THE CHEMISTRY AND METABOLISM OF BACTERIA
         BY J. HOWARD MUELLER
       Department of Bacteriology and Immunology
     Howard Medical School, and School of Public Health
              Boston, Massachusetts
It has been necessary to confine the material to be discussed in this
review to certain aspects only of the very broad subject. This course
has been dictated not only by the limitations of the reviewer but also
necessarily by the exigencies of the moment, and by the consideration
that certain portions of the subject will undoubtedly be reviewed under
other headings. Thus, many of the contributions to bacterial nutrition
and to the use of bacteria for purposes of bioassay can appropriately
be included in the more general subjects of vitamins, amino acids, etc.
The present review is limited to (a) a discussion of certain aspects of
the chemical composition of the bacterial cell and some of its products,
(b) that portion of the current work on bacterial nutrition which re-
lates to new and unidentified growth requirements, and (c) some
phases of chemotherapy.

        COMPLEXITY OF THE BACTERIAL CELL
One of the most remarkable properties shared by many varieties of
bacteria is the existence of a multiplicity of immunological types for
each species of organism. In a number of instances, for example, the
pneumococci in which there are mor.e than thirty such different types,
the differences have been found to be concerned with the existence of
chemically different polysaccharides which make up the mucoid cap-
sules surrounding these organisms. Moreover, under certain condi-
tions, these bacteria may undergo a degenerative change or "dissocia-
tion," in which the capsules and the immunological type specificity
disappear. With the loss of the capsule, virulence also disappears.
Colonies of such degenerated pneumococci are smaller than those
formed by the encapsulated cells and have lost the glossy surface char-
acteristic of the latter. The terms "smooth" (S) and "rough" (R) are
commonly used to describe the original form and its variant. In gen-
eral, rough forms from all types of pneumococci are indistinguishable
either culturally or immunologically.
By certain procedures it is possible to reverse this change and to
obtain smooth forms from the degenerate rough forms. Under normal

                     733


734                 MUELLER
conditions the original type specificity is regained, that is, a rough
Type II reverts to a smooth Type II. However, Griffith (1) in 1928
showed that a rough culture obtained from Type II could sometrmes
be converted to a smooth form with different specificity (e.g., Type
III) by injecting mice with a small inoculum of living rough Type II
and a large quantity of heat-killed smooth Type III pneumococci. The
Type III smooth, virulent organisms so obtained could be maintained
indefinitely on suitable media, and it thus appeared that a pneumo-
coccus of one type could be permanently changed to another type by
passing through an intermediate rough form. These experiments,
adequately confirmed, were extended by Dawson & Sia (2) who de-
vised an in vitro method of accomplishing the same result also using
living R forms and heat-killed S forms. Still further refinements were
introduced by Alloway (3) in which Berkefeld filtered, cell-free ex-
tracts of the S cells were substituted for the whole, killed organisms.
Avery, MacLeod & McCarty (4) have now announced the isola-
tion from smooth Type III pneumococci of a substance, provisionally
identified as a polymerized desoxyribonucleic acid, which in minute
amount possesses the property of inducing the R + S transformation
involving change in type specificity. As little as 0.003 ug. in 2.25 cc.
of culture fluid was effective. Ultracentrifugation and cataphoresrs
gave results supporting the conclusion that the nucleic acid polymer
was the active substance. Ultraviolet absorption spectra were charac-
teristic of nucleic acid. A tentative estimate of the molecular weight,
based on the physical data obtained, led to the figure 500,000. Solu-
tions of the substance were relatively devoid of precipitability by im-
mune sera capable of reacting in high dilution with pneumococcus pro-
tein or with Type III specific carbohydrate. In other words, it appears
that a polymer of a nucleic acid may be incorporated into a living, de-
graded cell, and will endow the cell with a property never previously
possessed, namely, the ability to produce a capsule composed of a com-
plex polysaccharide entirely different in structure from that produced
by the smooth organism from which the degraded form was originally
derived. When thus induced the function is permanent, and the nucleic
acid itself is also reproduced in cell division. The importance of these
observations can scarcely be overestimated and stimulates speculation
concerning such matters as the chemical basis for specificity in nu-
cleic acids, and the genetic implications presented by the ability to
induce permanent mutation in a cell by the introduction of a chemical
substance. Such speculation may well include considerations of the

         CHEMISTRY OF BACTERIA         735
relation of this phenomenon to the sequence of events following the
introduction of a filterable virus (or a bacteriophage particle) into a
susceptible cell. A brief consideration of the facts now accumulated
concerning the chemical composition of the viruses may bring this rela-
tion into perspective.

