therefore, a concentration of bacterial hydrol!vsate
that suitably limits the growth of ati\. mutant already isolated
should be appropriate for the isolation of new mutants.

Since the assumptions made here, that biosyntheses are not
markedly over- or underproducti\re and that losses of grol\Tth fac-
tors in preparing hydrolysates are uniform, hold only ver1. approxi-
mately for many factors, these generalizations are far front precise.
However, the technique has been successfully employed for the
isolation of mutants of E. co/i and of Azotobacter agilis (12) both
by using graded concentrations (6) and by using bacterial h~droly-
sates (19).

Comment by Bernard D. Davis

A possible advantage of limiting enrichment is that it does not require
the survival of isolated nongrowing cells, a consideration that may be im-
portant with organisms less hardy than E. coli.

    ISOLATION OF MUTASTS l3Y PESICILLIN (4a, 5, 22)
Considerable improvement can be achier-ed with penicillin. Its
application depends on the fact that the bactericidal action of
penicillin is only exerted on cells in a medium in which they
could grow in the absence of the antibiotic. In a synthetic medium
in which biochemical mutants are unable to grow, a differential
between the survival of mutants and prototrophs after treatment


-
-

      BIOCHEMICAL MUTANTS OF BACTERIA       9
with penicillin permits of considerable concentration of mutants.
With Salmonella and E. coli K-12, the logarithmic ratio is of the
order of 2; i.e., for each lo-fold reduction in viable mutants, the
prototrophs are diminished 100-fold. 10-G is the largest degree
of killing that leaves a com~enient number of survivors; this will
often result in lOOO-fold augmentation of the ratio of mutants to
prototrophs in the population treated in synthetic medium.

PROCEDC'RE

A suspension of bacteria (grown in a complete medium from
an irradiated inoculum to a density ca. 2 X 10" cells/ml) is
washed twice with sterile saline and diluted to 20 times the
original volume in minimal medium. A freshly prepared solution
of penicillin is added to yield a final concentration of 300 units/ml.
The tubes are incubated at 37 C on a shaker for 4-24 hr. Samples of
0.1, 0.01 and 0.001 ml are spread on complete medium plates.
After 24 hr incubation, isolated colonies are picked at random
and tested for growth on minimal medium. In successful runs,
half the colonies may show no growth on the 1st test, but 1/3 or
more of these supposed mutants may subsequently behave as
prototrophs after subculture on complete medium. No satisfac-
tory explanation has been found for this transitory behavior,
which parallels that of the "mutants" described by Peacocke and
Hinshelwood (27)  and of chilled cultures of Achromobacter
fischeri (26).

EvaZztatiolz.-The penicillin method is undoubtedly the most
efficient but the most erratic. There appears to be considerable
difference among strains of E. coli and Salmonella in sensitivity to
penicillin. Adjustments in the time of treatment and concentra-
tion of penicillin must be made empirically. For isolation of the
widest range of mutants, especially those with vitamin require-
ments, it may be desirable to use lower initial cell concentrations
(5) . However, E. coli mutants requiring thiamine, nicotinamide
and pantothenate have been isolated with the described pro-
cedure.

Freshly irradiated cells should not be subjected directly to the
penicillin selection. In our laboratory, cells grown several hours
or overnight in complete broth from irradiated inocula were
used uniformly on theoretical grounds, to permit of nuclear segre-
gation (8, 19). Davis (5), however, ascribed the beneficent effects
of preincubation to the exhaustion of biosynthetic mechanisms
which might persist for a time and permit growth after the occur-
rence of genetic changes that suppress syntheses. Probably both
effects play a role. At any rate, the caution that treated popula-


10        GEXETICS OF MICRO-ORG=WISMS
tions be gro\vn for some time after irradiation before applying
selective methods for isolating mutants has been empirically
justified.