It is generally recognized that the most outstanding characteristic
of filterable viruses as a group is their failure to multiply outside of the
host cell. Beyond this single common property there are wide differ-
ences in size and in chemical complexity. From the upper extreme
represented by the visible elementary bodies of vaccinia and psitta-
cosis viruses and (for the sake of uniformity of concept) the bacillary
forms called rickettsiae, down to the crystalline plant viruses and
such agents as poliomyelitis and foot and mouth disease viruses which
approach the dimensions of large protein molecules, there are repre-
sentatives of a wide range of sizes. Similarly, in regard to chemical
complexity, while all viruses contain nucleoprotein and the smallest
may consist of single molecules of this material alone, the larger ones

approach the bacterial cell in complexity, containing lipoid, polysac-
charides, and even certain enzymes. Thus as a group viruses may
reasonably be considered as living cells deficient in a range of vital
functions which must be supplied by a suitable host cell. For ex-
ample, the larger viruses may be devoid of oxidative mechanisms
only, but retain many synthetic capabilities. The latter drop out pro-
gressively as size and complexity decrease until nothing remains in
the smaller viruses but a large molecule of nucleoprotein multiply-
ing as a result of the combined vital processes of the host cell.
Considerable definite information relating to the chemical compo-
sition of the largest and the smallest of the viruses has been available
for some time. Information has now been provided by Taylor and
others about viruses of intermediate size (5, 6, 7)) namely influenza
viruses A and B and swine influenza virus. Lipoid components varied
from 21 to 24 per cent among the three, and included neutral fat
phospholipid, and cholesterol. The remainder consisted of polysacl
charide, protein, and desoxyribonucleic acid. Even these viruses of
intermediate size, therefore, are still relatively complex entities. It
would be interesting to know whether they still possess certain en-
zymes such as phosphatase which has been found to be present in
vaccinia virus (8) though absent from small viruses such as the poly-
hedral virus of Lywuzntria monacha L. (9), and tobacco mosaic and
bushy stunt viruses (10). W'll' 1 rams, Schlenk & Eppright (11) re-


736            MUELLER
norted the absence of the various components of the B vitamin com-
I
plex from tobacco mosaic, tobacco necrosis, and bushy stunt viruses
and from a strain of influenza A virus. They believe such absence
carries implications of the inanimate nature of viruses. In the light
of the above discussion it seems probable that the cellular functions
dependent upon these readily extractible substances may well be miss-
ing from even the largest viruses, which may yet retain so complex a
make up and properties so varied as to bring their inanimate nature
into considerable question.

The one component common to all viruses, therefore, is nucleo-
protein, while in the case of the substance inducing variation in the
pneumococcus, nucleic acid alone is involved. In the latter case a
synthetic and purposeful change is brought about, i.e., the ability to
produce a polysaccharide capsule rendering the cell resistant to cer-
tain destructive influences. On the other hand, so far as is known,
viruses lead to degenerative changes in the host cell and eventually to
its death. Conceivably, therefore, the. crucial difference between the
transplanted "gene " and the smallest virus may lie in the specificity
of the protein which is present in the latter, or to which the former
probably attaches itself in the cell. In the one case the protein heterol-
ogous to the cell, renders the particle a completely foreign parasitic
molecule, which eventually becomes injurious as it accumulates. In
the other instance, the cell's own protein combines with the new
nucleic acid, and the resulting homologous nucleoprotein takes its
place in the normal economy of the cell and confers a new property
upon it. In any case, these considerations again indicate the enormous
complexity of cellular and bacterial protoplasm, and emphasize our
complete ignorance of the many and varied metabolic processes which
proceed inside the cell wall.

As further illustrations of the great complexity of the bacterial
cell a few instances from the contributions of the current year may be
mentioned, selected primarily because of some bearing on medical
bacteriology.

Stockinger, Ackerman & Carpenter, for example, reported exten-
sive studies of the composition of the gonococcus (12) and its prod-
ucts (13). They described two nucleoprotein fractions obtained by
extraction of mass cultures. One of these contained considerable com-
bined lipoid. Carbohydrate was found in three different forms, but a
polysaccharide having type specific properties was not detected. A
variety of lipoidal constituents were present, among which a lecithin,

CHEMISTRY OF BACTERIA          737

a cephalin, and sphingomyelin were identified. From broth in which
the gonococcus had been grown, they isolated a protein, believed to be
a degradation product of the cellular nucleoprotein. It was moderately
toxic to animals and possessed immunological specificity as measured
by complement fixation. Boor & Miller (14) prepared carbohydrate-
lipid complexes from a number of strains of gonococcus and menin-
gococcus. These "glucolipids" were also moderately toxic for animals
and were antigenic in rabbits. Sera so produced precipitated both
the glucolipid itself and the carbohydrate component separated by
acid hydrolysis. These complexes, therefore, have properties similar
to compounds of the same sort prepared earlier by Boivin & Mesro-
beanu (15, 16) from a variety of gram negative bacilli.

Kabat, Kaiser & Sikorski (17) have prepared a polysaccharide
from Type I meningococci which is electrophoretically homogeneous,
and which is type-specific. It is weakly but definitely antigenic in
man. Direct comparison with an earlier product obtained by Scherp
& Rake (18) indicates that the avoidance of acid or alkali durinEr

the preparation of the more recent material has resulted in a some-
what purer and more nearly natural antigenic substance.
An antigenic capsular polysaccharide has been obtained from Cl.
perfringens by Svec & McCoy (31). It appears to be common to

                                   --               --- _-
most members of the perfringens group, regardless of their toxigenic
properties.

The tremendous amount of investigation on the chemistrv of tu-

                                                 ,     
berculin carried out in the last two decades has been reviewed during
the year by Seibert (19), particularly in regard to carbohydrates,
nucleic acid, and protein. Corper & Cohn (20) have compared the
tuberculoprotein obtained from autolyzed tubercle bacilli with that
present in culture filtrates, and found similar properties, together
with greater purity, in the former. Chargaff & Moore (32) described
the isolation of glycogen from tubercle bacilli. The molecular weight
of the material was shown to be of the order of 12 to 13 million. An-
derson and his collaborators (21, 22, 23) have added to an already
extensive investigation of the lipids of the tubercle bacillus, and car-
ried out similar studies (24, 25) with Plzytomonas tumefaciens, in-
cited by the possibility that some of its lipid material may be respon-
sible for the ability of the organism to induce plant galls. An unidenti-
fied fat acid with the formula &H,,O, was isolated. It is believed
to possess a branched chain.

The capsular material of certain hemolytic streptococci, identified


738                 MUELLER
some time ago as hyaluronic acid, has aroused considerable interest
as to its possible relation to the virulence of this organism. Some evi-
dence pointing in this direction has been adduced by Kass & Seastone
(26). Other bacteria and even some strains of hemolytic streptococci
may secrete an enzyme, hyaluronidase, capable of hydrolyzing this
acid. This enzyme appears capable of acting on the tissues of the
body at the site of infection in such a way as to lead to a rapid dis-
semination of particulate material (India ink, bacteria) and is an im-
portant member of a group of substances called "spreading factors"
which share this property. This subject was reviewed in 1942 by
Duran-Reynals (27). In the current year, however, Crowley (28)
has examined a considerable number of strains of hemolytic strepto-
cocci for hyaluronidase production, and finds no relation between this
substance and virulence. Similar results have been obtained by Hum-
phrey (29) for various pneumococci, and by Evans (30) for the
Welch bacillus. The significance, therefore, of the production of
hyaluronic acid by some bacteria, and of hyaluronidase by others is
not yet clear.