If specific classes of mutants are desirecl, the synthetic medium
can be supplemented with the irrele\.ant growth factors, so that
unwanted mutants are killed along with prototrophs.
The mutants obtained by the penicillin methods can be sub-
jected to a 2d treatment to produce double mutants (the penicillin
medium should, of course, now contain the growth factors needed
by the original mutant) . This is possible because, in general, the
mutant survivors of this technique are not resistant to penicillin.
The 300 units/ml concentration of penicillin is required be-
cause of the low penicillin sensitivity of organisms such as Salmon-
ella or Escherichia. Lower concentrations may be sufficient fol
other organisms.

Comment bll Bernard D. Dacis

In the penicillin method the chief source of sterilization of mutants ap-
pears to be the syntrophic effect of traces of grolvth factors excreted by the
wild-type organisms. In some cases the mutants show no sterilization by
penicillin until the wild-type population has reached densities exceeding
106 cells/ml. Since large inocula increase the number of mutants present
but decrease the efficiency of survival, \ve espose a variety of population
densities to penicillin for long periods, then plate out in the largest amount
compatible with elimination of the penicillin effect by dilution. \Ve usually
inoculate in 3 ml of minimal medium containing penicillin (300 units/ml),
1 O-l, I O-2 and IO--" ml of a turbid sucpension of tvashed cells, previously
irradiated and gro\\`n oi.ernight. \\`e incubate for 4-21 hours (without
shaking), then plate 0.1 ml from each tube in minimal and in enriched
medium. Pour plates seem preferable to surface inoculation because of the
danger of sterilization of mutant cells when exposed to nutrilites in the
presence of a high, though transient, local concentration of penicillin.
Colonies are usually picked only from those enriched plates M-hich shokv a
significantly higher colony count than the corresponding minimal plates,
although mutants are frequently obtained even when no such differential
is evident.

A modification of the method, which substantially improves its efficient)
(Sal, consists in preincubating the washed organisms for 4-6 hours in a
medium lacking nitrogen before placing them into penicillin. Prein-
cubation helps exhaust stored metabolites. The minimal medium is then
completed by addition of ammonium sulfate, and penicillin is added. The
preincubation particularly improves the recovery of certain vitamin-rcquir-
ing mutants.

Since crude penicillin may be contaminated by growth factors, it is
desirable to use only crystalline material. Penicillin solutions are stable in
the refrigerator for only a few days, but may be kept for at least a month in


      BIOCHEMICAL XIUTAXTS OI: B44CTERI.A       11
the freezing compartmerlt of a refrigerator. A salt-ice mixture ma) be nece.+
sar,. initially to induce freezing.

CH.-~R.XTERIZ.ATIOS OF BIOCHE\lIC.-\L MUTAXI (2)

-~`~,~o approaches are available for determining the requirements
of ,,utritional mutants: single-additions and single-omissions. By
the 1st method, possible growth factors are added singly and in
nlistures to determine minimal requirements for growth of the
nlUtalit. By the 2d approach, a comprehensive mixture of growth
factors that will support growth of the mutant is prepared, and
single factors or groups of factors are then omitted to determine
those whose removal prevents growth of the mutant. The single-
additions method fails when the mutant requires multiple supple-
men ts; the single-omissions when 1 growth factor can replace
another (e.g., glutamic acid and proline, or methionine and
cystine). For routine purposes, single additions are more eco-
nomical and convenient, especially if certain complex require-
ments, which have been discovered empirically, are kept in mind
(uide infm) .

hlost nlutants require 1 of the following substances: (n) amino
acids, (0) water-soluble vitamins, (c) purine bases and similar
nucleic acid components, or (d) further components, known or
unknown, of complex supplements such as malt or yeast estract.
In tracking down the specific requirement of a mutant, it is con-
venient 1st to classify it in 1 of 4 categories by inoculating the
mutant into minimal media supplemented as follows: (1) no addi-
tion; (2) 0.1 y0 acid-hydrolyzed casein (vitamin-free) plus 0.5 Pg/ml
tryptophane; (3) a mixture of water-soluble vitamins (see Table
2); ; (4) 0.1% alkali-hydrolyzed ).east nucleic acid, and (5) 0.3%

1.
2.
`3 . .
4.
3
6.
I.
s.
9.
IO.
:::

TABLE 2.-RFCOMMESDED LIST OF \I'ATER-SOLUBLE I'ITAVIXS U'ITH
      COSCESTRATIOSS~ SLMXFSTITD FOR E~cherichia coli
Thiamine                         0.001
Riboflavine                         03

p-nminobenzoic acid
Sicotinic acid

0.1   (0.001)
0.1

Pantothenic acid
Pyridosine
l'tero)-lglutamic acid
Choline
Inositolt
Biotin
"I'itamin K" (meth!-1-naphthnqtlinolle)
"l'itamin B1?" (Cobione)

0.1
3.1
0.01
2
tYooo1 (0.001)
IL (0.001)

-111 mg/l. Concentrations given are believed to be in considerable excess. Values given in
parentheses were found adequate for E. coli mutants in our laboratory.
?Inosi?ol is believed to be absent from cells of E. co/i.


12        GENETICS OF MICRO-ORGANISMS
yeast extract. According to personal preferences, one may conduct
these growth tests either in tubes of liquid medium or on agar
plates. Inocula are most conveniently taken by suspending a
loopful of growth from an agar slant and diluting so that the
suspension is only barely turbid. A drop or loopful of such a
suspension is then added to the liquid medium tubes or brushed
on the surface of the supplemented minimal agar plates. Usually
24 hr is a suitable time for incubation, but this will depend
on the organism. Since almost all of the supplements may be more
or less contaminated with traces of vitamins, a response to the
vitamin mixture takes precedence over the others. After a specific
vitamin requirement has been established, it is left to the in-
vestigator's judgment whether to regard additional responses to
other compounds as due to simple contamination of the chemicals
or to more subtle replacement mechanisms.

Once the major group response is established, it remains to
determine which compound or compounds within the group are
active. If an amino acid requirement is indicated by growth on
hydrolyzed casein, it is preferable not to proceed with tests on 28
individual amino acids, but instead to categorize the mutant lvith-
in an amino acid subgroup, The amino acids may be subdivided
arbitrarily, if desired, but Table 3 represents an attempt to

TABLE 3.-LIST OF AMINO ACIDS WITH RECOMMENDED GROUPISGS*

Lysine, arginine, methionine, cystine
Leucine, isoleucine, valine
Phenylalanine, tyrosine, tryptophane
Histidine, threonine, glutamic acid, proline, aspartic acid
Alanine, glycine, serine, hydroxyproline
(Optional miscellaneous): nor-online,? nor-Ieucine,~ alPha-amino-l)~lt!,ric
acid. cysteine

*It is suggested that all amino acids be administered at a concentration of 10 mg/l, except
cystine (50 mg) and threonine (20 mg). When available, the L- (natural) isomers are
preferable, but the DL- compounds are generally suitable for routine purposes (however,
see 6bb.

tProbably not naturally occurring amino acids.

group them to facilitate the characterization of mutants that may
have complex or alternative requirements. Once the amino acid
subgroup is settled, there is a much smaller number of single-
addition or single-omission tests to be made within the group.
In E. coli K-12, L-valine by itself is inhibitory and must be accom-
panied by isoleucine in growth tests." Also, in this strain (and
others), methionine plus lysine is a fairly common complex re-
quirement. Methionine or cystine, proline or glutamic acid, and
glycine or serine exemplify alternative requirements that have

*lIL-serinc inhibition of E. coli I\TIS found to be due to the D-isomer (6b).


     BIOCHEMICAL MUTANTS OF BACTERIA      15
frequently been found and for which there is an olx?ous bio-
chemical basis. The distinction of complex requirements from
double deficiencies, occasionally produced by radiation, has been
discussed by Davis (6a).

Table 4 lists the substances which may be tested in the nucleic
acid "breakdowns."