GROWTH REQUIREMENTS OF BACTERIA

As already stated, it is not proposed to review completely the
voluminous literature which continues to accumulate on bacteria1
growth requirements. The significant developments of the year will
quite surely be reviewed elsewhere in this volume. It may not be out
of place to point out at this time the accuracy of the prediction made
by the writer more than twenty years ago (33) that definite knowl-
edge of bacterial growth requirements would quite certainly supply
information of considerable value in fields other than bacteriology.
The incredibly close relation between the nutrition of animals and
that of microorganisms, and the present concept of certain types of
chemotherapeutic action more than justify the earlier optimism.
The bacterial culture still appears to offer the most convenient
tool for the definition of unrecognized nutritional substances, for the
existence of which there is ample evidence. The factor which seems
nearest to complete elucidation is "folic acid." The rather chaotic
facts bearing on the chemistry and physiology of this and related
substances have been recently reviewed (34). Probably nothing can
bring greater testimony to the change in status of bacterial nutrition
in the last twenty-five years than the present state of affairs in wl63
most of the pharmaceutical companies in the country are competing

           CHEMISTRY OF BACTERIA          739
for first place in clearing up this tangle. The writer recalls somewhat
grimly the difficulties encountered in 1920 while attempting to enlist
cooperation in getting. a hundred pounds of casein hydrolyzed with
sulfuric acid, from which the first of the unrecognized growth factors
for bacteria (methionine) was eventually isolated.
There are a number of observations of other probably new growth
factors. Their relationship to each other, to "folic acid," and to fac-
tors already more accurately defined must await chemical identifica-
tion. Happold and his collaborators (35, 36) have described a sub-
stance occurring in acid-hydrolyzed casein, and more abundantly in
liver extract, which is required for the growth of certain strains of
C. diphtheriae and of L. casei. From liver extract a considerable de-
gree of purification has been achieved, and certain of the properties
of the material have been defined. It is not replaced by folic acid. A
second factor greatly stimulating early growth of L. casei appears
also in liver extract, and behaves in such a manner as to lead the
authors to believe it may be identical with a growth factor for L. casei
described by Pollack & Lindner (37) as present in Wilson's bac-
teriological peptone. The latter authors describe the substance as
being stable at 100 between pH 2 and 11, but reduced in potency to
half its original strength by heating at pH 12 and destroyed by 0.7
N sulfuric acid. Happold's factor, however, is stated to be adsorbed
by norite at pH 3, whereas the substance in peptone is said not to be
adsorbed to any extent by charcoal (Darco G-60) nor by a variety of
other adsorbents between pH 3.0 and 8.0. A third factor for L. casei
is described by Happold as occurring in liver extract in a combined
and inactive form. Gentle hydrolysis splits it off in an active condition.
Smith (38) reported the presence in yeast extract of a growth
stimulant for Streptococcus lactis. It failed to precipitate with the
heavy metal salts and was not adsorbed by charcoal (Darco) or fullers'
earth.

Sprince' & Woolley (39) point out the similarity between the
second Happold factor (above) for L. casei, the Pollack & Lindner
factor for the same organism, the Smith factor for S. lactis, and a fac-
tor for the hemolytic streptococcus previously described by Woolley
(40). Some evidence for this similarity is presented, and the name
"streptogenin" is tentatively suggested for the substance. The efficacy
of a number of concentrates of "solubilized liver extract" prepared in
various ways was compared on the three varieties of bacteria and in
each case the relative recovery of active material was the same for all


740                 MUELLER

three organisms. One of the preparations employed was not made

from liver extract but from partially acid-hydrolyzed vitamin-free
casein. The "per cents of recovered activity" for the three organisms
are stated to be 17, 19, and 18 respectively. It is not clear to the re-
viewer what value, in this case, is taken as 100 per cent, since both
the original casein and its complete acid hydrolysate are said to be
inert. In any event decision on the relationship of these several growth
manifestations must await the isolation or better characterization of
an active material.