TUMBLE 4.--COhlPONESTS OF (YEAST) NUCLEIC ;\cID .~ND RELATED ESTR.KTIVES*

Purinest

xanthine, hyposanthine, adenine, guanine
Pyrimidinest
uracil, thymine, qtosine
Nucleosidesj-

[ribose] uridine, cytidine, santhosine, inosine, adenosine. guanosine
[desoxyribose] thymidine
Sucleotidesi

adenosine-3-phosphoric acid (yeast adenylic acid)
adenosine-5phosphoric acid (muscle aclen!lic acid)
santhylic acid, guanylic acid, uridylic acid, qtidylic acid

"Italicized compounds may he difficult to obtain from commercial sources.
fsuggested concentration, 10 mg/l.

Previous experience in biochemical genetics (cf. 3) has shol1.n
that many mutants can be characterized more precisely than in
terms of the end-metabolite if the stage at which the svnthesis is
blocked can be found. Table 5 @\-es some ad hoc sugg&tions for
tests on particular kinds of mutants which may help to distinguish
[hem further.

TABLE 5.-SLYXESTIOSS FOK FURTHER TESTi AFTER PRI~IARY CHAR \CII:KIZ\TIOS

IF ~IKTAXT RESPONDS
        TO:
Arginine
Tryptophane

Cystine or methionine

Proline
Tvosine
Thiamine

Ptero!-lglutamic acid
Sicotinamide

Pantothenic acid
l'yridoxine
Choline

Biotin
Isoleucine
\7itamin B,, or PXB;\

OF SUTRITI~NAL ;\IUTANTS

ALSO TEST (ox RETEST) :

Omithine, citrulline
Indole ;t serine, anthranilic acid, kyurenil~c, nico-
tinic aci
Sulfite, sulfide, thiosulfate, thioglycollate. homocystine,
cystathionine

Glutamic acid, ornithine, alpha-ketoglnt;\1 ic acid
Phenylalanine
"Vitamin-thiazole;"  "Vitamin-pyrimicline." methio-
nine, thioformamide, Buta-3-one-l-01 (ncetopropanol)
p-aminobenzoic acid
Sicotinic acid, hydrosyanthranilic acid. ;tnJ corn--
pounds listed with tryptophane
Panto>- lactone, beta-alanine, aspartic acid
Pyridosal, pyridosamine, thiamine
Ethanolamine, A'-methylaminoethanol, S-clitncth!l-
aminoethanol
Desthiobiotin, pimelic acid
Xlpha-aminobutyric acid
>\lethionine


14       GENETICS OF hUCRO-ORGAXISMS

Comment by Bernard D. Daris
Attention has naturally been concentrated mainly on mutants I\.itl: abso-
lute requirements. Mutants of 2 other classes are, ho\vever, quite common:
those with relative requirements, \\hich grokv slowly. on minimal medium
and rapidly with the proper supplement, and "slow-gro\vers," lvhich cannot
be hastened by any available supplements. As might be expected, such
mutants are isolated less frequently by the penicillin method than by the
methods in which small colonies are selected. Mutants of these types are also
frequently found among the reversions, spontaneous or ultraviolet-induced:
from strains with absolute requirements.
Into$retn2ion of 7~e~ntiw 1.cslfIls.--Frequentl~., mutants are
found which respond to yeast extract or e\.en to hydrolyzed casein
but which cannot readily, be classified further. Before such a
mutant is labeled as requn-mg a new or unkno~vn gro!vth factor,
the following possibilities should be considered.
1. A requirement for a balanced amino acid combination. E.g.,
certain Neurospora mutants requiring arginine are very sensitive
to inhibition by lysine; another mutant requires isoleucine and
valine in a very precise ratio (3) . A single-omission test series
usually will give the necessary clues as to the compounds required.
It may be necessary to test a number of amino acids in varying
proportions before optimal growth responses are obtained.
2. X requirement for fat-soluble factors. Sterols, unsaturated
fatty acids and carotenoids are essential for certain organisms and
should be considered as possible requirements for bacterial mu-
tants, although such requirements have not been encountered.
Such compounds can be incorporated into aqueous media with
the aid of non-ionic detergents such as Tween SO (concentration
q%).
3. I\`ater-soluble factors not previousl), listed. Some of these-
glutamine, glutathione and ascorbic acid-are \.ery heat-labile and
should be filter-sterilized before addition to autoclal-ed medium.
Other compounds include ethyl alcohol, organic acids (acetic,
malic, fumaric, succinic, lactic, pyruvic), strepogenin, p)Gdosal
phosphate, putrescine, hemin, hexose phosphates, cocarboxylase
(thiamine pyrophosphate) and cozymase (diphosphopyridine
nucleotide) . It is impossible to offer an cshaustive list of potential
gro\\.tli factors.
4. The carbon, sulfur or nitrogen sources offered may bc un-
suitable for the mutant; e.g., some E. coli mutants cannot grow
on glucose (7). Such a mutant would probably grow in yeabt
extract or in casein hydrolysate media, but in few with single
synthetic supplements in small quantities. The solution is either
to determine whether the carbon source offered is attacked in a