Evidence for the existence in tomato juice of an unidentified stimu-
lant for growth of L. arabinoszls has been given by Kuiken and col-
laborators (41). It is prepared by adsorption on charcoal and elution
with a mixture of pyridine, alcohol, and water. It is stable to strong
acid hydrolysis, but its properties are not further described.
Ballentine et al. (42) have studied a growth factor for CL per-
fringens occurring in yeast extract. After precipitation by lead hy-
droxide, the substance was recovered with hydrogen sulfide and im-
purities removed with the resin Amberlite IR-4 followed by norite
adsorption. It was stated to be precipitable by mercury and by picric
acid but not by a variety of other reagents. It was stable to cold normal
acid and alkali though inactivated by both in five minutes of boiling.
Welton, Stokinger & Carpenter (43) have presented the com-
position of a synthetic medium for the growth of the gonococcus. One
of the components is indole-3-acetic acid, and thus by implication this
substance is essential for the growth of the organism and must be
considered to be a potential bacterial growth factor. So far as the
reviewer is aware, this is the first available evidence that a plant auxin
may function as a growth stimulant for bacteria, and the observation
deserves further amplification.

Gould (44) has found that certain passage strains of the gonococcus
develop a requirement for glutathione. This appears to be the first
instance of the identification of this substance as a bacterial growth
factor. The strains which depend upon the presence of this material
for growth are inhibited by low concentrations of cystine, possibly
through a blocking or competitive action in some essential system.
Finally, the presence of two chloroform-soluble components of
liver extract which promote the growth of Lnctobacillm helvetims
and Streptococcus la.ctis has been described by Barton-Wright, Emery
k Robinson (45). These substances are not destroyed by nitrous
acid, or by acetylation or benzoylation. They are unique among the

CHEMISTRY OF BACTERIA          741

factors mentioned in this review because of their solubility in chloro-
form. The authors promise a further description. It is possible that
fatty acids or other lipid material may be at least partially responsible
for the effects noted. Oleic acid and oleates are known to act as
growth stimulants or depressors (according to concentration) for
the diphtheria bacillus (46) and the tetanus bacillus (47). Moreover,
Kodicek & Worden (48) have recently directed attention to a variety
of changes in the growth of L. helveticus obtainable with materials
which are chloroform-soluble and almost certainly occur in liver prep-
arations. Their results showed that growth of the organism is aug-
mented by palmitic and stearic acids, though inhibited by oleic, linoleic,
and linolenic acids. The effect of linoleic acid is reversed by either
cholesterol or lecithin.

CHEMOTHERAPBJTIC ACTION

Evidence continues to accumulate in support of the view, first
expressed by Fildes (49)) that chemotherapeutic substances operate
by blocking some specific chemical grouping necessary for the success-
ful growth of the microorganism. The empirical observation of the
value of sulfanilamide and other related drugs as therapeutic agents
was followed by the discovery of a specific inhibitory substance in
tissue extracts, and later by the provisional identification of this sub-
stance as p-aminobenzoic acid (50). Subsequently this compound was
shown to be essential for the growth of certain bacteria, and therefore
presumably to function as a component of some metabolic system.
Sulfanilamide appears to be sufficiently similar in structure to combine
at the same point, but the next step in the reaction fails to occur and
the metabolism of the cell is arrested. The relatively low toxicity of
the sulfonamides for animals may be due either to production in the
cells of sufficient p-aminobenzoic acid to prevent the blocking or to
the ability of tissues to replace.the blocked function by some alternative
mechanism.

A wide variety of compounds of the general nature of p-amino-
benzoic acid and sulfanilamide, but with various substituent groups
at different positions, have been tested both as bacteriostatic substances
and as inhibitors of sulfonamide stasis (51 to 55), and certain general
relationships appear to have been established. The nature of the
process which normally iflvolves p-aminobenzoic acid remains unde-
termined. There is evidence, however, that two of the sulfonamides,