     BIOCI-IEh/llCXL hIUTAX;TS OF B.4CTERI.4       15
complete medium 01 to supply an a1ternatit.e carbon source (e.g.,
asparagine). In Neurospora, mutants ha1.e been described which
can assimilate ammonia but not nitrate, but ammonia is the usual
nitrogen source in most media for bacteria. E. coli mutants have
been described which cannot reduce either sullate or sulfite, but
I\vhich respond to sulfide (13). 0 wing to the preparatilre methods,
many amino acids may be contaminated tt,ith sufficient sulfide to
give confusing responses, but such mutants arc usually first picked
up as requiring cystine. Sulfide should be tried in any instance of
a broad response to many amino acids. H2S is sufficiently lrolatile
to contaminate media autoclaved together Gth sulfide-supple-
mented media. Difco agar, unless repeatedly rinsed Tvith water, is
also contaminated with materials that allow growth of sulfide-
requiring mutants.
5. Physical conditions. A supplemented medium might faIror
growth of a mutant by virtue of physical characteristics, such as
pH, rH, ionic strength or osmotic pressure rather than its chemical
constituents. These factors must be considered in analyzing mu-
tants rvhich do not give well defined responses to specific chemicals.
Also, some mutants may have unique temperature responses which
cause confusion if this parameter is not controlled. The usual pat-
tern (25, 10) is biochemical deficiency (for known or unknown
substances) at a higher temperature and competence at a lower.
Since it is impossible to predict the characteristics of mutants so
far not isolated, the choice of temperatures at which to conduct
these tests is arbitrary. TVe have had fortunate results with 40
and 30 C as the differential temperatures in studies on E. co/i and
Salmonella mutants.
`~lodi/icnlions.-Choice of procedures depends largely on per-
sonal preferences, because the methods have not been documented
precisely. The details of nutritional characterization tests-inoc-
ulum size, volume of medium, temperature and time of incuba-
tion, use of liquid vs. solid medium, etc.-must all be left to indi-
\.idual judgment.
The nzrxttrzop.cr$hic rrlctlrod, devised by Beijerinck, has certain
adlxntages (16) . Here, washed cells of the inutant are heavily
(about IO"--10s cells/ml) and uniformly seeded in a thin top layer
of minimal agar, over a prepourcd minimal agar plate. after the
agar has hardened and dried for 2 hr, the different supplements
are dropped at different points on the agar surface. The supple-
ments may be used either in solution or in the form of a few
crystals. Sterile technique is unnecessary, but gross contamination
should be avoided. A growth response is indicated by turbidity in
a circular or annular zone where the growth factors hn1.e diffused.


16        GENETICS OF MICRO-ORGANISMS
Since this zone is usually less than 2 cm in diameter, there is
usually sufficient room for a complete characterization of a
mutant on a single plate. The IllaLes can be read 6-10 hr after sup-
plementation, and unsupplemented areas of the same plate can
be used for single additions after the major grouping has been
established. The acl\rantages ol' this procedure are (a) erraticgrowth
responses due to re\.ersion are eliminated, since reverse mutations
in the agar give rise to single colonies; (b) the diffusion gradient
esposes the cells to a wide range of concentrations; (c) comples
requirements can be detected as tlirbidit!, at the common boundar),
of 2 zones (to take full advantage of this, the single supplements
should be strategicall!. placed in relation to each other), and (d)
the supplements need not be sterilized. I\`hen small inocula, of the
order of 1000 cells /plate, are used with a single supplement /plate,
this method is the most reliable to study the requirements of
unstable, relrersible mutants.

Comment 618 Bernard D. DaLIis

Although agar has been suspected of contamination \\-ith interfering
amounts of vitamins, \ve have found only negligible impurities. Unll.ashed
Difco agar provides no growth stimulation for mutants of E. coli requiring
pantothenate and thiamine and permits only microscopic to barely visible
gro\vth of streaks of mutants requiring pyridosine, PABA, biotin, niacin
and p-alanine. This slight background grolvth can be largely eliminated h)
\\-ashing the ;rgar.

Alany or most nutritional mutants ha\-e the potentiality oC
"adapting" so that thcv can grow in minimal medium in tlic
absence of' the ~ubstanc.e I\.hich thw originally required. Although
this process supcrficiall>~ suggests a direct action of the environ-
nient in educating the nilltant bacteria to dispense lvitli tlicil
cxst~vhilc recjuirciiicnts, a more thorough anal!xis indicates that
the aclal>lation is probably tile reslilt of spontaneous mutations
toward nutritional competence, the reverted mutants being
strongly selected for by the miniinal medium (31, 32). Limited
in\,estigation in Kceuros~~oi~a atid F1. co/i has shoddy that these mu-
tations arc usually l,ack-nlLltationc;ls restoring the wild-type concli-
tion geneticall\, as M-e11 as ph~~siologicall~~; some apparent rcl-er-
sions, lio~~ever, arc the result of "niodifiel:" or "suppressor" mu ta-
tions, iii\-011.ing further gent mutations I\.liicli merely miinic the
wild type (11).

The possibilit), of reversion IIILISL be considered in all nutritional
and metabolic studies on micro-organisms, be they artificial mu-
tants or microbes that carry nutririonnl deficiencies, probably


-


I

      BIOCHEMICAL MUTANTS OF BACTER1.A      Ii
originated by mutation, when isolated from nature. This inter-
ference is best countered by carrying mutant stocks on adequate
Illedium, so that random reversions will not be selected for; by
{I-equent reisolations, to get rid of accumulated reversions, and,
;,l)o~.e all, by verifying all critical or doubtful responses by reinocu-
l;,tion into minimal medium to insure the persistence of the nutri-
t icjljal requirements.

SYXTROPI-IIS;\I (16)

oven lvhen biochemical and genetic information is not avail-
able, mutants with similar nutritional requirements can often be
distinguished by syntrophism (mutual feeding) , As has been
demonstrated most clearly in Neurospora work, genetic blocks in
the biosynthesis of an end-product may result in the accumulation
and excretion of the intermediate whose further utilization is
blocked. The excreted intermediate may be capable of stimulating
the growth of another mutant which is unable to make the same
end-product but which is blocked at an earlier step in the reaction
chain,. The ability of a mixture of mutants to grow on minimal
medium is a definite indication of physiologic differences bet\\-een
them and, by inference, of genetic nonidentity.

PROCEDURE

The most sensitive test for syntrophic interaction is probably
a quantitative comparison of the turbidity of separate and mixed
cultures which are supplied with concentrations of the gro\<-th
factor requirement which limit the individual mutants to about
10% of optimal. Syntrophism can also be demonstrated by streak-
ing the 2 cultures across or near each other on a minimal agar
plate.

Evalrtntion.-This test is roughly comparable to the hetero-
caryon test for "allelism" in Neurospora (p. 61). A syntrophic
reaction indicates physiologic difference, and probably, though
not necessarily, nonallelism. Failure to obtain effecti1.e syn-
trophism does not prove physiologic or genetic similarit!-. Lam-
Ilen et crl. (14) have successfully applied this method to the classi-
fication of a group of cystine- and methionine-requiring mIltants
of E. coli.