742                 MUELLER
sulfapyridine, and sulfathiazole, also block a second function which
is concerned -with a nicotinamide-stimulated respiratory process (56,
57, 58). This latter effect is not prevented by p-aminobenzoic acid.
Moreover, in recent work, Reed, Orr & Reed (59) describe differences
between in vitro and in tivo effectiveness of the sulfonamides on the
gas gangrene group of bacteria and intimate that evidence is to be
forthcoming that two types of drug inhibition may exist.
Differences between species of bacteria in relation to sulfa drug
susceptibility are of considerable interest. That within the same
species may be found both susceptible and resistant strains, and that
the former can apparently acquire the properties of the latter either
i~ vitro or in viva, are facts of the utmost practical importance. Re-
sistance to the action of one of the sulfonamides usually extends to
all the others, and once established, the property seems to be perma-
nent. In the test tube, the change may be brought about by exposure
to sublethal concentrations of the drug. The same mechanism probably
operates in the body. It is consequently not surprising that evidence
has already begun to accumulate that certain human infections origi-
nally yielding in most instances to drug therapy may become in-
creasingly refractory to this type of treatment. The widespread usi
of small doses of sulfadiazine for the prophylactic checking of such
bacteria as the meningococcus, gonococcus, and streptococcus would
appear especially suited to the elimination of susceptible strains and
their replacement by others, equally virulent, but so altered met-
abolically as to be highly resistant to the sulfonamides. That this is
more than a theoretical possibility is illustrated by the fact (60) that
in 1940-41 about 70 per cent of cases of gonorrhea treated in one

Boston clinic were promptly cured by sulfonamide therapy, while the
remaining 30 per cent were unaffected. At the present time the pro-
portions are approximately reversed, and but for the timely introduc-
tion of penicillin it seems probable that the chemotherapy of this infec-
tion would shortly have become relatively ineffective.
  Experimental demonstration of the acquisition of drug resistance
in vitro has been made by numerous workers (e.g., 61, 62, 63). The
possibly complex nature of the change is indicated by the observation
(64) that whereas pneumococci which have acquired a high degree of
resistance to sulfonamides appear to remain permanently in that state,
partial resistance may be lost rather promptly when contact with sul-
fonamide is discontinued. In some instances, at any rate, resistance
appears to be due to increased production of p-aminobenzoic acid

            CHEMISTRY OF BACTERIA       743

(65), but not all naturally resistant organisms produce extracellular
inhibitors (66). Fortunately resistance to the sulfonamides does not
parallel that against penicillin (67). Information on the mode of
action of this antibiotic is not yet available.

A peptide containing ten or twelve glutamic acid residues linked
to the carboxyl group of p-aminobenzoic acid has recently been iso-
lated from yeast (68). It is not antagonistic to the sulfonamides until
hydrolyzed by acid.

In view of the clinical success attending the use of the sulfonamide
drugs, and of the development of a well supported theory of their
mode of action, it is not surprising that a number of attempts have
been made to explore the possibility of using derivatives of other known
growth factors as chemotherapeutic agents. Thus, McIlwain (69)
has shown that pyridine sulfonic acid inhibits the growth of organisms
requiring nicotinic acid or nicotinamide and recognized three "types"
of inhibition depending upon the individual circumstances. The sul-
fonic analogue of pantothenic acid, "pantoyltaurine," has been pre-
pared by Snell (70, 71) and. shown to inhibit the growth of bacteria
which require pantothenic acid. The inhibition was reversed by
additions of the latter. Similar observations were reported inde-
pendently by Kuhn, Wieland & Moller (72) who also observed the
specific antagonistic effect of pantothenic acid and, in addition, noted
a partial annulment by p-alanine. McIlwain (73) has obtained similar
results and shown that the effect is inhibited by pantothenic acid and
by p-alanine. Certain strains of streptococci were resistant to the
action of pantoyltaurine. This property was entirely unrelated to
sulfonamide resistance (74). Neither was it related to the requirement
of pantothenic acid for growth, which was common to all strains
examined. Resistant strains could be rendered susceptible by salicylate
and it is suggested that this fact ,may be evidence for the existence
of alternative mechanisms for the same function, one involving panto-
thenic acid, the second blocked in some way by salicylate. In the case
of the diphtheria bacillus, McIlwain was able to effect the transposition
of strains susceptible to the action of pantoyltaurine into resistant
forms by "training" those originally requiring pantothenic acid to
produce their own (75). This was accomplished by gradual with-
drawal of pantothenic acid and demonstrates that drug resistant strains
may develop without the presence of the drug itself. Certain of the
above observations have been extended by quantitative studies of the
fermentation of glucose by streptococci under circumstances in which


MUELLER

pantothenic acid and pantoyltaurine can be shown to exert antagonistic
effects (76, 77).