Cornmolt by Bernard D. Dacis

The increased grotvth of a mixture of mutants, compared with their
separate growth, is usually predominantly or exclusively an increase in the
xro\\4~ of 1 component at the expense of an intermediate accumulated by
the other component. If the term "syntrophism" is used to mean the feeding
of 1 strain by another, mutual s\ntrophism is a special case, occurring, for

I
i  -


18       GESETICS OF h,IIC:RO-ORGr~i\;ISMS
example, with certain tyrosine- and phenylalanine-requiring mutants. \Yc
have found parallel streaking to be the most convenient and useful test for
syntrophism, since it tells at a glance which strain is being fed (Ga). -4
possibly more sensitive test for syntrophism is the satellite phenomenon
observed in a pour plate containing lo-100 feeder cells surrounded by a
large number of cells of the strain to be fed ( 1 O-`--IO").

FER\fENT,-\TIOS IVUT.ASTS

Fermentation mutants ha1.e pro~~~l useful in genetic and them-
ical work in E. co/i (17, 20, 7). Thev are readily obtained and
classified and are usually distinctive and independent in beha\-ior.
Their most useful attribute is their ability to be classified by
colony appearance on a plate lvithout further test. This permits
of their application to population. segregation and other studies,
\i.here counts of the relative proportions of 2 genetic t?-pes must
be made.

The essential for finding and using fermentation mutants i$ an
indicator medium which will permit of colony classification.
Eosin-methylene blue agar (see Table 1, 1~. 5) is the most reliable
and informative indicator medium tried. On this medium, colonies
oreven sectors of colonies can be scored for fermentative capacit),,
even on a crowded plate. Colonies -cvhich ferment the sugar take
up a dense purplish black coloration, often with a green sheen,
while nonfermenters remain of a white or pink color, later some-
times turning a transparent blue, which indicates alkali produc-
tion. Slow or late fermenters may assume \.arying shades of purple,
all against the dark but transparent orange-purple background.
Particularly when the colonies are crowded, the contrast does not
falror the finding of nonfermenters, but close examination will
usually permit their discovery.

PROCEDURE

The most convenient method of obtaining fermentation mu-
tants has consisted in spreading a drop (about 0.05 ml) of a fully
grown culture of E. coli in any complete broth over the surface
of each of a number of EMB plates lvith appropriate sugars. Each
plate is then exposed to ultraviolet light long enough to reduce
the colony survival to 200-400. 1\`ith a Hanovia high pressure
lamp, 125 w at 15 cm distance, this requires about 7 sec. *After
18-24 hr the plates are examined for mutant colonies and sectors.
With lactose, maltose, galactose, etc., yields of 0.02-O.lOyO of non-
fermenting mutants are found after this heavy dose of radiation.
The mutants often occur as sectors, indicating that a mutated


      BIOCHEMICAL MUTANTS OF BACTERIA      19
nucleus may segregate from an unmutated 11uc1eus in the forma-
tion of the colony, as suggcstcd earlier. The mutants are tested
and purified by streakin g them out on EXfB agar. After they arc
purified, they should be tested for their fermentation reactions
on other sugars, since different patterns of effects may occur (`i).

.\lociiJirntions.-For large scale platings, triphenyltetrazoliunl
cllloride may be a useful indicator in place of EMB (18) . The
indicator is added (after being sterilized separately) to a 0.0570
concentration in nutrient agar-sugar medium. The acid produced
1~1, fermenters inhibits the intracellular reduction of the indicator
td a colored, water-insoluble formazan, which gives a brilliant
red color to the nonfermenters. Since the background is neutral,
the contrast here favors the mutants. Unfortunately, although
most TUIIS with tetrazolium have given excellent results, occasion-
all!. all colonies on a batch of plates, fermenters or not, take up
enough pigment to obscure the results. For further study and
verification, it is adl.isabIe to transfer suspected fermentation
mutant, to EMB agar.

APPLICATIOSS OF BIOCHEMIC.SL RlUTASTS

7`1~ most generally interesting use of biochemical mutants is
probably in the stud!, of biosyntheses of growth factors. Alost
work of this type has been done with mutants of fungi (3) and
Tvitli naturally occurring, esacting organisms such as lactobacilli,
but bacterial mutants ha1.e begun to assume a prominent role
in this type of work. I\`e may cite studies on the synthesis and
utilization of proline (33), ' tyrosine (34), purines (9), sulfur
amino acids (14), p-aminobenzoic acid (13) and isoleucinc (cf.
3). In addition, mutants 1lal.e been used in the analysis of such
diverse processes as light production in luminous bacteria (24),
nitrogen fixation (37) and synthesis of starch from maltose in
E. co/i (17). There is every reason to believe that nutritional mu-
tants could compete fal.orably with the lactobacilli in the search
for new vitamins, especially since most workers who have pro-
ducecl bacterial mutants have accumulated cultures that respond
to !`east extract but not to any known pure substance. However,
this expectation remains to be realized.

AJJong the same lines, mutants should be suitable for micro-
biologic assays of various vitamins, amino acids and nucleic acid
derir-atilres. However, this al'enue has been pre-empted by the
painstaking work alreadv done with lactic acid bacteria and lvith
Seurospora mutants. E&ept for substances for which suitable
assay methods are not \.et a\.ailable, the reward does not seem


20        GENETICS OF MICRO-ORGANISMS
to justify the tedious investigations needed to perfect this potential
application.
Aside from these more biochemical uses, biochemical mutants
are indispensable in genetic studies on bacteria. In the first place,
they provide excellent "markei-5" for studies in natural selection
and experimental evolution, i.e., in population dynamics. Fer-
mentative mutants are especially useful here, as the!, can be
enumerated by inspection on indicator media.
Second, nutritional markers ha\re proved especialI), useful for
the genetic analysis of the sexual cycle that has been discoi.ered in
E. coli, strain K-12 (36). The necessity for genetic methods to
investigate the question of sex in bacteria is clearly shown by the
ambiguity and inconclusiveness of the morass of previous c!,tologic
work. In principle, any sufficiently stable genetic markers can be
used to look for the recombination of characters, which is the
function and the proof of sexual reproduction; nutritional require-
ments have the special advantage that they allow for the efficient
selection of minute numbers of recombination types amidst large
populations of the parents.
The selection for recombinants can be accomplished by using,
as test parents, complementary nutritional mutants, such that each
is competent to synthesize the requirements of the other, e. g., a
biotin-methionineless and a threonine-leucineless pair. Double
mutants of this sort must be used to obviate ambiguity of results
caused by reverse mutation. If genetic recombination occurs
between these strains in a mixed culture, some of the resulting
recombinants should be prototrophic. The prototrophs can, of
course, be readily selected by plating washed suspensions of the
mixed culture into minimal agar medium.
The recovery of prototrophs (at the rate of about 1 for each
million mutant cells inoculated) from mixed cultures, and not
from the parents kept separately, was presumptive evidence for
recombination in strain K-12 (36). Much more pertinent evi-
dence was provided, however, with the help of other markers such
as phage resistance and sugar fermentations, which are not directly
affected by selection for prototrophs. If the prototrophs are the
result of recombination, unselected markers should likewise be
reassorted among the prototrophs, and this was found to be the
case.
The segregation behavior of unselected markers has led to the
inference of a linear linkage system, or chromosome, in this bac-
terial strain. The typical life cycle resembles that of the asco-
mycetes, with a transient diplophase and a vegetative haplophase
(21). Mutant stocks have been found, however, in which the diplo-


      BIOCHEMICAL XiIUTANTS OF BACTERIA      21
+ase may be prolonged, thus paralleling a transition from the
Zygosaccharomyces to the Saccharomyces type of life cycle. How-
ever, the diplophases currently available show abnormalities in
their segregation behavior whose basis is not yet clearly under-
stood, but which preclude a facile application to genetic prob-
lems (20).

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