McIlwain & Hawking (78) have shown that in rats, pantoyltaurine
acts as a moderately effective chemotherapeutic agent against hemo-
lytic streptococci. It is ineffective in mice, which have a higher level
of pantothenic acid in the blood. The possibility is pointed out that
pantoyltaurine may be of use in human streptococcus infection, since
the concentration of pantothenic acid in human blood is even lower
than in rat blood.

Nielsen & Johansen (79,80) have shown that with a yeast, stimu-
lation of growth induced by p-alanine is prevented by p-amino butyric
acid and by isoserine, but not by taurine.

The pyridine analogue of thiamine, "pyrithiamine," shown by
Woolley & White (81) to induce symptoms of thiamine deficiency
when fed to mice, was later found by the same authors (82) to inhibit
the growth of bacteria requiring thiamine or its component moieties.
Again, the effect was reversible by the natural vitamin. A resistant
strain of yeast was obtained by growth in the presence of the inhibitor
(83). This strain, however, still required thiamine or its pyrimidine
portion as a growth factor.

Studies on possible modifications of biotin are beginning to appear.
Desthiobiotin, described by du Vigneaud and his associates (84) can
replace biotin for growth of a yeast but not of L. casei. It was later
shown (85) that in the case of the latter organism, the substance in
suitable concentration inhibits growth, acting as a specific antibiotin.
Biotin sulfone and imidazolidone caproic acid exert a similar effect
(86) while a number of other derivatives and related compounds
either partially replaced biotin or were inert. The ability of certain
of these compounds to replace biotin fro.m its union with avidin is
also reported. Lilly & Leonian (87) investigated the effect of desthio-
biotin on a large number of microorganisms, which they were able
to classify as to their behavior toward the compound. Antibiotin
activity was manifested toward some but not all varieties unable to
use desthiobiotin.

Modification of riboflavin by substitution of two methyl groups by
chlorine yielded a product showing bacterial growth inhibitory prop-
erties for certain organisms (88). The inhibition was reversed by
riboflavin.

That modifications even of the amino acids may result in com-
pounds which interfere with normal bacterial growth is evident from

           CHEMISTRY OF BACTERIA          745
McIlwain's (89) experiments with sulfonic analogues of glycine,
alanine, valine, leucine, and aspartic acid. Delay or inhibition of
bacterial growth resulted from their presence, but the effect was readily
reversed by natural a-amino acids, regardless of specific structure.
Fox, Fling & Bollenbach (90) obtained considerable inhibition of
growth of L. arabinosus, which requires I-leucine, by means of the
unnatural isomer. This observation should be amplified, since syn-
thetic amino acids have been universally employed in bacterial growth
experiments. In the work cited the proportion of d to 1 form is given
as 200 to 1. Since general experience has led to the belief that the
forms not occurring in nature are sometimes used quite as readily as
the natural forms, in other instances are inert and occasionally have
exhibited anomalous behavior (see for example 91-93) a further
quantitative as well as qualitative investigation of the phenomenon is
desirable.

The facts thus far available in connection with the effect of chemo-
therapeutic agents on diseases due to filterable viruses are generally
in harmony both with the commonly held explanation of sulfonamide
action and with the view that viruses are dependent upon host cells
for many of their functions. Thus, while it has been known for some
time. that the sulfonamides were moderately effective against such
large viruses as psittacosis and lymphogranuloma, they are completely
inert in the case of the smaller agents (94 to 97). The typhus fever
rickettsia appears to be moderately susceptible to penicillin, and, curi-
ously, to p-aminobenzoic acid (98), a fact for which no ready expla-
nation seems obvidus. It is possible that as more knowledge of the
vital processes remaining to intracellular parasites becomes available,
some form of systematic approach to their chemotherapy may be
found. Thus far, the empirical attack has been entirely negative al-
though a wide variety of organic compounds of various types has been
investigated (99).


746                  MUELLER                                               CHEMISTRY OF BACTERIA          747

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748                 MUELLER

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     DEPARTMENT OF BACTERIOLOGY AND I~~MUNOLOGY
  HARVARD MEDICAL SCIIOOL AND SCHOOL OF PUBLIC HEALTH
           BOSTON, MASSACHUSETTS